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Mechanosensitivity and Mechanotransduction

Mechanosensitivity in Cells and Tissues

Volume 4

Series Editors

A. Kamkin
Department of Fundamental and Applied Physiology, Russian State Medical
University, Ostrovitjanova Str. 1, 117997 Moscow, Russia

I. Kiseleva
Department of Fundamental and Applied Physiology, Russian State Medical
University, Ostrovitjanova Str.1, 117997 Moscow, Russia

For further volumes:


http://www.springer.com/series/7878
Andre Kamkin Irina Kiseleva
Editors

Mechanosensitivity and
Mechanotransduction

Foreword by Holger Scholz

123
Editors
Prof. Andre Kamkin Prof. Irina Kiseleva
Department of Fundamental and Applied Department of Fundamental
Physiology and Applied Physiology
Russian State Medical University Russian State Medical University
Ostrovitjanova Str. 1 Ostrovitjanova Str. 1
117997 Moscow 117997 Moscow
Russia Russia
Kamkin.A@g23.relcom.ru Kamkin.A@g23.relcom.ru

Editorial Assistant
Dr. Natalia E. Lapina
Division of Neurosurgical Research
Medical Faculty Mannheim
Ruprecht-Karls-University Heidelberg
Theodor-Kutzer-Ufer 1-3
D-68167 Mannheim
Germany
Natalia.Lapina@medma.uni-heidelberg.de

ISBN 978-90-481-9880-1 e-ISBN 978-90-481-9881-8


DOI 10.1007/978-90-481-9881-8
Springer Dordrecht Heidelberg London New York

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Foreword

Mechanosensitivity and Mechanotransduction edited by Andr Kamkin and Irina


Kiseleva describes and discusses the latest research findings in the field of cellular
biomechanics. Internationally recognized experts contributed to this comprehen-
sive and visionary work, which represents the accomplishment of a series of four
related books. Mechanosensitivity describes the capacity of cells and tissues to
perceive mechanical stimuli and translate them into biological signals. As such,
mechanosensing and mechanotransduction are fundamental physiological mecha-
nisms allowing cells to react to physical forces. Considering the importance and
widespread distribution of mechanosensitive cells in many tissues of the body
one can anticipate that a failure of normal mechanosensation may have severe
consequences for various disease processes.
The book is composed of seven coherent and well-arranged parts covering the
multiple aspects of cellular mechanosensation. Part I highlights the role of the
cytoskeleton in mechanotransduction. Special attention is given to the function of
integrin receptors as mechanotransducers in the myogenic response of blood vessels.
Importantly, signalling of integrins in response to enhanced mechanical forces has
been implicated in vascular remodelling and arterial hypertension. Another focus is
on the significance of the cytoskeleton for mechanosensing in the bone and artic-
ular cartilage. As underlined, the integrity of the cytoskeleton is essential for the
modulation of gene expression in osteoblasts by mechanical stimuli. The topic is
resumed in Chapters 12 and 13 discussing that mechanical loading of bone causes
movements of interstitial fluid. This in turn generates fluid shear stress that stimu-
lates anabolic activity in bone cells. Thus, a failure of normal mechanosensing by
osteoblasts and chondrocytes may impair the ability of bone tissue and joint carti-
lage to adjust to the dynamics of biomechanical forces. Such maladaptation bears
the risk of mechanical instability and onset of diseases of the skeletal system, i.e.
osteoarthritis.
The second part of this book discusses recent findings regarding the molecular
control of mechanically gated ion channels (MGCs). This work includes the regu-
lation of MGCs in the heart by nitric oxide and the signalling pathways involved
in the myocardial response to mechanical stimulation. Substantial progress in the
analysis of molecular MGC gating has recently been made with the use of GsMTx4,
a gating-modifier peptide obtained from spider venom.

v
vi Foreword

Mechanical stretch resulting from pulsatile blood flow can modulate vascular cell
differentiation and function. Proteoglycans have a crucial role in mechanobiological
responses of the arterial system. The issue of mechanosensing and mechanotrans-
duction in vascular cells is discussed in Chapters 8 and 9. Emphasis is put on the
importance of haemodynamic forces for the maintenance of homeostasis in the
cardiovascular system. It is pointed out that a detailed knowledge of the molecu-
lar mechanisms underlying mechanosensing and mechanotransduction in vascular
cells is essential to develop novel concepts for the prevention and therapy of
cardiovascular diseases.
Mechanotransduction in the lung, the topic highlighted in part IV, is estab-
lished through the transient receptor potential vanilloid-4 (TRPV4) non-selective
cation channel. Activation of TRPV4 initiates increased vascular permeability in
response to high airway pressure or vascular pressure in the lung. Understanding
the molecular regulation of this ion channel may therefore provide novel insights
into various pulmonary disease processes including ventilator induced lung injury
and pulmonary oedema.
Primary cilia are organelles that have recently been recognized as mechanosen-
sors. Cilia can sense the movement of body fluids in various tissues including
endolymph in the inner ear, urine in renal tubules, bile in the hepatic biliary sys-
tem, and digestive fluid in the pancreatic duct. Mechanosensing by primary cilia is
discussed in Chapter 14. As outlined here genetic defects of the molecular compo-
nents of primary cilia, i.e. polycystins and other interacting proteins, can give rise
to various disorders such as polycystic kidney disease.
The final chapter addresses the role of mechanosensing and mechanotransduc-
tion in blood cells. Blood cells encounter substantial mechanical stimuli due to
variable haemodynamics. Large-conductance background K+ channels (LKbg ) have
been identified as mechanosensors in mouse B lymphocytes and their molecular
regulation by phospholipase C-dependent signalling pathways is discussed.
In summary, Mechanosensitivity and Mechanotransduction compiles in-depth
reviews of the current knowledge of mechanosensing and mechanical signal trans-
duction in various tissues. The readers attention is attracted by the profound and
clearly written contributions. Articles cover the many facets of cellular mechanosen-
sation ranging from the molecular gating of mechanosensitive ion channels to
perspectives for the future treatment of diseases.

Institut fr Vegetative Physiologie Holger Scholz


Charit - Universitstsmedizin Berlin
Hessische Strasse 3-4
10115 Berlin
Editorial

Basic Principles of Mechanosensing


and Mechanotransduction in Cells
Andre Kamkin and Irina Kiseleva

Both mechanosensitivity and mechanotransduction are fundamental physiological


processes which are responsible for sensing of mechanical forces and their transfor-
mation into electrical or (and) biochemical signals. The cell membrane interfaces
with the external medium or with neighbouring cells and gets mechanical stress
mainly in the form of stretch or compression due to general tissue deforma-
tion. Mechanical stimuli trigger different electrophysiological and biochemical
responses. Mechanically gated ion channels (MGCs), reacting to the membrane ten-
sion, were shown to play the key role in one of the mechanisms, through which the
cell responds to mechanical stimuli. Sachs and Morris (1998) noted that MGCs
(originally called MSCs: mechanosensitive channels) are channels that recognize
mechanical deformation as a proper physiological signal and react to mechanical
stimulation with changes in kinetics. But MGCs are only a link (probably final from
the point of view of changed ion transport and electrophysiological responses of
the cell) in big chains of reception and transmission of mechanical stress. In living
tissues, mechanical sensing and stress-induced responses in cells are defined by sev-
eral cellular components including extracellular matrix, integrins, the cytoskeleton,
and MGCs. The linkage between these cellular components undoubtedly plays a
critical role.
Mechanical forces are transmitted to cells, obviously, through the physical inter-
actions of the cell with the surrounding extracellular matrix (Geiger et al., 2001;
Matthews et al., 2006; Janmey and Weitz, 2004). The extracellular matrix is phys-
ically linked to the cytoskeleton via cell surface receptors primarily of the integrin
family (Carver and Fuseler, 2010). It was shown, that alterations in the mechan-
ical environment can be transmitted from the extracellular matrix to the cell via
integrin receptors (Bershadsky et al., 2006; Sanchez-Esteban et al., 2006). Integrins
are the main receptors for extracellular matrix proteins like collagen, fibronectin and
laminin. Mechanical deformation, applied to extracellular matrix proteins, which are
ligands for integrins, triggers the assembly and growth of focal contacts (Bershadsky
et al., 2003) and activation of several downstream second messenger systems,
including Rho GTPases, serine/threonine kinases, phosphatases, MAP kinases, Akt,

vii
viii Editorial

and PKC (Lal et al., 2010). For example, in fibroblasts (Kumar et al., 2006; Zhao
et al., 2007; Zhang X et al., 2007), endothelial (Mathur et al., 2007), or vascular
smooth muscle cells (Na et al., 2007) stress-stiffening has been attributed to an
integrin-Rho-dependent activation of stress fibers. Stretch-induced conformational
changes in the ECM may alter integrin structure, resulting in activation of liganded
integrin receptors and focal contact-associated secondary messenger pathways in
the cell, such as FAK, Src family kinases, Abl and integrin-linked kinase ILK (Li
et al., 1999; Liu et al., 2000). Many studies have demonstrated that the physical net-
work involving the extracellular matrix, integrins and the cytoskeleton is critical for
the response of cells to mechanical forces (see for review Carver and Fuseler, 2010).
Thus, during the deformation of the whole cell the possibility of the transmission of
membrane forces into the cell is realized from the extracellular matrix through inte-
grin receptors to the cytoskeleton and then to channel proteins (see for review Boriek
and Kumar, 2008). It is beyond any doubt that integrins are major mechanosensors
or mechanotransducers in many different cell types because of their unique abil-
ity to transduce extracellular mechanical signals exerted by extracellular matrix into
intracellular signals. Integrins and cytoskeletal proteins act together as mechanosen-
sors (see for review Thampatty and Wang, 2008). Despite of the achieved progress
in addressing a number of questions, in many respects the mechanisms by which
different cells sense, transduce and convert mechanical stimuli into cellular signals
remain to be completely revealed.
For this reason the first part of this volume is devoted to a network involving
the extracellular matrix, integrins and the cytoskeleton. The first part of the volume
is beginning with an article devoted to the role of integrin-mediated mechanotrans-
duction by Yip et al. (2011). Vascular smooth muscle cells are used as a model for
a discussion of the main issues concerned with integrins: Integrins and their asso-
ciated kinases, integrins as mechanotransducers, mechanisms of coupling integrin
activation to myogenic constriction. Depending on the types of integrin involved
and the specific vascular beds, integrin dependent myogenic vasoconstriction can
be mediated through Ca2+ dependent mechanisms of L-type Ca2+ channel poten-
tiation, activation of global and local Ca2+ release from ryanodine sensitive Ca2+
stores; as well as Ca2+ independent mechanisms of FAK, c-Src, MAPK, and ILK
activation and reactive oxygen species release (Yip et al., 2011).
The second article is devoted to the role of the actin cytoskeleton in mechanosen-
sation (Luo and Robinson, 2011). The actin cytoskeleton composed of actin fila-
ments, myosin motors, and actin crosslinking proteins plays a critical role in force
propagation and in response to deformation. The chapter discusses the microstruc-
tures and deformations of the actin cytoskeleton, functions of the actin cytoskeleton
in mechanosensation. There are numerous overlaps between the mechanotrans-
duction and traditional chemical signal transduction pathways. Because of the
overlap between these pathways, mechanical-chemical coupling and feedback loops
are a natural consequence of this system integration. Because the actin cytoskele-
ton is structurally integrated with nearly every aspect of the cell, mechanical
inputs can be transmitted quickly throughout the cell. Challenges for understanding
mechanosensation through the actin cytoskeleton include revealing how proteins
Editorial ix

function cooperatively over short nanometer-length-scales and fast sub-second


time-scales.
The following two articles discuss the role of the cytoskeleton in the
mechanosensitivity of genes in osteoblasts (Fu et al., 2011) and the involvement of
the cytoskeletal elements in articular cartilage mechanotransduction (Blain, 2011).
The osteoblast is an important mechanosensitive cell in bone tissue (Fu et al.,
2011). This article reviewed the roles of the cytoskeleton in the mechanotransduc-
tion of genes in osteoblasts, summarizes how cytoskeleton integrity is essential for
the expression of bone formationrelated genes in osteoblasts, and concludes that
cytoskeleton reorganization inhibition can enhance the mechanosensitivity of some
genes in osteoblasts. The article of Blain (2011) focuses on the organization and
function of the three major cytoskeletal networks in articular cartilage chondrocytes.
Articular cartilage is a major load-bearing tissue of the synovial joint; it is well
known that the cytoskeleton acts as a physical interface between the chondrocytes
and the extracellular matrix in sensing mechanical stimuli.
We are yet far away from a clear understanding of how mechanical deformation
of a cell can modulate MGCs. One model postulates that the mechanical energy
is transferred though changes in lipid bilayer tension. However, the seal of a patch
pipette isolates most of the stress in the patch from that in the cell, making a direct
energy transfer through the lipid bilayer rather unlikely (Ursell et al., 2008). The sec-
ond model postulates that mechanical sensing and stress-induced response in cells
are defined by several cellular components including extracellular matrix, integrins,
the cytoskeleton, and MGCs. The issues related to discussing this model are briefly
mentioned above and are discussed in detail in the first Part of the Volume.
The discussions presented in this Part of the Volume are devoted to the role,
structure and function of the extracellular matrix itself. Extracellular matrix can be
connected with certain structural qualities of integrins and here their interaction with
extracellular matrix is discussed. The integrin receptor has a long N-ending extra-
cellular transmembrane and very short S-ending cytopasmatic domains. Various
combinations of - and -subunits define the specific properties of the binding of
the extracellular receptors domain to certain ligands of the extracellular matrix.
The majority of integrin receptors can bind several ligands. On the other hand
one and the same ligand, e.g., laminin can bind to several integrins. Cellular inte-
grin receptors bind to those glycoprotein parts of the extracellular matrix that
contain the aminoacid sequence Arg-Gly-Asp. The integrin receptors intracel-
lular domain binds with cytoskeleton microfilaments through a chain of various
interconnected cytoplasmatic proteins. This is the modern vision of how the
structural connection between the extracellular matrix and the cytoskeleton is
formed.
Interactions with extracellular matrix, cytoskeletal and intracellular signaling
cascades enables integrins to mediate both outside-in and inside-out signaling
(Hynes, 2002; Ross, 2004). Binding of integrins to extracellular ligands produces
intracellular signals (outside-in signals) such as changes in intracellular signaling
events and cytoskeletal reorganization that critically influences cell shape, migra-
tion, growth, and survival (Hynes, 2002). Inside-out signaling occurs when specific
x Editorial

intracellular signals impinge on integrin cytoplasmic domains, triggering changes


in conformation and ligand-binding affinity in the extracellular domain.
Although the first Part is formally devoted to the interaction of extracellular
matrix, integrins and the cytoskeleton, practically all parts of the Volume contain
discussions of physical networks which are mobilized by mechanosensitivity and
mechanotransduction in cells from different organs and tissues like the cardiovas-
cular system (Kazanski et al., 2011; Guo et al., 2011; Shyu, 2011; Baker, 2011), the
lung (Parker and Townsley, 2011; Rubacha and Liu, 2011), bone and joint tissues
(Young and Pavalko, 2011; Sanchez et al., 2011), sensor systems (Nauli, 2011), and
blood cells (Kim and Nam, 2011).
The Second Part of the Volume is devoted to molecular mechanisms of mechan-
otransduction and ion channel modulation. This part of the volume begins with an
article devoted to the role of nitric oxide in the regulation of mechanically gated
channels in the heart (Kazanski et al., 2010a, b; Kazanski et al., 2011). The article
discusses experimental data recorded from isolated ventricular myocytes of mouse,
rat and guinea pig by means of patch-clamp in the whole-cell configuration about
the role of NO in the regulation of MGCs. The presented data demonstrate that NO
donors lead to MGCs activation and the appearance of MG-like currents in unde-
formed ventricular myocytes while in stretched cells with activated MGCs the NO
donors lead to their inactivation and inhibition of the conductivity of these chan-
nels. The NO scavenger PTIO causes inactivation of all MGCs. Application of
non selective inhibitors of NO syntases, L-NAME or L-NMMA, resulted in com-
plete blockade of MGCs. In ventricular myocytes of wild-type mice, NOS1/ and
NOS2/, stretching of cells results in the activation of typical MG-currents. On
the contrary, in cells from NOS3/ mice stretch does not activate MG-currents.
The results suggest that NO plays an important role in activation and inactivation
of MGCs in cardiomyocytes and demonstrate that NOS3 dominate as the NO ori-
gin (Kazanski et al., 2010a, b; Kazanski et al., 2011). Different studies report that
besides direct activation of MGCs by mechanical forces (via cytoskeleton or bilayer)
fast indirect modulation of MGCs by pharmacological compounds is also possi-
ble. Moreover some of those compounds are capable of activating MGCs in the
absence of actual cellular stretch, others of MGCs inactivation despite the continu-
ous presence of cellular stretch. If this is true for compounds beside nitric oxide, this
provides an excellent opportunity for the development of new drugs for treatment
of mechano-induced arrhythmias.
The role of signaling pathways in the myocardial response to biomechani-
cal stress and in mechanotransduction in the heart is discussed in the review
by Guo et al. (2011). The biochemical signals derived from mechanical stim-
uli activate both acute phosphorylation of signaling cascades, such as in the
PI3K, FAK, and ILK pathways, and long-term morphological modifications
via intracellular cytoskeletal reorganization and extracellular matrix remodelling.
Mechanotransduction plays a fundamental role in cardiac (and vascular) function
and involves interaction between extracellular matrix and intracellular cytoskeletal
proteins via cell adhesion complexes, which are modulated by PI3Ks. Loss of PI3K
signaling enhances the susceptibility to biomechanical stress while the loss of its
Editorial xi

negative regulator, PTEN, is associated with a wide variety of adaptive mechanisms


necessary to resist the progression of maladaptive ventricular remodelling and heart
failure.
The third review in this part is Atomistic molecular simulation of gating modifier
venom peptides: Two binding modes and effects of lipid structure and is devoted to
GsMTx4 (Nishizawa, 2011). This drug is a gating-modifier peptide obtained from
tarantula venom. It is a valuable tool for the investigation of gating mechanisms of
mechanosensitive channels. Free energy profile analyses suggests that these toxins
exhibit two modes of binding to lipid membranes, namely, the shallow mode and
the deep mode. These toxins favor the deep mode, especially in membranes rich in
saturated lipid acyl chains, which make the headgroup layer tight. It is hypothesized
that in the case of HaTx the deep mode is the action mode, while for GsMTx4 the
two modes can explain the concentration-dependent (biphasic) effect of GsMTx4
that has recently been reported. The possibility that such toxins seek out specific
types of lipid molecules is discussed. Simulation results support the view that the
channel/GsMTx4 (or HaTx)/lipids make a tertiary complex crucial to the effective-
ness of the toxin and therefore binding of the toxin to channels occurs only in the
presence of lipid molecules with appropriate structures (Nishizawa, 2011).
The following parts of the Volume are devoted to the mechanosensitivity and
mechanotransduction in vascular cells (Part III), in the lung (Part IV), in bone and
joint tissues (Part V), in sensor systems (Part VI) and in blood cells (Part VII).
Part III begins with a review devoted to molecular effects of mechanical stretch
on vascular cells (Shyu, 2010). The vascular endothelium plays an important role
by sensing alterations in biological, chemical, and physical properties of blood flow
to maintain homeostasis. Mechanical stretch can modulate cell alignment and dif-
ferentiation, migration, survival or apoptosis, vascular remodeling, and autocrine
and paracrine functions in smooth muscle cells. Laminar shear stress exerts anti-
apoptotic, anti-atherosclerotic, and anti-thrombotic effects on endothelial cells.
Knowledge of the impact of mechanical stretch on the cardiovascular system is
vital to the understanding of the pathogenesis of cardiovascular diseases and is
also crucial to provide new insights in the prevention and therapy of cardiovascular
diseases.
The role of proteoglycans in vascular mechanotransduction is discussed in the
next chapter (Baker, 2011). This review focuses on the role of proteoglycans in
vascular mechanobiological responses of the arterial system. Proteoglycans are pro-
teins that are post-translationally modified with polysaccharide glycosaminoglycan
chains. These molecules are intimately involved in controlling cellular organization,
proliferation and migration. Several studies have demonstrated that the expres-
sion of syndecan-1 and syndecan-4 are increased in mechanically stimulated cells.
The syndecans are good candidates for mechanotransduction pathways as they
have multiple interactions with integrins, focal adhesion, cytoskeletal elements and
growth factor signaling. While the last decade has yielded many new insights into
the basic biology of proteoglycans, future studies will hopefully be able to shed
additional light on the roles these molecules play in mechanotransduction and
vascular remodeling.
xii Editorial

Two chapters are devoted to the mechanosensitivity and mechanotransduction


in the lung. The transient receptor potential vanilloid-4 (TRPV4) non-selective
cation channel and its effect on the lung is discussed in the first review of Part
IV (Parker and Townsley, 2011). Increased pulmonary venous pressure and venti-
lation with high peak inflation pressures increase endothelial calcium influx, nitric
oxide production, and vascular permeability in a TRPV4 dependent fashion in intact
lungs. Calcium influx occurs with relatively modest increases in pulmonary vascular
or airway pressures, and the channel is essential for initiating the increased vascu-
lar permeability induced by high vascular and airway pressures. Mechanical gating
is mediated by epoxyeicosatrienoic acids, whereas heat and phorbol ester activa-
tion occur through different mechanisms. TRPV4 is a major mediator of injury to
pulmonary microvascular endothelium, which lacks the store operated TRPC chan-
nels of macrovascular endothelium. Several potential regulatory sites on the TRPV4
molecule have been identified for phosphorylation by Src family kinases, PKA, and
PKC. However, the mechanisms for epoxyeicosatrienoic acid dependent regulation
of TRPV4 or amplification of TRPV4 by phosphorylation in intact lungs subjected
to mechanical stress have not been clarified (Parker and Townsley, 2011).
The second review of this part deals with the role of protein-protein interac-
tions in mechanotransduction, and with particular implications in ventilator induced
lung injury (Rubacha and Liu, 2011). Under discussion are the issues of basic lung
mechanics vs. mechanical ventilation, ventilator induced inflammatory response,
protein-protein interactions, unfolding of p130Cas as a mechanosensor, AFAP as
an activator of Src PTK, Src PKT activation by multiple physical forces, block-
ing Src PTK as a potential therapy for VILI. VILI is a complex problem that will
likely require a multifaceted approach to solve including work from basic scientists,
clinicians, and even engineers. In this chapter the authors discuss this novel mech-
anism for mechanosensation and mechanotransduction, and propose to inhibit Src
protein tyrosine kinase activation as a potential therapy for ventilator induced lung
injury.
The next Part of the Volume is devoted to bone and joint tissues. Two chapters
presented in this Part describe cellular and molecular mechanisms of mechanosen-
sitivity and mechanotransduction. The cells within bone, i.e. osteocytes and
osteoblasts, are responsible for detecting and responding to mechanical loading. The
first review discusses several of the key mechanisms used by bone cells to convert
mechanical signals into altered biochemical responses (Young and Pavalko, 2011).
Understanding how bone cells sense and respond to mechanical signals has been the
focus of considerable research. The review deals with a number of important top-
ics. It includes detection of mechanical stimuli and in this context focal adhesions
with the mechanosome hypothesis, ion channel and purinergic signaling in bone.
The propagation of mechanical signals in bone cells is discussed in detail. In this
context are considered focal adhesion kinase (FAK), Wnt/-catenin/sclerostin, Gap
junctions, NFAT, Nitric oxide cGMP-dependent kinases, and Nmp4/CIZ (Young
and Pavalko, 2011). Recent research has resulted in significant progress toward the
identification of the key molecular components used by cells to detect mechanical
stimuli and to propagate those signals through the cytoplasm and into the nucleus.
References xiii

The second chapter also demonstrates that joint tissues, including cartilage,
bone, meniscus, tendon, ligament and synovial membrane, are exposed to high
levels of mechanical stimulation. This review is devoted to the mechanosensitiv-
ity of cells in joint tissues. Under discussion is the role of the mechanosensitivity
in the pathogenesis of joint diseases (Sanchez et al., 2011). The chapter reviews
the issues of mechanical stimuli and cartilage matrix remodeling and chondrocyte
mechanotransduction. The discussion covers mechanical stimuli and subchondral
bone and other joint tissues mechanosensitivity.
The following Part presents the review devoted to primary cilia as mechanosen-
sory organelles in vestibular tissues (Nauli, 2011). In this chapter the author
describes cilia as newly recognized mechanosensory organelles. The author dis-
cusses ciliary classification, function and disease relevance in mammals. Besides,
structural and functional ciliary proteins based on their ciliary domains are pre-
sented. The importance of sensory cilia in other organ systems is yet to be
discovered, and many more cilia-related diseases are still to be identified.
The final Part of the Volume presents the review Mechanosensitive K+ channels
in mouse B lymphocytes: PLC-mediated release of TREK-2 from Inhibition by PIP2
(Kim and Nam, 2011). There is a limited amount of data regarding mechanosensi-
tivity and mechanotransduction in blood cells, therefore this chapter is of special
interest to our readers. Blood cells can encounter significant mechanical stim-
uli in variable flow. In mouse B lymphocytes and their cell line WEHI-231, the
authors found large-conductance background K+ channels (LKbg ) that show sig-
nificant mechanosensitivity. The biophysical characteristics of LKbg were similar
to those of TREK-2. The activity of TREK-2 is under tonic inhibition by PIP2 ,
and a relatively mild membrane stretch relieves this inhibition via activation of PLC
hydrolysing PIP2 . Apart from the mechano-biochemical signaling mechanism, more
direct regulation by stronger membrane stretch is also suggested.
Thus, this book is a unique collection of reviews outlining current knowledge and
future developments in this rapidly growing field. Currently, investigations of the
molecular mechanisms of mechanosensitivity and mechanotransduction are focused
on several issues. The majority of studies investigate intracellular signaling path-
ways. Knowledge of the mechanisms which underlie these processes is necessary
for the understanding of the normal functioning of different living organs and tissues
and allows to predict changes, which arise due to alterations of their environment,
and possibly will allow to develop new methods of artificial intervention. The book
brings up the problem closer to the experts in related medical and biological sci-
ences as well as practicing doctors besides just presenting the latest achievements
in the field.

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Contents

Foreword by Holger Scholz . . . . . . . . . . . . . . . . . . . . . . . . . v


Editorial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Andre Kamkin and Irina Kiseleva
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Part I The Role of Cytoskeleton in Mechanosensitivity


and Mechanotransduction
1 Integrin-Mediated Mechanotransduction in Vascular
Smooth Muscle Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kay-Pong Yip, Lavanya Balasubramanian,
and James S.K. Sham
2 The Role of the Actin Cytoskeleton in Mechanosensation . . . . . . 25
Tianzhi Luo and Douglas N. Robinson
3 Effect of Cytoskeleton on the Mechanosensitivity of Genes
in Osteoblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Qiang Fu, Yiping Zhang, Yajuan Xu, Yourui Li, Ling Guo,
and Minfeng Shao
4 Involvement of the Cytoskeletal Elements in Articular
Cartilage Mechanotransduction . . . . . . . . . . . . . . . . . . . . 77
Emma J. Blain

Part II Molecular Mechanisms of Mechanotransduction


and Ion Channels Modulation
5 The Role of Nitric Oxide in the Regulation of Mechanically
Gated Channels in the Heart . . . . . . . . . . . . . . . . . . . . . 109
Victor Kazanski, Andre Kamkin, Ekaterina Makarenko,
Natalia Lysenko, Natalia Lapina, and Irina Kiseleva

xvii
xviii Contents

6 Role of Signaling Pathways in the Myocardial Response


to Biomechanical Stress and in Mechanotransduction
in the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Danny Guo, Zamaneh Kassiri, and Gavin Y. Oudit
7 Atomistic Molecular Simulation of Gating Modifier Venom
Peptides Two Binding Modes and Effects of Lipid
Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Kazuhisa Nishizawa

Part III Mechanosensitivity and Mechanotransduction


in Vascular Cells
8 Cellular and Molecular Effects of Mechanical Stretch
on Vascular Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Kou-Gi Shyu
9 Role of Proteoglycans in Vascular Mechanotransduction . . . . . . 219
Aaron B. Baker

Part IV Mechanotransduction in the Lung


10 Control of TRPV4 and Its Effect on the Lung . . . . . . . . . . . . 239
James C. Parker and Mary I. Townsley
11 The Role of Protein-protein Interactions
in Mechanotransduction: Implications in Ventilator
Induced Lung Injury . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Matthew Rubacha and Mingyao Liu

Part V Mechanosensing and Mechanotransduction in Bone


and Joint Tissues
12 Cellular Mechanisms of Mechanotransduction in Bone . . . . . . . 277
Suzanne R.L. Young and Fredrick M. Pavalko
13 The Mechanosensitivity of Cells in Joint Tissues: Role
in the Pathogenesis of Joint Diseases . . . . . . . . . . . . . . . . . 297
Christelle Sanchez, Marianne Mathy-Hartert,
and Yves Henrotin

Part VI Mechanotransduction of Sensor System


14 Primary Cilia are Mechanosensory Organelles in Vestibular
Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Surya M. Nauli, Hanan S. Haymour, Wissam A. Aboualaiwi,
Shao T. Lo, and Andromeda M. Nauli
Contents xix

Part VII Mechanosensitivity and Mechanotransduction


in Blood Cells
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes:
PLC-Mediated Release of TREK-2 from Inhibition by PIP2 . . . . 353
Sung Joon Kim and Joo Hyun Nam
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Contributors

Wissam A. Aboualaiwi Department of Pharmacology, MS 1015; The University


of Toledo; Health Science Campus, HEB 274; 3000 Arlington Ave., Toledo, OH
43614, USA
Aaron B. Baker Harvard-MIT Division of Health Sciences and Technology,
Massachusetts Institute of Technology, 77 Massachusetts Avenue, E25-442,
Cambridge, MA 02139, USA, abbaker@mit.edu
Lavanya Balasubramanian Department of Molecular Pharmacology and
Physiology, University of South Florida, Tampa, FL 33612, USA
Emma J. Blain Connective Tissue Biology Laboratories, Biomedical Sciences
Building, School of Biosciences, Cardiff University, Museum Avenue, Cardiff,
CF10 3AX, UK, Blain@cardiff.ac.uk
Qiang Fu Department of Prosthodontics, Guanghua School & Hospital of
Stomatology, Sun Yat-Sen University, Guangzhou 510055, Guangdong, PR China,
fuqiangds@yahoo.com.cn; fuqiangds@gmail.com
Ling Guo Department of Prosthodontics, Guanghua School & Hospital of
Stomatology, Sun Yat-Sen University, Guangzhou 510055, Guangdong, PR China
Danny Guo Division of Cardiology, Department of Medicine, Mazankowski
Alberta Heart Institute, University of Alberta, Edmonton, AB, T6G 2S2, Canada
Hanan S. Haymour Department of Pharmacology, MS 1015; The University of
Toledo; Health Science Campus, HEB 274; 3000 Arlington Ave., Toledo, OH
43614, USA
Yves Henrotin Bone and Cartilage Research Unit, University of Lige, Institute of
pathology level 5, CHU Sart-Tilman, 4000, Lige, Belgium, yhenrotin@ulg.ac.be
Andre Kamkin Department of Fundamental and Applied Physiology, Russian
State Medical University, Ostrivitjanova 1, Moscow 117997, Russia,
Kamkin.A@g23.relcom.ru

xxi
xxii Contributors

Zamaneh Kassiri Mazanokowski Alberta Heart Institute, and Department of


Physiology, University of Alberta, Edmonton, AB T6G 2S2, Canada
Victor Kazanski Department of Fundamental and Applied Physiology, Russian
State Medical University, Ostrivitjanova 1, Moscow 117997, Russia
Sung Joon Kim Department of Physiology, Ischemic/Hypoxic Disease Institute,
Kidney Research Institute, Seoul National University College of Medicine, 103
Daehangno, Jongno-gu, Seoul 110-799, Korea, sjoonkim@snu.ac.kr;
physiolksj@gmail.com
Irina Kiseleva Department of Fundamental and Applied Physiology, Russian State
Medical University, Ostrivitjanova 1, Moscow 117997, Russia,
Kamkin.A@g23.relcom.ru
Natalia Lapina Department of Fundamental and Applied Physiology, Russian
State Medical University, Ostrivitjanova 1, Moscow 117997, Russia; Division of
Neurosurgical Research Medical Faculty Mannheim, Ruprecht-Karls-University
Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167, Mannheim, Germany,
Natalia.Lapina@medma.uni-heidelberg.de; lapina_n@rambler.ru
Yourui Li Department of Prosthodontics, Guanghua School & Hospital of
Stomatology, Sun Yat-Sen University, Guangzhou 510055, Guangdong, PR China
Mingyao Liu Faculty of Medicine, University of Toronto, 101 College Street
Toronto Medical Discovery Tower, Room 2-814, Toronto, ON M5G 1L7, Canada,
mingyao.liu@utoronto.ca
Shao T. Lo Department of Pharmacology, MS 1015; The University of Toledo;
Health Science Campus, HEB 274; 3000 Arlington Ave., Toledo, OH 43614, USA
Tianzhi Luo Department of Cell Biology and Department of Pharmacology and
Molecular Science, Johns Hopkins University School of Medicine, 725 N. Wolfe
Street, Baltimore, MD 21205, USA
Natalia Lysenko Department of Fundamental and Applied Physiology, Russian
State Medical University, Ostrivitjanova 1, Moscow 117997, Russia,
fraunata@yandex.ru
Ekaterina Makarenko Department of Fundamental and Applied Physiology,
Russian State Medical University, Ostrivitjanova 1, Moscow 117997, Russia,
kmakarenko@yandex.ru
Marianne Mathy-Hartert Bone and Cartilage Research Unit, University of Lige,
Institute of pathology level 5, CHU Sart-Tilman, 4000 Lige, Belgium
Joo Hyun Nam Department of Pharmacology and Research Center for Human
Natural Defense System, College of Medicine, Yonsei University, Seoul, Korea
and Department of Physiology, Dongguk University College of Medicine,
Gyeongju, 780-714, Korea
Contributors xxiii

Andromeda M. Nauli Department of Health Sciences; East Tennessee State


University; College of Public Health; Johnson City, TN 37614, USA
Surya M. Nauli Department of Pharmacology, MS 1015; The University of
Toledo; Health Science Campus, HEB 274; 3000 Arlington Ave., Toledo, OH
43614, USA, Surya.Nauli@UToledo.Edu
Kazuhisa Nishizawa Department of Laboratory Medicine, Teikyo University
School of Medical Technology, Kaga, Itabashi, Tokyo 173-8605, Japan,
kazunet@med.teikyo-u.ac.jp
Gavin Y. Oudit Division of Cardiology, Department of Medicine, and
Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, AB, T6G
2S2, Canada, gavin.oudit@ualberta.ca
James C. Parker Department of Physiology and Center for Lung Biology, MSB
3074, University of South Alabama, Mobile, AL, 36688, USA,
jparker@usouthal.edu
Fredrick M. Pavalko Department of Cellular and Integrative Physiology, Indiana
University School of Medicine, 635 Barnhill Drive, MS 346A, Indianapolis, IN
46202, USA, fpavalko@iupui.edu
Douglas N. Robinson Department of Cell Biology and Department of
Pharmacology and Molecular Science, Johns Hopkins University School of
Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA; Department of
Chemical and Biomolecular Engineering, Johns Hopkins University Whiting
School of Engineering, 3400 N. Charles St, Baltimore, MD 21218, USA,
dnr@jhmi.edu
Matthew Rubacha Faculty of Medicine, University of Toronto, 101 College Street
Toronto Medical Discovery Tower, Room 2-814, Toronto, ON, M5G 1L7, Canada,
mprsmc.rubacha@utoronto.ca
Christelle Sanchez Bone and Cartilage Research Unit, University of Lige,
Institute of pathology level 5, CHU Sart-Tilman, 4000, Lige, Belgium
Holger Scholz Institut fr Vegetative Physiologie, Charit Universittsmedizin
Berlin, Hessische Strasse 3-4, 10115, Berlin, Germany, holger.scholz@charite.de
James S. K. Sham Division of Pulmonary and Critical Care Medicine, Johns
Hopkins Medical Institutions, 5501 Hopkins Bayview Circle, Baltimore, MD
21224, USA, jsks@jhmi.edu
Minfeng Shao Department of Prosthodontics, Guanghua School & Hospital of
Stomatology, Sun Yat-Sen University, Guangzhou 510055, Guangdong, PR China
Kou-Gi Shyu Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital,
Taipei, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine,
Taipei Medical University, Taipei, Taiwan, shyukg@ms12.hinet.net
xxiv Contributors

Mary I. Townsley Department of Physiology and Center for Lung Biology, MSB
3074, University of South Alabama, Mobile, AL, 36688, USA,
mtownsley@usouthal.edu
Yajuan Xu Department of Prosthodontics, Guanghua School & Hospital of
Stomatology, Sun Yat-Sen University, Guangzhou 510055, Guangdong, PR China
Kay-Pong Yip Department of Molecular Pharmacology and Physiology,
University of South Florida, Tampa, FL 33612, USA, dyip@health.usf.edu
Suzanne R.L. Young Department of Cellular and Integrative Physiology, Indiana
University School of Medicine, 635 Barnhill Drive, MS 346A, Indianapolis, IN
46202, USA
Yiping Zhang Department of Prosthodontics, Guanghua School & Hospital of
Stomatology, Sun Yat-Sen University, Guangzhou 510055, Guangdong, PR China
Part I
The Role of Cytoskeleton
in Mechanosensitivity
and Mechanotransduction
Chapter 1
Integrin-Mediated Mechanotransduction
in Vascular Smooth Muscle Cells

Kay-Pong Yip, Lavanya Balasubramanian, and James S.K. Sham

Abstract Myogenic response is an intrinsic property of vascular smooth muscle


cells (VSMCs) by which VSMCs contract when transmural pressure is increased,
and dilates when transmural pressure is decreased. Myogenic response in resis-
tance arterioles is one primary mechanisms for blood flow autoregulation. There
is emerging evidence that the dynamic interactions between specific extracellular
matrix (ECM) and integrins are required for mechanotransduction in myogenic
response. Studies in VSMCs and microvessels show that myogenic response is
linked to Ca2+ influx as well as local and global Ca2+ release from ryanodine-gated
stores. Electrophysiological evidences suggest that Ca2+ -activated channels might
provide the link between activation of integrins and membrane potential modulation
in myogenic response. Aside from myogenic vasoconstriction, the elevated mechan-
ical stress imposed on the integrin signaling complex might play important roles in
vascular remodeling and hypertension.

Keywords Myogenic response Calcium spark Calcium-activated


channels Integrin-linked kinase Vascular remodeling

1.1 Introduction

Mammalian blood pressure has been shown to spontaneously fluctuate up to 40%


of the mean arterial blood pressure (Marsh et al., 1990). Blood vessels are therefore
constantly receiving mechanical stimuli from the ebb and flow of blood through
these conduits. A steady blood flow is vital for the physiological functions in many
organs, like the glomerular filtration rate in kidney and cerebral blood flow. One of

K.-P. Yip (B)


Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL
33612, USA
e-mail: dyip@health.usf.edu

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 3


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_1,

C Springer Science+Business Media B.V. 2011
4 K.-P. Yip et al.

the major mechanisms preventing the conversion of pressure fluctuations into fluc-
tuations in blood flow is the myogenic response of vascular smooth muscle cells
(VSMCs). Myogenic response is an intrinsic mechanism of VSMCs by which the
vasculature constricts on elevation and dilates on reduction of perfusion pressure
(Bayliss, 1902). It is necessary for maintaining constant blood flow and capillary
hydrostatic pressure (Davis and Hill, 1999). End organ damage in hypertension
has been attributed to the transmission of increased blood pressure into the kid-
ney (Bidani et al., 1987; Karlsen et al., 1997; Abu-Amarah et al., 2005). Impaired
myogenic response in fawn-hooded hypertensive rats showed glomerular damage
(Simons et al., 1993; van Dokkum et al., 1999) highlighting the importance of nor-
mal myogenic response in maintaining a constant GFR and hence renal integrity.
However, the mechanisms of how VSMCs sense the changes in the perfusion
pressure are not well defined (Davis and Hill, 1999).
Mechanotransduction of VSMCs is not only involved in acute adaptation of
vascular resistance in myogenic response, but also in chronic adaptation such
as vascular remodeling in hypertension. Vascular remodeling is closely related
the VSMCs proliferation and extracellular matrix (ECM) accumulation. Genetic
hypertension and angiotensin II induced hypertension are associated with changes
in integrins and ECM expression in resistance arterioles (Intengan et al., 1999;
Intengan and Schiffrin, 2000; Mori and Cowley, 2004; Seubert et al., 2005).

1.2 Potential Candidates for the Sensor of Mechanotransduction

In order to alter the vascular resistance in response to changes in perfusion pressure,


mechanisms for sensing vascular wall stress/tension should be present (Johnson,
1980; Davis and Hill, 1999). Such mechanosensors should be conveniently seated
on the plasma membrane to transduce the external force into the intracellular milieu.
Their proximity to other signaling molecules like kinases and local Ca2+ stores
is essential to cause an immediate local response. The potential candidates for
mechanosensing fall into three major categories. They include ion channels which
modulate membrane potential; transmembrane proteins which link the ECM to the
cytoskeleton; and kinases which phosphorylate proteins involved in contractility and
proliferation.

1.2.1 Stretch-Sensitive Ion Channels


It is believed that the non-selective stretch-sensitive cation channels (NSCCs) act
as the sensors in mechanotransduction. An increase in transmural pressure stretches
the vascular wall and opens the stretch-sensitive channels, which initiates contrac-
tion by depolarizing the VSMCs leading to the activation of voltage-gated Ca2+
channels (VGCCs) and hence an increase in the intracellular calcium concentra-
tion ([Ca2+ ]i ) (Kirber et al., 1988; Davis et al., 1992; Meininger and Davis, 1992).
Stretch-activated whole cells currents have been recorded in many types of VSMCs
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 5

(Wellner and Isenberg, 1994; Wellner and Isenberg, 1995). The stretch-induced
depolarization could be explained by the activation of mechanosensitive ion chan-
nels promoting Na+ or Ca2+ influx, Cl efflux, or inhibiting K+ efflux (Davis and
Hill, 1999). Ion transport pumps in the plasma membrane have also been suggested
as a plausible sensor. Stretching the VSMCs showed an increase in the expression
and translocation of the -subunit of the Na+ , K+ ATPase to the plasma membrane
(Sevieux et al., 2003).
Another group of NSCCs that has been implicated in myogenic response is the
transient receptor potential (TRP) superfamily of channels (Inoue et al., 2006). Most
TRPC channels can be activated by PLC pathway, which can likely be triggered
by the increase in intravascular pressure. In frog oocytes, TRPC1 forms stretch
sensitive cation channel (Maroto et al., 2005), and TRPC6 has been shown to
regulate myogenic response by their involvement in pressure-induced membrane
depolarization in rat cerebral arteries (Welsh et al., 2002; Spassova et al., 2006).
Increasing evidence suggests that TRPM4 channels play a major role in myogenic
response. Suppression of TRPM4 expression using antisense oligodeoxynucleotide
decreased pressure-induced depolarization and myogenic response in vitro, and
altered autoregulation in vivo in rat cerebral arteries (Earley et al., 2004; Reading
and Brayden, 2007). Another study proposed that TRPM4 may actually affect myo-
genic response by stretch-induced Ca2+ release via ryanodine receptors (Morita
et al., 2007). Besides cerebral arteries, TRPC3 and TRPC6 are highly expressed
in rat renal resistance vessels (Facemire et al., 2004; Inoue et al., 2006); TRPC1,
TRPC6, TRPM4, TRPM8, TRPV2, and TRPV4 are also abundant in pulmonary
arterial smooth muscle cells (Lin et al., 2004; Yang et al., 2006). However, their
roles in myogenic response in these vascular beds are unclear. Moreover, TRPV1
and TRPV2 have been implicated as mechanosensitive channels in aortic smooth
muscle cells; but their involvement in myogenic response is debatable (Roman,
2002; Muraki et al., 2003). It is noteworthy that even though stretch-sensitive
membrane bound channels may operate as mechanosensors accounting for the ini-
tiation of myogenic vasoconstriction, they could not provide an error signal to
sustain myogenic constriction because of the abrogation of stretch after myogenic
constriction.

1.2.2 Integrins and Their Associated Kinases

The other plausible sensors for the myogenic phenomenon are integrins, the het-
erodimeric receptors of ECM. They are considered as stress sensing and transducing
elements in many cell types. Of the 24 known integrins, 16 have been reported in
various aspects in vascular biology. Among them, 1 1 , 2 1 , 3 1 , 4 1 , 5 1 ,
6 1 , 7 1 , 8 1 , 9 1 , v 1 , v 3 , v 5 , and 6 4 are expressed in VSMCs
(Glukhova et al., 1994; Moiseeva, 2001). They connect the ECM to the internal
cytoskeleton (CSK) via the short cytoplasmic tail of the -subunit and trans-
duce both outside-in (modification of cellular events on integrin ligation) and
inside-out signals (modification of the ECM in response to integrin ligation). An
6 K.-P. Yip et al.

increase in perfusion pressure might provide the mechanical signal to activate the
VSMC integrins via attachment between native ECM and integrin during passive
dilation. Stretch of ECM may induce conformational changes in integrin resulting
in the activation of focal adhesion associated secondary messenger pathways, such
as focal adhesion kinase (FAK), Src family kinases, and integrin-linked kinase (ILK)
(Lal et al., 2009).
FAK is a cytosolic soluble tyrosine kinase, which plays a major role in integrin-
mediated signaling. It consists of an N-terminal functional domain that interacts
with -integrin and growth factor receptors, a catalytic domain for tyrosine kinase
activity, and a focal-adhesion targeting domain that localizes FAK to focal adhe-
sions and binding sites for associated signaling molecules (Schaller, 2001). Integrins
induce auto-phosphorylation of FAK Tyr397 creating binding sites for cellular-
sarcoma (c-Src) family of tyrosine kinase. These FAK/c-Src complexes trigger
other downstream signals including stimulating mitogen-activated protein kinases
(MAPKs). Activation of MAPKs like extracellular signal-regulated kinase 1/2 (ERK
1/2) lead to cell proliferation and/or trigger other vascular remodeling signaling
in response to mechanical stimulus. ERK1/2 has been shown to be activated by
mechanical stretch in a time and strength-dependent manner in the VSMCs isolated
from rat aorta (Hu et al., 1998). Moreover phosphorylation of ERK 1/2 is essen-
tial for integrin-mediated phenotypic change of cultured aortic smooth muscle cells
(Roy et al., 2001).
ILK is a serine/threonine protein kinase that binds to the cytoplasmic tail of
the integrin subunit. It plays a central role in translating the external mechani-
cal stimulus in to intracellular biochemical signals. ILK has been shown to involve
in Ca2+ -independent phosphorylation of smooth muscle myosin light chain, hence
it may provide an alternative pathway through which integrin activation regulates
vascular resistance independent of Ca2+ (Deng et al., 2001). It has been shown as
an essential component of mechanical stretch sensor by controlling the contractility
in the zebrafish heart (Bendig et al., 2006); and deletion of ILK in zebra fish using
antisense morpholino oligonucleotides results in marked patterning abnormalities
of the vasculature and is lethal (Friedrich et al., 2004). Furthermore, inhibiting ILK
using siRNA was shown to inhibit vascular oxidative stress mediated by hyper-
tension (Vecchione et al., 2009). In addition, integrins is known to mediate the
activation of the serine/threonine protein kinase Akt through FAK-dependent and
independent mechanisms (Chen et al., 1996; Velling et al., 2004), and members
of Rho GTPases, including RhoA and Rac1, which interact with a wide-variety of
downstream effectors to engage in specific vascular responses.

1.3 Integrins as Mechanotransducers in the Vascular Cells


There is considerable evidence suggests that integrins can transduce mechanical
force across plasma membrane and initiate intracellular signaling (Ingber, 1990;
Sadoshima and Izumo, 1993; Wang et al., 1993; Vuori, 1998; Boudreau and
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 7

Jones, 1999; Davis et al., 2001). Extracellular mechanical force can be transmit-
ted across the plasma membrane via integrins to initiate intracellular signaling in
non-muscle cells (Ingber, 1990; Wang et al., 1993; Martinez-Lemus et al., 2003;
Katsumi et al., 2004). It is known that an intact ECM- integrin-CSK axis is essential
for mechanosensing and mechanotransduction in the endothelial cells and fibrob-
lasts. The tensegrity model proposed by Ingber suggests that cellular tension or
prestress is balanced and stabilized by the forces within the intracellular acto-
myosin cytoskeleton network (Ingber, 1997, 2000, 2003a, b; Wang et al., 2001).
Disturbances in external mechanical stress can restructure the microtubules in the
cytoskeleton to offset the tension in adherent cells. This transduced force and
restructuring of microtubules can then initiate secondary messengers and signal-
ing cascades including an increase in the [Ca2+ ]i causing contraction. Accordingly,
a change of perfusion pressure in an intact blood vessel may alter the stress in the
attachment sites between native ECM, and VSMC integrins, triggering downstream
signaling events for myogenic response.

1.3.1 Evidence in Endothelial Cells and Fibroblasts

The participation of integrins in mechanotransduction in endothelial cells was


demonstrated by directly applying force to integrins using magnetic twisting cytom-
etry technique with integrin ligand-coated ferromagnetic beads (Wang and Ingber,
1994; Wang, 1998). Force applied to 1 integrin induced focal adhesion forma-
tion and supported a force-dependent stiffening response. The cytoskeletal stiffness
increased in direct proportion to the applied stress and required intact microtubules,
intermediate filaments, and microfilaments. Mechanical force transduced via dif-
ferent integrins increased cytoskeletal stiffness to different degrees; for example,
1 integrin had the maximum effect (Wang et al., 1993; Wang and Ingber, 1995).
These observations indicate that integrins serve as the mechanoreceptors to trans-
duce force across the plasma membrane and trigger stiffening of cytoskeleton by
inducing structural rearrangements within the tensionally integrated cytoskeletal
network in endothelial cells. Magnetic force transduced via integrins using RGD-
coated beads also increased the expression of endothelin-1 in endothelial cells (Chen
et al., 2001). This integrin dependent transcriptional response is dependent on intact
CSK. Inhibition of myosin ATPase, myosin light chain kinase, and disruption of
actin filament abolished the twist-induced endothelin-1 upregulation. Glogauer and
colleagues showed an increase in the [Ca2+ ]i when fibroblasts were pulled using
collagen coated ferromagnetic beads (Glogauer et al., 1995). This process was pre-
sumably mediated via 1 -, 2 -, and 3 -integrins of collagen receptors, and was
dependent on stretch-activated cation channels, whose sensitivity could be mod-
ulated by filamentous actin. Moreover, integrin dependent stretch-induced ERK2
activation was seen in rat cardiac fibroblasts (MacKenna et al., 1998). These find-
ings highlighted the importance of integrin-CSK axis in transducing mechanical
force into different physiological processes, including cellular contraction, Ca2+
signaling, gene regulation, and vascular remodeling.
8 K.-P. Yip et al.

1.3.2 Evidence in Vascular Smooth Muscle Cells


Using a Fluorescence Resonance Energy Transfer (FRET)-based cytosolic Src
reporter in live smooth muscle cells, RGD- and 1 - integrin antibody coated beads
rapidly (< 0.3 s) activated Src when force was applied through magnetic twist
cytometry (Na et al., 2008). While forces applied with beads coated with non-
adhesion ligand or non-adhesion integrin antibody did not elicit a response. These
observations confirmed that mechanical force transduced by 1 integrin activates
Src. Application of cyclic mechanical force with fibronectin-, vitronectin-, and
3 integrin antibody coated beads induced activation of ERK 1/2; 2 - and 1 - inte-
grin antibody coated beads did not significantly activate the kinase (Goldschmidt
et al., 2001).
Using atomic force microscopy (AFM), Meininger and colleagues detected
micromyogenic events in VSMCs that counteract the pulling force when these
cells were pulled with fibronectin coated bead fused to the AFM probe tip (Sun
et al., 2008). These microevents were suggested to be equivalent to the myo-
genic response observed in VSMCs in intact arterioles. These micromyogenic
events were abolished in the presence of function blocking antibodies to 5 1 and
V 3 integrins (Sun et al., 2008). 5 1 integrins are the primary receptors for
fibronectin in VSMCs. Using traction force microscopy to monitor the changes
in traction force of individual renal VSMC in response to pulling of fibronectin-
coated beads, it was found that the traction force remained elevated even after the
pulling force was terminated (Balasubramanian et al., 2008a). However, there was
no residual traction force when beads coated with the non-adhesion ligand low-
density-lipoprotein (LDL) were used to apply this pulling force. The persistence of
elevated traction force might be interpreted as the sustained contraction triggered by
the integrin-mediated mechanotransduction.
Integrin mediated mechanotransduction has also been studied in isolated arte-
rioles. Exogenous RGD-containing peptide impaired myogenic response in cre-
master muscle arterioles and rat afferent arterioles (Martinez-Lemus et al., 2005;
Balasubramanian et al., 2007). Myogenic response was also significantly inhibited
when cremaster arterioles (Martinez-Lemus et al., 2005) and renal afferent arte-
rioles were treated with either anti-5 , anti-1 or anti-3 integrin antibodies (Yip
et al., unpublished data). The ability to exhibit vasoconstriction is not compro-
mised as seen by the response to KCl and norepinephrine. Hence, blocking integrins
only impaired the pressure-induced vasoconstrictor response in the arterioles. These
observations strongly suggested that the interactions between ECM and integrins are
essential for myogenic constriction.

1.4 Mechanisms of Coupling Integrin Activation to Myogenic


Constriction

1.4.1 Calcium-Dependent Mechanisms

Mechanotransduction through integrins may lead to myogenic constriction by Ca2+


dependant and independent mechanisms. The Ca2+ dependent mechanism may
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 9

involve multiple Ca2+ influx and release pathways. Activation of integrins has been
implicated in the modulation of L-type Ca2+ channels. Early studies in rat cre-
master VSMCs showed that beads coated with fibronectin or 5 integrin antibody
enhanced, whereas v 3 ligands or 3 antibody inhibited L-type Ca2+ currents (Wu
et al., 1998). Another study showed that the activation of 4 1 integrins also trig-
gered an increase in Ca2+ influx via L-type channels (Waitkus-Edwards et al., 2002).
Leu-Asp-Val (LDV) peptide, a sequence of an alternatively spliced fibronectin vari-
ant, caused vasoconstriction and increase in [Ca2+ ]i in cremaster arterioles; and
the response could be blocked by nifedipine and anti-4 antibody. The 5 1 and
4 1 integrin induced L-type Ca2+ current potentiation is mediated through tyro-
sine phosphorylation involving Src proteins in native VSMCs (Wu et al., 2001;
Waitkus-Edwards et al., 2002). It was confirmed in a heterologous expression sys-
tem that 5 1 integrin regulates L-type Ca2+ channels (Cav 1.2) by PKA and c-Src
dependent phosphorylation of 1C C-terminal residues Ser1901 and Tyr2122 , respec-
tively (Gui et al., 2006). Furthermore, activation of 5 1 integrin using antibodies
or fibronectin also potentiated BK channel activity in rat cremaster VSMCs through
both Ca2+ and c-Src dependent mechanism (Wu et al., 2008), suggesting negative
feedback regulation of Ca2+ entry through L-type Ca2+ channel. In cerebral arteries,
TRPC6 and TRPM4 channels have been shown to be involved in myogenic response
(Welsh et al., 2002; Earley et al., 2004; Spassova et al., 2006; Reading and Brayden,
2007). But the connection between these TRP channels and integrin has not been
established.
In renal VSMCs, integrin mediated mechanotransduction triggers a global
increase in [Ca2+ ]i . Application of Arg-Gly-Asp (RGD)-containing peptide initi-
ated local increase in [Ca2+ ]i which propagated as recurrent Ca2+ waves across
renal VSMCs (Chan et al., 2001; Balasubramanian et al., 2008b). However, the
RGD-induced Ca2+ mobilization in renal VSMCs was ryanodine-sensitive, but
not inhibited by nifedipine or removal of extracellular Ca2+ . Moreover, mechani-
cal force applied by pulling freshly isolated renal VSMCs with fibronectin-coated
beads triggered ryanodine-sensitive local Ca2+ release (Ca2+ spark) which was
followed by global Ca2+ increase (Balasubramanian et al., 2007). In intact arte-
rioles, stepwise increase of pressure from 80 mm Hg to 120 mm Hg induced
an increase in the frequency of Ca2+ sparks (Yip et al., 2007; Balasubramanian
et al., 2008a). Preincubation with ryanodine or RGD-containing peptide inhibited
pressure-induced myogenic constriction (Balasubramanian et al., 2007). These find-
ings support the concept that ligation of integrin modulate myogenic constriction in
renal arterioles by activating Ca2+ release from the ryanodine-gated Ca2+ stores.
Similar to renal VSMCs, a study in VSMCs of rat pulmonary arteries showed
that the GRGDSP peptide (Gly-Arg-Gly-Asp-Ser-Pro) triggers Ca2+ release from
ryanodine-sensitive stores and lysosome-like organelles; and Ca2+ influx is not
required for the integrin mediated Ca2+ response (Umesh et al., 2006).
It is interesting that myogenic response and integrin activation trigger both global
and local Ca2+ response. Global mobilization of [Ca2+ ]i activates Ca2+ /calmodulin
dependent myosin light chain kinase to initiate actin-myosin interactions and
smooth muscle contraction; whereas local Ca2+ events may modulate ion channels
and Ca2+ effectors in local vicinity to regulate vascular reactivity. In cerebral
arterioles, increase in intraluminal pressure elicits myogenic response and activates
10 K.-P. Yip et al.

Ca2+ sparks (Nelson et al., 1995; Jaggar et al., 1998), which are local Ca2+ release
events originate from clusters of ryanodine receptors. It is well established that
Ca2+ sparks causes large increase in local [Ca2+ ] (10100 M) in subsarcolem-
mal microdomains and activate nearby Ca2+ activated K+ channels (KCa ), leading
to membrane hyperpolarization, reduction of Ca2+ influx via L-type Ca2+ channels
and vasodilatation. They operate as the negative feedback modulators of membrane
potential in cerebral arterioles. In contrast, the integrin mediated Ca2+ sparks in
renal arterioles contribute to the myogenic response because their inhibition with
ryanodine reversed the active myogenic vasoconstriction to passive vasodilatation
(Yip et al., 2007; Balasubramanian et al., 2008a). Integrin induced Ca2+ sparks in
renal arterioles may support vasoconstriction by functioning as elementary Ca2+
events underlying the global Ca2+ transients (Cheng et al., 1993). Alternatively,
Ca2+ sparks may activate Ca2+ activated Cl channels (ClCa )(Gordienko et al., 1999)
to initiate myogenic response via membrane depolarization and the subsequent Ca2+
entry through voltage gated Ca2+ channels. The latter is supported by the obser-
vations of spontaneous transient inward ClCa currents activated by Ca2+ sparks in
airway (Kotlikoff and Wang, 1998; ZhuGe et al., 1998; Bao et al., 2008), portal vein
(Mironneau et al., 1996), and corpus cavernosum smooth muscle cells (Williams
and Sims, 2007). A significant contribution of Ca2+ sparks in vasoconstriction has
also been suggested in rat pulmonary arterial smooth muscle cells (Remillard et al.,
2002; Zhang et al., 2003). Nevertheless, the roles of local Ca2+ signaling in integrin
mediated myogenic response require further investigations.

1.4.2 Ca2+ Independent Mechanisms

By examining the temporal and steady-state relationships between lumen diam-


eter and vascular smooth muscle [Ca2+ ]i in isolated arterioles of hamster cheek
pouch exposed to step changes in perfusion pressure, DAngelo et al. has suggested
the presence of a Ca2+ -independent regulatory system in the mechanotransduction
of myogenic response (DAngelo et al., 1997). As aforementioned, integrins are
associated with kinases which phosphorylate proteins pertinent to smooth muscle
contraction. Integrin-linked kinase (ILK) is an integrin associated protein which
binds to the cytoplasmic tail of 1 and 3 integrins (Dedhar, 2000; Wu and Dedhar,
2001). ILK is a serine/threonine kinase with a low basal kinase activity, which is
stimulated by cell-ECM interactions and certain growth factors (Dedhar, 2000).
Mechanical stress evoked by high blood pressure in carotid artery increased the
expression and activity of ILK (Vecchione et al., 2009). ILK can function as an
Ca2+ -independent myosin light chain kinase to phosphorylate 20-kDa light chain
of myosin and to induce contraction of vascular smooth muscle (Deng et al., 2001;
Wilson et al., 2005). ILK also inhibits myosin light chain phosphatase via phos-
phorylation, and thus increases the Ca2+ sensitivity of VSMCs (Muranyi et al.,
2002). It has been shown that pressure induced myogenic constriction in mouse
tail artery required the generation of reactive oxygen species (ROS) via NADPH
oxidase pathway (Nowicki et al., 2001; Keller et al., 2006). ILK activation might
attribute to the activity of NADPH oxidase (Vecchione et al., 2009).
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 11

1.4.3 Integrins as Part of a Larger Mechanotransduction Complex


In addition to direct linkage to ECM and CSK, integrins could interact with other
mechanosensory elements on the surface of VSMCs to form a larger mechanosen-
sory complex. It has been suggested that cellular responses and signaling cascades
initiated by mechanosensitive channels may be regulated more effectively if such
channels were organized around integrins, kinases and cytoskeletal complexes
(Shakibaei and Mobasheri, 2003). One possible mechanosensory protein that might
interact with integrin in renal VSMCs is epithelial Na+ channel (ENaC). Members
of the degenerin/Epithelial Na+ channel (DEG/ENaC) family of proteins are known
to function as mechanosensitive channels in C. elegans. ENaC message and pro-
tein expression in interlobar arteries of mouse kidney have been confirmed by
RT-PCR, Western blotting and immunofluorescence (Drummond et al., 2004;
Jernigan and Drummond, 2005). Inhibiting DEG/ENaC with amiloride and ben-
zamil also impaired myogenic constriction in mouse interlobar arteries, suggesting
that their presence is vital for myogenic response in renal circulation (Drummond
et al., 2004; Jernigan and Drummond, 2005). Similarly amiloride and benzamil
impaired myogenic constriction in juxtamedullary afferent arterioles of rat kidney
(Guan et al., 2009). Afferent arterioles are the major resistant arterioles in renal
circulation. However, RT-PCR studies did not detect mRNA for -, -, or - sub-
units of ENaC in rat afferent arterioles (Wang et al., 2008). It has been speculated

Fig. 1.1 Potential interactions among integrins, Ca2+ sparks, ion channels in myogenic response.
cADP cyclic adenosine diphosphate; ClCa calcium activated chloride channel; DEG/ENaC
degenerin/epithelial Na+ channel; ECM extracellular matrix; ERK extracellular signal-
regulated kinase; FAK focal adhesion kinase; KCa calcium activated potassium channel; KV
voltage gated potassium channel; MAPKs mitogen-activated protein kinases; NKCC sodium
potassium chloride cotransporter; NCX sodium calcium exchanger; NSCC non-selective
stretch-sensitive cation channel; RGD Arg-Gly-Asp motif; RyR ryanodine receptor; SR
sarcoplasmic reticulum; Src sarcoma family of tyrosine kinases; TRP transient receptor
potential superfamily of channels; VGCC voltage gated calcium channel
12 K.-P. Yip et al.

that ENaC subunits form the pore and interact with integrins, ECM and CSK to
form a larger mechanosensitive complex (Shakibaei and Mobasheri, 2003; Jernigan
and Drummond, 2005; OHagan et al., 2005; Drummond et al., 2008). There is
no direct evidence for these mechanosensitive complexes in VSMCs yet. However,
1 integrin subunit has been shown to co-localize with ENaC in mouse limb-bud
chondrocytes by co-immunoprecipitation and immunofluorescence (Shakibaei and
Mobasheri, 2003; Fig. 1.1).

1.5 Integrin-Mediated Mechanotransduction in Hypertension


and Vascular Remodeling

1.5.1 Roles of Integrins in Systemic Hypertension

Aside from myogenic vasoconstriction, the elevated mechanical stress imposed on


the integrin signaling complex may play important roles in vascular remodeling and
hypertension. Mechanical stimuli are initially transmitted via ECM-integrin-CSK
axis to regulate VSMC functions (Dajnowiec et al., 2007); and vascular cells then
react reciprocally by remodeling their surrounding ECM (Dajnowiec and Langille,
2007). Many integrins have been implicated in differentiation, migration, hypertro-
phy and proliferation of VSMCs, as well as ECM synthesis and deposition. These
processes are essential for the inward eutrophic remodeling in resistance vessels,
and outward hypertrophic remodeling in conduit vessels in hypertension.
It has been reported that the expression of V 3 and 5 1 integrins in mesen-
teric arteries were upregulated in adult spontaneous hypertensive rats (SHR)(Bezie
et al., 1998; Intengan et al., 1999); 1 , 5 , 8 , 1 , and 3 integrin expressions
were enhanced in the aorta and carotid arteries of angiotensin II-induced hyper-
tensive rats and mice, respectively (Brassard et al., 2006; Louis et al., 2007); and
8 integrin immunoreactivity was increased in VSMC of renal vasculature in des-
oxycorticosterone acetate (DOCA) induced hypertensive rats (Hartner et al., 2002).
V 3 and 5 1 integrins are known to play major role in myogenic response. Their
upregulation in SHR may account for the enhanced myogenic tone in these animals
(Falcone et al., 1993; Shibuya et al., 1998; Falcone and Meininger, 1999). The sus-
tained vasoconstriction caused by vasoconstrictors or myogenic tone in resistance
arterioles is believed to initiate eutrophic inward remodeling by stimulating auto-
relengthening and repositioning of VSMCs, cross-linkage formation with ECM,
structural rearrangement of matrix materials, and entrenchment of vascular wall
(Bakker et al., 2002; Martinez-Lemus et al., 2004; Dajnowiec and Langille, 2007).
Evidence from the TGR(mRen2)27 hypertensive rat model suggests that V 3 inte-
grin is involved in vascular remodeling (Heerkens et al., 2006). In these animals,
hypertension was associated with upregulation of V subunit and prominent arterio-
lar inward eutrophic remodeling. Inhibition of V 3 integrin with a specific peptide
inhibitor abolished inward remodeling but enhanced vascular smooth muscle hyper-
trophy. Inward eutrophic remodeling in isolated rat skeletal muscle arterioles in vitro
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 13

was also inhibited with an antibody directed to 3 integrins (Bakker et al., 2004).
Since V 3 integrin is associated with myogenic response (Martinez-Lemus et al.,
2005), VSMC migration (Jones et al., 1996) and ECM remodeling (Bendeck et al.,
2000; Sajid and Stouffer, 2002), these observations are consistent with the notion
that ECM-V 3 interactions can trigger eutrophic remodeling.
In addition, the collagen receptor 1 1 integrin was upregulated in conduit
arteries of angiotensin induced hypertensive rats and mice (Brassard et al., 2006;
Louis et al., 2007). Evidence from 1 integrin knockout mice suggested that 1 1
integrin play a significant role in smooth muscle cell hypertrophy in conduit arter-
ies of hypertensive mice through the FAK and p38 MAPK signaling pathways
(Louis et al., 2007). Furthermore, the tissue-type transglutaminase, a cross-linking
enzyme that associates with 1 and 3 integrins to form co-receptors for fibronectin
(Akimov et al., 2000), also contributed significantly to enthothelin-1 induced inward
eutrophic remodeling in small resistance arteries (Bakker et al., 2005), suggesting
that associated protein partners of integrin are involved in the remodeling process.

1.5.2 Roles of Integrins in Pulmonary Hypertension

Integrin expression has been reported in pulmonary vasculatures (Damjanovich


et al., 1992; Schnapp et al., 1995; Buck et al., 1996; Gotwals et al., 1996; Jones
et al., 1997b, 1999; Yao et al., 1997; Medhora, 2000; Singh et al., 2000). Our
survey with RT-PCR showed the presence of 1 -, 2 -, 3 -, 4 -, 5 -, 7 -, 8 -,
v -, 1 -, 3 -, and 4 - integrin mRNA in rat pulmonary arteries, similar to those
expressed in rat aorta (Umesh et al., 2006). Their relative expression were in
the orders of 8 >v >5 >7 >1 and 1 >3 >4 (Umesh and Sham, unpublished);
and 5 , v , 1 , and 3 integrin proteins were also detected in rat pulmonary
arteries and aorta. Compare to systemic circulation, the basal vascular resistance
and tone of pulmonary circulation are low and myogenic response is not evi-
dent under normal conditions except in fetal circulation. However, many studies
have reported major contributions of ECM and integrins in vascular remodel-
ing associated with pulmonary hypertension, which is characterized with outward
hypertrophic and hyperplasia remodeling in larger vessels and neomusculariza-
tion in small arterioles of all forms of pulmonary hypertension (Stenmark et al.,
2006, 2009). Increased deposition of extracellular matrix components, particularly
collagen, elastin, tenascin-C, and fibronectin have been reported in pulmonary vas-
culature of human and animal models of the disease (Botney et al., 1992; Jones et al.,
1996, 1997a, 2001; Novotna and Herget, 1998; Ihida-Stansbury et al., 2006). It is
attributed to the increase in the activity of serine elastase, and a change in the bal-
ance of matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase
(TIMP) activity (Rabinovitch, 1999; Cowan et al., 2000; Zaidi et al., 2002; Hassoun,
2005). The increase in serine elastase activity activates MMPs to degrade native
type I collagen, causing clustering of 3 containing integrins and increase tenascin-
C transcription. Tenascin-C in turn interacts with v 3 integrin in pulmonary arterial
smooth muscle cells to amplify the proliferative response by promoting epidermal
14 K.-P. Yip et al.

growth factor receptor clustering and phosphorylation (Jones et al., 1997b, 1999).
The evidence that v 3 integrin blockade with selective antagonists induced apopto-
sis of pulmonary arterial smooth muscle cells and reduced medial wall thickness in
organ culture of pulmonary arteries of monocrotaline-treated rats (Merklinger et al.,
2005) supports an important role for v 3 integrin in hyperplasia and hypertrophic
remodeling.
The expression of integrins in pulmonary vasculature with respect to pul-
monary hypertension has not been systemically examined. Our recent prelim-
inary study found significant changes in the expression of multiple and
integrins in pulmonary arteries of chronic hypoxia and monocrotaline-induced
pulmonary hypertensive rats (Umesh and Sham, unpublished). These changes
in integrin expression could be related to the phenotypic changes of VSMCs
occur during vascular remodeling. It is noteworthy that mild myogenic tone
was observed in pulmonary microvessels of pulmonary hypertensive animals
(Broughton et al., 2008), but the involvement of integrins has not yet to be
determined.

1.5.3 Roles of Integrins in Vascular Remodeling Caused


by Mechanical Injury

Integrins are also major contributors in vascular remodeling and neointimal forma-
tion in vascular injury, atherosclerosis, and restenosis. 1 1 , 5 1 , V 3 , 7 and
3 integrin expression were increased (Gotwals et al., 1996; Stouffer et al., 1998;
Pickering et al., 2000; Kappert et al., 2001; Sajid and Stouffer, 2002; Chao et al.,
2004) and the expression of 8 and 1 integrins were reduced in vascular cells of
neointima formed after vascular injury (Gotwals et al., 1996; Zargham and Thibault,
2005). Among these integrins, V 3 integrin has attracted most attention. Increased
expression of V 3 integrin in the medial and neointimal layers have been observed
following mechanical injury in various animal models, including those in mice, rats,
rabbits, baboons, and pigs, and in human atherosclerotic graft and arteriopathic
coronary arteries (Hoshiga et al., 1995; Sajid and Stouffer, 2002; Sadeghi et al.,
2004; Zhang et al., 2005). Many potential ligands of V 3 , including osteopontin,
thrombospondin, prothrombin, fibronectin, vitronectin, MMP-2 were found at the
sites of injury with increased abundance. Studies using V 3 antagonists to inhibit
neointima formation and vascular remodeling suggested a central role of V 3 inte-
grins in neointima formation through multiple mechanisms including reduction of
VSMC proliferation and migration, MMP production, and increased smooth mus-
cle cell apoptosis (Slepian et al., 1998; van der Zee et al., 1998; Bendeck et al.,
2000; Dufourcq et al., 2002; Honda et al., 2005). Similar to V 3 antagonists, 3
integrin blockade with peptide inhibitors or anti-3 antibodies reduced neointimal
thickening, VSMC migration and proliferation, MMP activity in injured arteries
(Slepian et al., 1998; Stouffer et al., 1998; Bendeck and Nakada, 2001). The 3 inte-
grin mediated VSMC migration could be related to activation of 3 -FAK signaling
1 Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells 15

pathway by osteopontin (Yue et al., 1994; Wang et al., 1996; Han et al., 2007). In
contrast to pharmacological inhibition of 3 -integrin, deletion of 3 -integrin gene in
mice provides no protection to transluminal probe induced mechanical injury, but
inhibited VSMC accumulation in neointima after milder injury induced by carotid
ligation (Smyth et al., 2001; Choi et al., 2004). The discrepancy could be explained
by species differences in the response to vascular injury, mechanistic differences
between 3 -integrin deficiency and blockade with antagonists, and compensatory
mechanisms in 3 -integrin knockout animals.
Other integrins, such as 1 1 , 5 1 , 7 1 and 8 1 , may also contribute to neoin-
tima formation. For example, the expression of 8 1 integrin is downregulated in
the medial layer in the early stage and is upregulated in the intima later during
inward constrictive remodeling after balloon angioplasty (Zargham and Thibault,
2005; Zargham et al., 2007). It has been shown that 8 integrin is required for main-
taining the contractile, differentiated phenotype of VSMCs (Zargham and Thibault,
2006). Gene silencing of 8 integrin increased VSMC migration and expression of
smooth muscle cell de-differentiation markers; and overexpression of 8 integrin
attenuated smooth muscle cell migratory activity and restored contractile pheno-
types (Zargham et al., 2007a, b). The alteration in 8 1 integrin expression, hence,
correlated with de-differentiation of VSMCs for migration during early neoin-
tima formation, and the contractile phenotype of VSMCs during late constrictive
remodeling in restenosis.

1.6 Conclusion and Perspectives

It is beyond any doubt that integrins are major mechanosensors or mechanotrans-


ducers in many different cell types because of their unique properties of transducing
extracellular mechanical signals exerted by ECM into intracellular signals via CSK,
kinases and other associated proteins. In this chapter, we have reviewed the major
evidence pertinent to the pivotal role of integrins as the mechanosensors of myo-
genic response in resistance arteries. Depending on the types of integrin involved
and the specific vascular beds, integrin dependent myogenic vasoconstriction can be
mediated through Ca2+ dependent mechanisms of L-type Ca2+ channel potentiation,
activation of global and local Ca2+ release from ryanodine sensitive Ca2+ stores; as
well as Ca2+ independent mechanisms of FAK, c-Src, MAPK, and ILK activation
and reactive oxygen species release. Furthermore, integrin-mediated mechanotrans-
duction of VSMCs is not only involved in acute adaptation of vascular resistance,
but it also actively participates in chronic adaptation such as hypertension and
vascular remodeling. Since integrins are ubiquitous transmembrane proteins, their
role and significance in mechanotransduction should be pursued beyond myogenic
response and vascular remodeling.
Acknowledgements This work is supported by the NIH grants R01-HL071835, R01-HL075135,
and American Heart Association.
16 K.-P. Yip et al.

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513 (Pt 3):711718
Chapter 2
The Role of the Actin Cytoskeleton
in Mechanosensation

Tianzhi Luo and Douglas N. Robinson

Abstract Cells are capable of sensing mechanical stimuli and translating them into
biochemical signals. This ability allows cells to adapt to their physical surround-
ings by remodeling their cytoskeleton, activating various signaling pathways, and
changing their gene expression. These phenomena involve two essential processes
mechanosensing and mechanotransduction. In these processes, force or deformation
needs to be transmitted from the outside environment to the proteins and organelles
inside the cell. The actin cytoskeleton composed of actin filaments, myosin motors,
and actin crosslinking proteins plays a critical role in force propagation and in
response to deformations. Cellular adaptation to these deformations is often asso-
ciated with feedback loops, and proteins in the actin cytoskeleton accumulate and
function cooperatively in response to mechanical stimuli. Mutations in these pro-
teins cause failure in cellular mechanosensing, which eventually leads to cellular
errors associated with disease progression.

Keywords Mechanosensing Mechanotransduction Actin cytoskeleton


Actin-crosslinking proteins Myosin II

2.1 Introduction

A mechanotransduction system requires at least one sensor and one transducer.


The sensors, commonly located next to the outer layer of the cell membrane,

D.N. Robinson (B)


Department of Cell Biology, Department of Pharmacology and Molecular Science, Johns Hopkins
University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA; Department
of Chemical and Biomolecular Engineering, Johns Hopkins University Whiting School of
Engineering, 3400 N. Charles St, Baltimore, MD 21218, USA
e-mail: dnr@jhmi.edu

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 25


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_2,

C Springer Science+Business Media B.V. 2011
26 T. Luo and D.N. Robinson

sense the mechanical stimuli, such as force, pressure and flow speed, and trans-
mit the mechanical signals into the inside of cells. Ion channels, protein kinases,
integrins, membrane glycocalyx, G proteins, intercellular junction proteins and
other membrane-associated signal-transduction molecules are capable of sensing
mechanical stimuli as shown in Fig. 2.1 (Ingber, 2006; Vogel and Sheetz, 2006;
Wang et al., 2009). The transducers, mainly situated inside the cell membrane
or inside the cytosol, convert the mechanical signals to chemical and biological
signals.
Physiological examples of mechanosensation include hearing (Fettiplace and
Hackney, 2006), blood flow regulation in the circulatory system (Chien, 2007), and
bone remodeling (Robling et al., 2006). Auditory sensing occurs in the inner ear
in a region known as the cochlea that is covered with hair cells characterized by
stereocilia. The mechanoelectrical transduction (MET) ion channels in one stere-
ocilium are linked to a neighboring stereocilium through tip linkers. These linkages
are then coupled internally to the core actin bundles through myosin I motors. When
one stereocilium is flexed due to sound vibrations, this induces tension along the
tip link, leading to channel opening and conversion of the sound into an electrical
signal. Subsequent adaption and associated closing of the METs occurs partially
through the unbinding of myosin I from the actin bundles. In the circulatory sys-
tem, endothelial cells sense the shear and stretch forces due to blood flow and
activate a number of mechanosensors, such as membrane proteins, integrins, G
proteins and ion channels. The mechanosensing triggers a cascade of signaling
pathways and consequently modulates gene expression, resulting in cytoskeleton
remodeling and cell realignment. Bone adapts its structure to mechanical stimuli
and repairs structural damage through remodeling. At bone surfaces, osteoclasts and
osteoblasts control the bone resorption and formation, respectively. However, the
osteocytes, which are embedded deep within the bone, appear to have the primary

Fig. 2.1 Schematic cartoon of mechanosensation and mechanotransduction in cells


2 The Role of the Actin Cytoskeleton in Mechanosensation 27

job of sensing where the bone needs to be remodeled and then relaying this infor-
mation to the osteoblasts and osteoclasts located on the bone surface. One appealing
model suggests that the osteocytes sense changes in fluid flow inside canaliculi and
the associated viscous drag creates tensile forces along the central actin filament
bundle in the osteocytes. The osteocytes then release second messengers such as
prostaglandins and nitric oxide, which activate the osteoblasts and osteoclasts. At
the molecular level, the bone mechanotransduction is also thought to involve ion
channels, focal adhesions, and G protein-coupled receptors, but detailed molecular
mechanisms are still unclear.
The actin cytoskeleton composed of actin filaments, actin crosslinking proteins
(ACLPs) and myosin II motors, is unambiguously involved in these mechanosens-
ing processes. To understand how mechanical stimuli are propagated through actin
filaments and how the actin cytoskeleton responds to these stimuli, it is essential
to understand the mechanical behaviors of the individual players, the reconstituted
networks in vitro and the whole network in vivo.

2.2 Microstructures and Deformations of the Actin Cytoskeleton

Cellular mechanics originate from mechanical features of the cytoskeleton that


traverse many length- and time-scales. On the molecular level, the mechanical
properties of proteins are determined by their molecular structures, such as pep-
tide sequences, folding states and assembly states. On the next complexity level,
the mechanical properties of the actin network depend not only on its morphol-
ogy/microstructures, such as mesh size, filament length, bundle diameter and
homogeneity, but also on the binding strength between the actin filaments and dif-
ferent crosslinkers and motor activities. In the past few decades, tremendous effort
has been invested in characterizing the mechanical behaviors of ACLPs and motor
proteins using single molecule techniques, and many attempts have also been made
to study the mechanical properties of reconstituted actin networks. However, the
understanding of individual ACLPs is still far from complete due to the complexity
of these proteins and the limitations of the techniques. Additionally, the mechanical
strength of reconstituted actin networks are often several orders lower than that of
the intact cells.
The central core protein of the actin cytoskeleton is monomeric actin, a 5-nm
diameter, 42 kDa globular protein (G-actin) found in all eukaryotic cells. Each
monomer is organized into four subdomains, flanking an internal cleft that binds
ATP and magnesium ions. The end of G-actin close to the base of the cleft is
called the plus end and the opposite end is called the minus end. G-actin monomers
undergo polymerization and form microfilaments (F-actin) upon the addition of salt.
Since the plus end of one G-actin is connected to the minus end of the neighboring
G-actin, microfilaments also have well-defined plus and minus ends. The inclusion
of ATP assists in G-actin stability and dramatically reduces the critical concentration
for assembly. The 8-nm wide actin filament can be considered to have a left-handed
28 T. Luo and D.N. Robinson

helical morphology with 13 actin monomers per pseudo-repeat and a pseudo-repeat


length of 37 nm. Alternatively, the actin filament can be considered to have a right-
handed helical structure with two strands slowly twisting around each other. Each
actin monomer is rotated 166 rotation with respect to its two nearest neighbors
across the strand (Holmes et al., 1990). Within the strand, subdomains 2 and 4 con-
tact subdomains 1 and 3 in the next monomer in the strand, and each monomer
reaches across to the other strand through a hydrophobic plug that links the two
strands together. The persistence length Lp of pure F-actin is around 17 m. The
intrinsic bending stiffness, b = Lp kB T, and the elastic Youngs modulus E are
7 1026 Nm2 and 2.6 109 N m2 , respectively (Gittes et al., 1993; Kojima
et al., 1994). The effective stretching stiffness is 44 pNnm1 provided the cross-
sectional area of a fully filled filament is 25 nm2 , and the torsional rigidity is
8 1026 Nm2 (Tsuda et al., 1996).
Non-muscle myosin II, a member of the conventional myosin II superfamily, is
composed of functional hexameric monomers, consisting of two heavy chains, two
essential light chains (ELCs) and two regulatory light chains (RLCs), which com-
bine to form a 500 kDa complex (shown in Fig. 2.2). The amino-terminal motor
domain consists of the catalytic core, which is structurally conserved with the Ras-
family small GTPase and includes the switch I and switch II helices and a Walker
p-loop-family nucleotide-binding pocket. This catalytic core is functionalized with
an actin-binding interface and a converter domain. The converter domain connects
to an 8-nm long -helix, which is wrapped by an ELC and RLC, forming the lever
arm. The lever arm links to the carboxyl-terminal coiled coil domain (Warrick et al.,
1987). Upon binding to actin, the motor domain remains rigid, whereas the lever
domain is rotated through a 70 angle as the products of ATP hydrolysis are
released; this lever arm rotation leads to the translation of the actin filament relative
to the coiled coil of the myosin.
To generate force and to bear load, myosin II must assemble into bipolar thick
filaments (BTFs). These assemblies can range from as few as eight (Acanthamoeba)

Fig. 2.2 Domain structure of myosin II. HMM and LMM represent heavy meromyosin and light
meromyosin, respectively
2 The Role of the Actin Cytoskeleton in Mechanosensation 29

to as many as 400 hexamers (mammalian skeletal muscle). Most mammalian non-


muscle myosin IIs (nonmuscle heavy chain IIA, IIB and IIC) assemble into BTFs
ranging from 1030 monomers, and Dictyostelium discoideum myosin II is thought
to assemble into BTFs with up to 70 monomers. For Dictyostelium, the assembly
process is thought to occur through the formation of two monomers into a paral-
lel dimer and two of these parallel dimers join together to create an anti-parallel
tetramer. Once the anti-parallel tetramer is formed (the stable nucleus), the BTF
grows through side-by-side (lateral) addition of dimers, resulting in no extra elonga-
tion of the BTF as more dimers are added (Fig. 2.3) (Mahajan and Pardee, 1996). In
the thick filament, myosin II molecules are thought to stack their rod tails in parallel
with a small overlap where the subunits are held together through electrostatic inter-
actions (Hostetter et al., 2004). Therefore, unlike muscle myosin II thick filaments
which grow in length as they are assembled, the Dictyostelium myosin monomer is
250 nm whereas the thick filaments are just 400 nm long, independent of the num-
ber of monomers assembled. The assembly process is regulated by myosin heavy
chain kinases (MHCKs), which phosphorylate three critical threonines in the tail
region (Liang et al., 1999). This phosphorylation prevents the myosin monomer
from assembling into BTFs, which is necessary to maintain a free pool (8090% of
total myosin II) of monomeric myosin in the Dictyostelium cell.
ACLPs can organize actin filaments into bundles and branched networks,
depending on their molecular structures (as shown in Fig. 2.4), kinetic proper-
ties, and concentration (Revenu et al., 2004). Bundle forming ACLPs include
fascin, forked, villin, fimbrin, espin, scruin, plastin, cortexillin and dynacortin,
whereas examples of meshwork forming ACLPs are filamin, Arp2/3, gelsolin and
ERM (ezrin, radixin, and moesin) proteins. Some of these proteins have addi-
tional properties. For example, Arp2/3 also nucleates actin assembly and ERMs
link actin filaments to the membrane. However, this classification of bundlers ver-
sus meshwork formers is an oversimplification as some ACLPs, such as -actinin
and dynacortin, can form bundles or meshworks depending on concentration and
actin:crosslinker ratios. Some of these ACLPs, such as villin, espin, forked, fimbrin

Fig. 2.3 Illustration of the


assembly process of
non-muscle myosin II
30 T. Luo and D.N. Robinson

Fig. 2.4 Schematic graph of a few actin crosslinking proteins:(a) human filamin A; (b) -
actinin; (c) cortexillin-I. In all cases, N and C represent the N-terminus and C-terminus,
respectively

and fascin, are monomeric with at least two actin-binding motifs, allowing them
to crosslink as monomers. By contrast, other ACLPs, such as filamin A, -actinin,
dynacortin, and cortexillin are dimeric, and filamin A, cortexillin, and most likely
dynacortin form parallel dimers while -actinin forms anti-parallel dimers. Actin-
binding domains are localized at the amino-terminus of filamin A and -actinin. Due
to the differences in their structures, Y-shaped filamin A dimers can only crosslink
actin filaments into nearly orthogonal networks while -actinin forms either net-
works or loose bundles. Some of the most common filamentous actin-binding
domains (ABDs) are the calponin homology (CH) domain, the Wiskott-Aldrich syn-
drome homology region 2 (WH2 ) domain, the gelsolin homology (GH) domain, and
the formin homology (FH) domain (Sjblom et al., 2008). Different ABDs generally
associate with different surfaces on the actin monomer and with different apparent
affinities.
These network structures may experience four types of deformations (Vogel,
2006; Ferrer et al., 2008): (1) deformation in actin filaments, (2) deformation of
the binding between actin filaments and myosin-II, (3) deformation of the bind-
ing between actin filaments and ACLPs and (4) intramolecular deformation of
ACLPs. In other words, intermolecular and intramolecular deformations exist at
the same time within the same network. One additional feature of how these net-
works respond to deformation is the relationship of the time-scale of the deformation
to the time-scale of the turnover/remodeling of the network. For example, pro-
filin binds monomeric actin, inducing it to exchange its nucleotide and shuttling
the actin monomer to sites where elongation has been stimulated. By contrast,
cofilin binds cooperatively to sever the actin filaments, which has a complicated
effect on the actin network. Severing provides free actin plus ends that may
grow whereas the free minus ends can disassemble. These proteins influence the
time-scales over which the actin polymers can grow and disassemble, modulat-
ing the time-scales of remodeling of the actin network. These features of network
remodeling may provide a phenomenological viscous character to the network,
allowing it to flow and remodel over the long time-scales of many biological
processes.
2 The Role of the Actin Cytoskeleton in Mechanosensation 31

2.2.1 Intermolecular and Intramolecular Deformations


Intermolecular interactions, including hydrogen bonds, electrostatic interactions,
van der Waals interactions and hydrophobic interactions, are non-covalent in nature.
Based on the transition state theory, there are two popular bond models to describe
the single-bond behaviors of intermolecular interactions: the catch-bond model and
the slip-bond model. A simplified definition of the catch-bond model is the life-
time of the bond increases as the stretching force increases. In contrast, a slip-bond
shows the opposite behavior, i.e., the bond lifetime decreases as the force increases.
A schematic diagram of the energy landscapes of the two different models are shown
in Fig. 2.5. In the spirit of Bell (Bell, 1978), both models can be described by the
unified formula (Evans and Ritchie, 1997; Evans, 2001):
 
f x
koff = koff
0
exp , (2.1)
kB T
  
0 = exp G k T is the unbinding rate in the absence of force,
where koff B
is the vibration frequency, G is the energy difference between the transition state
and the bound state, kB is Boltzmanns constant, T is temperature, f is the external
force that pulls the two molecules apart, and x is the bond length change along

D Energy
slip bond pathway catch bond pathway

bound
state

unbound unbound
state state

Force Direction

Fig. 2.5 A schematic diagram of the force dependence of protein-protein interaction strength:
(a) catch-bond model; (b) slip-bond model; (c) three typical bond behaviors; (d) two pathways
between bound state and unbound state. Red and blue lines are energy landscapes in the presence
and absence of external load, respectively
32 T. Luo and D.N. Robinson

the force direction. When the force pulls the system close to the transition state,
i.e., f x > 0, Eq. (2.1) describes the slip-bond model; however, when f x < 0,
it refers to the catch-bond model. Equation (2.1) can only be applied to a single
pathway (either catch-bond or slip-bond). However, sometimes there are multiple
pathways between the bound state and unbound state, and catch-slip and slip-catch
transitions usually occur (as shown in Fig. 2.5). In that case, the unbinding rate is a
superposition of the rates of two different pathways:
   
f xs f xc
koff = ks0 exp + kc0 exp , (2.2)
kB T kB T

where the subscript s and c represent slip and catch, respectively.


To characterize intermolecular interactions, two kinds of experiments are often
conducted: the measurement of the bond life-times at a constant force and the
measurement of rupture forces at a constant loading speed. Under constant force
conditions, the surviving probability P (t) of a bond in the bound state satisfies
(Thomas, 2008)

dP (t)
= koff (f ) P (t) , (2.3)
dt

where koff (f ) is described by Eq. (2.1). Apparently, P (t) decreases exponentially


over time and the slope of P (t) in log scale is koff (f ). For the slip-bond model, the
surviving probability shows a larger negative slope when force increases; whereas
for the catch-bond model, it shows the opposite trend. Under constant loading rate
conditions, the rupture force is
 
kB T x kB T  
f = ln 0 + ln rf , (2.4)
x koff kB T x

where rf is the loading rate and f (t) = rf t (Ackbarow et al., 2007). Equation (2.4)
predicts a linear relationship between the rupture force and logarithm of the constant
loading
 rate. If the intermolecular interaction involves multiple bonds, the f ln
rf will show different slopes associated with different bond energies.
Intramolecular interactions include covalent bonds, hydrogen bonds, electrostatic
interactions, van der Waals interactions, and hydrophobic interactions. Mechanical
deformation can cause twisting and stretching of a single bond or the whole protein,
general conformational changes and the unfolding of domains. Since intramolecular
interactions are much more complicated than intermolecular interactions, there is no
simple mathematic model to describe how mechanical force affects the intramolec-
ular interactions even though protein unfolding can still be probed experimentally
using similar techniques. However, for a linear polyprotein, such as an actin filament
and titin, the force-extension relationship can be predicted using a worm-like-chain
(WLC) model that was originally developed to describe the mechanical behaviors of
double stranded DNA molecules (Bustamante et al., 1994; Marko and Siggia, 1995):
2 The Role of the Actin Cytoskeleton in Mechanosensation 33

kB T 1 x 1
f =   2 + , (2.5)
Lp 4 1 x Lc Lc 4

where Lp is the persistence length, Lc is the contour length, and x is the exten-
sion. Another popular model of the force-extension of proteins is the free-joint-chain
(FJC) model (Bueche, 1962):

 
fl kB T
x = Lc coth , (2.6)
kB T fl

where l is the Kuhn monomer size. In the WLC model, the force is written as a
function of the chain extension while in FJC model the extension is written as a func-
tion of the force. However mathematically, both models give similar force-extension
curves.

2.2.1.1 Force Generation Associated with Actin Polymerization


Actin polymerization, like microtubule polymerization, is force generating. During
actin polymerization, actin monomers bound with a nucleotide assemble into helical
filaments. The nucleotide binding cleft sits between subdomain 2 and subdomain 4,
which faces the minus or pointed end of the actin filament. The opposite end is the
fast growing so-called barbed- or plus-end of the filament. The plus and minus ends
have different affinities for ADP-actin or ATP-actin monomers with the plus end
having the highest overall affinity for ATP-actin monomer. After some time delay
after the ATP-actin monomer incorporates into the polymer, the ATP is hydrolyzed
to ADPPi . Subsequently, the Pi is released, leaving behind an ADP-actin monomer
within the actin filament. The effect of these kinetics is that a density gradient of
ATP-, ADPPi -, then ADP-actin monomers extends from the plus- to minus-ends of
the actin polymer. In comparison to ATP-monomers, ADP-monomers have different
conformations and are less stable in the filament form. Therefore, at concentrations
between the critical concentrations of the two ends, net polymerization at the plus
end and net depolymerization at the minus end lead to treadmilling: the net flow
of actin monomers from plus end to the minus end. During polymer assembly, the
filament can generate a force, measured at 1 pN, which is close to the theoretical
estimate (for a given concentration of G-actin) according to
 
kB T k+ cA
f = ln , (2.7)
k

where is the length increment of one monomer addition, k+ (k ) is the on(off)


rate of polymerization at the plus end, and cA is actin concentration (Kovar and
Pollard, 2004; Footer et al., 2007). Therefore, filament assembly by itself is force
generating, and compressive forces can suppress filament growth. Furthermore, the
34 T. Luo and D.N. Robinson

theoretical estimate implies that simply by increasing the free actin concentration,
greater forces may be generated by filament assembly. However, this process is
limited by the flexural rigidity of the filament, which sets a force limit beyond
which the filament buckles. However, actin-associated proteins may increase the
flexural rigidity, increasing the range of forces that might be generated by filament
assembly.

2.2.1.2 Force-Dependent Behaviors of Actin-Myosin Binding


Myosin II is one type of an actin-based motor that converts chemical energy into
mechanical work by amplifying a small conformational change associated with ATP
hydrolysis in its motor domain and translating it into the relative movement of the
myosin II and the actin filament. The actin-activated myosin II ATPase cycle is
shown in Fig. 2.6 (Spudich, 2001). Initially, ATP binds to the nucleotide-binding
pocket in the myosin head (motor domain), which results in the unbinding of the
motor from an actin filament. Upon ATP hydrolysis, the myosin rotates its lever
arm, moving the head into the pre-stroke state. In this state, the motor can weakly
sample the actin filament in search of its binding site. Upon binding, the motor locks
on tightly and releases the Pi as the head begins to swing the lever arm through its
working stroke (a 70 rotation). Upon completing the full working stroke, the
ADP-bound motor remains locked onto the actin filament. Subsequently, the motor
releases the ADP, forming the so-called rigor state. ATP can then rebind, starting
the cycle over again, which will continue until ATP is depleted. The time the motor
spends tightly bound to the actin filament is the strongly bound state time. The ratio
of the strongly bound state time to the entire ATPase cycle time gives rise to a duty
ratio, which specifies the fraction of time each motor domain spends tightly bound
to actin and correspondingly, the fraction of motor heads tightly bound to actin at
any time. Because the motor generates force as it translates the actin filament, the

Fig. 2.6 Myosin II ATPase cycle


2 The Role of the Actin Cytoskeleton in Mechanosensation 35

motor performs work as it undergoes its conformational change. For most myosin
family members (with myosin VI having some unique and exquisitely complicated
twists on this theme (Phichith et al., 2009)), the head region is highly rigid while
the lever arm is considered to be elastic. Furthermore, the ADP-release step itself
is not force-sensitive for most myosin isoforms. Rather the conformational changes
that precede the formation of the ADP-bound post-stroke configuration are force-
sensitive. Therefore, as the motor swings the elastic lever arm through its power
stroke, resistive tension can lead to deformation (strain) of the lever arm, inhibit-
ing the lever arm swing and locking the motor in the load-bearing, transition state.
Moreover, the step-size of the myosin is related to the length of its lever arm, and this
relationship has been shown to be linearly proportional to the lever arm length for
Dictyostelium myosin II. A further hypothesis is that the maximum force depends
on the length of the lever arm (Uyeda et al., 1996). Lower force is required to stall a
motor with a long lever arm whereas the same motor with a short lever arm powers
through greater loads before being stalled by load, implying that the strain on the
lever arm prevents the full conformational change needed to allow for the motor
to acquire the conformation where it can release the ADP. This load-dependency
is consistent with a catch-slip-like behavior, which can be interpreted using
Eq. (2.4).

2.2.1.3 Force-Dependent Binding Between Actin Crosslinkers and Actin


Filaments
In order to crosslink or bundle two actin filaments, an ACLP must have a mini-
mum of two actin-binding domains associated with each functional unit through
which the ACLP contacts the actin filaments. This can occur by either dimeriz-
ing a monomer that contains one ABD or by having two or more ABDs within a
monomer. Furthermore, the strength of the actin interactions and the conformation
of the actin network (meshwork or bundle) varies for different ABDs and the dif-
ferent conformations of ABDs within the ACLP. For example, the ABDs may be
closely linked through short spacers (e.g. fimbrin), resulting in tightly packed actin
bundles or there can be elongated spacers (e.g. -actinin and spectrin), which can
form relatively loose networks (Bauelos et al., 1998). The most common ABD
module is composed of two tandem CH domains; this module is found in ACLPs
such as -actinin, spectrin, dystrophin, fimbrin, filamin, plectin and cortexillin. For
comparison, the dissociation constants of Acanthamoeba -actinin, chicken smooth
muscle -actinin and Dictyostelium -actinin are 4.7, 0.6 and 3 M, respectively
even though the first two have very similar structures (Wachsstock et al., 1993).
In single molecule measurements, filamin A and rabbit muscle -actinin have actin-
unbinding energies of 4.3 kB T and 3.6 kB T, respectively, while displaying increasing
rupture forces with increasing loading rates (Ferrer et al., 2008). The loading rate
dependence of the rupture forces of these crosslinkers is indicative of catch-bond
behavior.
36 T. Luo and D.N. Robinson

2.2.1.4 Force-Dependent Intramolecular Deformation of ACLPs


During the deformation of the actin cytoskeleton, ACLPs undergo intramolecular
deformations, which involve domain unfolding and shearing and stretching between
two actin-binding domains. The unfolding process can be assessed experimentally
by single molecule stretching, and the resulting saw tooth-like force-extension curve
usually agrees well with the WLC model. Molecular dynamics simulations can also
be used to computationally pull on proteins from various directions at relatively
high pulling speeds to reveal detailed unfolding schemes. The most common struc-
tures of interest typically include bundles of -helices or -sheets (Rohs et al., 1999;
Ackbarow et al., 2007; Buehler and Keten, 2008). Two studied examples are filamin
and -actinin, both of which form anti-parallel homodimers. However, filamin is
constructed from multiple -sheet-like immunoglobulin (Ig) domains, the number
of which differs between various family members. By contrast, -actinin includes
multiple spectrin repeats, each of which consists of a bundle of three -helices.
Dictyostelium filamin (DdFLN) consists of an ABD at the amino-terminal end fol-
lowed by a rod domain, containing six Ig domains. Single molecule stretching of
DdFLN revealed that the sequence of the fourth Ig domain is unique because it has
a lower unfolding force (Schwaiger et al., 2004). Additionally, it was found that
the fourth Ig domain has an intermediate unfolded state in the low force regime.
Based on the WLC model, the persistence length is 0.5 and 0.9 nm for the high
force regime and the low force regime, respectively. The corresponding unfolding
force ranges from 50250 pN and the periodicity of extension also ranges from
1417 nm. Similarly, the human endothelial filamin A has one ABP and 24 Ig
repeats plus two flexible hinges. However, despite the diversity in the structures
between the two filamin family members, the force extension curve reveals a sim-
ilar persistence length of 0.33 nm and an unfolding force, ranging from 50 to 200
pN (Furuike et al., 2001). The unfolding of spectrin repeats requires an unfolding
force of 3050 pN, a persistence length of 0.8 nm and an extension period of
31 nm (Rief et al., 1999). In the Ig domains, -strands are arranged almost in anti-
parallel fashion with small twist angles mainly through hydrogen bond interactions.
During mechanical unfolding, two anti-parallel strands slide in opposite directions
while breaking the hydrogen bonds between them (Lu and Schulten, 2000; Keten
and Buehler, 2008). Many studies have shown that the unfolding of -sheet pro-
teins depends highly on the pulling directions (Brockwell et al., 2003; Nome et al.,
2007; Dietz et al., 2006; Bertz et al., 2009). In each spectrin repeat, three antiparal-
lel -helices linked by loops are folded into a left-handed coiled coil (Pascual et al.,
1997; Djinovic-Carugo et al., 1999). Despite the elasticity of coiled-coil structures,
the unfolding of these repeat domains mainly relies on the stretching of the linkage
between different helices (Altmann et al., 2002). The linkage dependent unfolding
has also been observed in other proteins (Carrion-Vazquez et al., 2003). In summary,
the full force-extension relationship of individual proteins can be obtained by linear
superposition of the force-extension of each domains and linkages while consider-
ing the corresponding folding and unfolding probability at certain forces (Li et al.,
2002).
2 The Role of the Actin Cytoskeleton in Mechanosensation 37

2.2.2 Mechanical Properties of an Actin Network


The mechanical properties of an in vitro assembled actin network depend on the
average length of actin filaments, the mesh size of the actin network, the concentra-
tion of myosin II, the assembly states of myosin II thick filaments, the concentration
of ACLPs, the binding strength between ACLPs and actin filaments, the mechani-
cal properties of each ACLP and the heterogeneity of the actin network (Gardel
et al., 2004a; Wagner et al., 2006; Bausch and Kroy, 2006; Ferrer et al., 2008).
The addition of myosin II can alter the fundamental character of the actin network.
In response to mechanical stimuli, the actomyosin system undergoes continuous
remodeling of its microstructure. The remodeling of the actin-myosin II contractile
system includes assembly/disassembly of actin filaments and myosin II thick fila-
ments and bundling/unbundling of actin filaments by ACLPs. During remodeling,
the whole network is more or less out of mechanical equilibrium, which leads to
transient behaviors within the network (Mizuno et al., 2007; Wilhelm, 2008).

2.2.2.1 Mechanical Properties of Pure Actin Gels


Polymers can be divided into three groups based on two length scales: the persis-
tence length Lp and the contour length Lc . A filament is considered rigid if Lp >> Lc
or flexible if Lp << Lc . Otherwise if Lp Lc , the polymer behaves as though it is
semi-flexible. In vitro, actin filaments assembled from 1-M monomer display an
exponential length distribution ranging from 270 m with a mean length of 22
m (Kaufmann et al., 1992). It should be noted, however, that the length distribu-
tion of many in vivo networks is much smaller. For example, Dictyostelium cells
have an actin filament length distribution that appears to be broadly distributed but
with a mean length of only 100 nm despite that the total (monomer plus poly-
mer) actin concentration is 250 M (Reichl et al., 2008). Nevertheless, for the in
vitro networks and the actin polymer persistence length of 1017 m, the poly-
mers are semi-flexible so that the response to deformation depends on bending and
compression of the filaments. The free energy has the form

Lc b  2  2 f
E= u + (u)2 dz, (2.8)
0 2 2

where u (z) is the transverse deviation of the filament away from a straight con-
formation along the z-axis (MacKintosh et al., 1995). Based on the equipartition
theorem, the force-extension of a single actin filament is


Lc2  f
L = Lc  , (2.9)
Lp
2 n n fb + f
2 2
n=1

where fb = 2 b Lc2 is the threshold force for the Euler buckling instability and
L is the end-to-end distance. In the low force regime, the force is approximately
38 T. Luo and D.N. Robinson
 
a linear function of extension, i.e. f k2 (Lc L) b TL4 (Storm et al., 2005).
 2
However, the force-extension relationship diverges nonlinearly as f 1 (Lc L)
in high force regimes where L Lc . Therefore, the stress increases non-linearly
with increasing strain. That is the F-actin shows strain-stiffening at high stress or
high strain, which is an essential property of many biological materials.
Semi-flexible polymers are viscoelastic in nature, i.e., their deformation is
time-dependent (frequency-dependent) and loading-history dependent (pre-stress-
dependent). The complex modulus G () can be measured by applying an oscil-
latory shear strain sin (t) to the actin solution and by measuring the stress
sin (t + ), where is the frequency and is the phase shift in the range of 0
2 with =0 and = 2, corresponding to a Hookean solid and Newtonian fluid,
respectively. The shear and loss moduli are defined as G () = |G ()| cos ( ())
and G ()=|G ()| sin ( ()), respectively. The complex modulus of F-actin has
a very weak frequency dependency in the low frequency regime 0.0110 Hz whereas
it shows a strong frequency dependency (G 3/4 ) in the high frequency regime
(1010,000 Hz) (Gittes et al., 1997; Gisler and Weitz, 1999; Crocker et al., 2000).
The shear modulus also shows a concentration dependency in which the concentra-
tion determines the mesh size of the actin network and le is the distance between

entanglement points. The 2D density of filaments then is 2 and 0.3 acA ,
where a is the actin monomer size and cA is the actin concentration (Schmidt et al.,
1989). If the extension is assumed to be linearly proportional to le , then the shear
modulus of the actin filament network at small strains is

b
G = , (2.10)
kB T 2 le3

where and are stress and strain, respectively (MacKintosh et al., 1995). 
Furthermore, the fluctuating segment of length le occupies a volume of kB TLe4 b
and the shear modulus can be written as a function of cA such that G b
  2 5
b kb T / (acA )11/5 , i.e., G cA / . On the other hand, considering the excluded
11 5

volume of a filament from an entropy point of view, one expects G c / (Hinner


75
A
et al., 1998; Gardel et al., 2003). Therefore, the moduli of actin networks have
a power low dependence on actin concentration and the corresponding exponent
is in the range between 7/5 and 11/5. Experiments of actin solutions with con-
trolled filament length (Hinner et al., 1998) and uncontrolled filament length (Gardel
et al., 2003) showed G cA/ and G cA/ , respectively. However, this power
75 95

law dependence breaks down when the thermal fluctuation of actin filaments is
severely depressed. For example, in a confined volume  such as a spherical aque-
ous droplet, the entropic effect gives G kb Tlp/ D7/2 when lp < D, where
12

D is the diameter of the droplet (Claessens et al., 2006b). Therefore, the modu-
lus is as much a function of the size of confinement as it is a function of actin
concentration.
2 The Role of the Actin Cytoskeleton in Mechanosensation 39

2.2.2.2 Effects of Crosslinking Proteins on the Microstructures


and Mechanical Properties of Pure Actin Networks
ACLPs microstructurally crosslink actin filaments to form bundles and/or isotropic
meshworks, which generally raises the shear modulus three orders of magnitude
from 0.1 Pa to 100 Pa. In comparison to actin networks without ACLPs at a
specific actin concentration, bundled actin networks have a larger mesh size and
increased bending modulus for each bundle while an isotropic meshwork has a
decreased mesh size. For bundled filaments, if the ACLPS are short and rigid, the
bending of all of the filaments inside the same bundle is coupled and the bending
modulus of each bundle shows a quadratic dependence on the number of filaments.
However, if the ACLPs are long and flexible, the filaments are incompletely cou-
pled, and the bending modulus of each bundle has a linear dependence on the
number of filaments (Claessens et al., 2006a). Based on Eq. (2.10), the effects
of bundling proteins are two-fold: first, they increase and le and second, they
enhance the effective bending modulus of each filament. This two-fold impact can
be observed in filaments bundled by the fimbrin isoform plastin, which shows this
linear dependence, while that of the bundles generated by depletion forces induced
by polyethylene-glycol (PEG) displays a quadratic dependence on filament number.
However, more complex relationships are also observed. The bending moduli of
fascin and -actinin bundles transition from linear to quadratic dependencies with
increasing ACLP concentration. This two-phase behavior may be due to the increas-
ing resistance to the relative shearing between two filaments during bending with
increasing ACLP concentrations (Claessens et al., 2006a). To eliminate the effect of
the deformation of bundling proteins, scruin (a very rigid tight-packing crosslinker)
was used to bundle actin filaments. Here, G () shows a quadratic dependence
on the crosslinking density (the ratio between the crosslinker concentration and
actin concentration) and G c2.5A (Gardel et al., 2004a, b). The corresponding fil-
ament bundle diameter scales as D 0.3 and 0.2 as shown in Figs. 2.7 and
2.8 (Shin et al., 2004). For fascin-bundled F-actin, G correlates strongly with the
microstructures: above a critical , G 1.5 and G c2.4
A ; below that, G
0.1

and G cA (Lieleg et al., 2007). The corresponding bundle diameter scales as
1.3

0.27 . Similarly, engineered proteins containing different repeats in hisactophilin


and disulfide bond-linked hisactophilin dimers exhibit G 0.6 and G 1.2 ,
respectively (Wagner et al., 2006), which indicates that the interaction between two
dimers is also very important. It was proposed that G cA / (6x+15y)/5 , where
11 5

x is the bundling exponent and y is the crosslinking exponent for bundled F-actin
(Shin et al., 2004). G () and G () show a weak frequency dependency in the
low frequency regime. However, both scruin and biotin-avidin crosslinked actin
networks have G () G () 3/4 in the high frequency regime (Gardel
et al., 2004b; Koenderink et al., 2006). By contrast, fascin-bundled actin shows
G () G () 0.5 (Lieleg and Bausch, 2007). Apparently, actin networks
crosslinked by scruin and biotin-avidin interactions, but not fascin, display a similar
power-law dependency of the elastic modulus on frequency at these high frequencies
as that of pure-actin gels.
40 T. Luo and D.N. Robinson

Fig. 2.7 Microstructural images of actin networks crosslinked by ACLPs seen by confocal
microscopy. The actin bundle size increases with the [scruin]/[actin] ratio R as shown in a.
Pure actin-fascin ([fascin]/[actin] = 0.05) and pure actin-filamin ([filamin]/[actin] = 0.1) are shown
in b and c, respectively. d shows the actin composite crosslinked by fascin and filamin at
[fascin]/[actin] = 0.01 and [filamin]/[actin] = 0.1. An illustration of the structural features domi-
nated by individual ACLP when its concentration is dominant is shown in e. Scale bars denote
10 m in all panels. Figure 2.7a is reproduced from Shin et al. (2004) with permission from Proc
Natl Acad Sci USA and the remaining images are reproduced from Schmoller et al. (2008) with
permission from the American Physical Society

Fig. 2.8 ACLP concentration-dependence of bundle size and mechanical properties of actin net-
works crosslinked by scruin. Panels a and b show the increasing of bundle size and mesh size as
a linear function of R ([scruin]/[actin]). The corresponding microstructures are shown in Fig. 2.7.
Elastic modulus (panel c) and critical strain (panel d) increases and decreases respectively with
increasing R. The scale of R in all panels is the same as shown in panel D. Reproduced from Shin
et al. (2004) with permission from the Proc Natl Acad Sci USA

For isotropic meshworks where le , the storage modulus shows a slightly


different dependency on actin concentration such that G b2 (acA )5/2 /(kB T)
(MacKintosh et al., 1995). DdFLN (Wagner et al., 2006) and -actinin (Tseng
and Wirtz, 2001) display G 0.4 and G 1.7 , respectively, when
2 The Role of the Actin Cytoskeleton in Mechanosensation 41

is beyond a certain threshold. Heavy meromyosin (HMM) crosslinked F-actin


exhibits G 1.2 (Tharmann et al., 2007). Therefore, G depends signifi-
cantly on the crosslinker density, which determines the mesh size. Similar to
pure F-actin networks, actin networks crosslinked by filamin A, DdFLN, and
their mutants display a weak frequency dependency of G and G in the low fre-
quency regime (Gardel et al., 2006; Wagner et al., 2006) while filamin A-linked
F-actin meshworks have G () G () 0.17 in the high frequency regime
(Shin et al., 2004). Furthermore, the rupture force of hinged filamins is 10-fold
higher than that of non-hinged filamins (Gardel et al., 2006), suggesting that
the linkages between domains of ACLPs contribute to the strength of the actin
cytoskeleton.
The whole F-actin network shows a decline of G , i.e. a catastrophe, when the
imposed strain exceeds a critical value . One interpretation of the mechanical
failure of a crosslinked network is the unbinding of the ACLPs from actin filaments.
Thus, the interaction strength between ACLPs and actin may determine the mechan-
ical strength of the whole network. It was found that 0.6 for scruin-bundled
networks as shown in Fig. 2.8 (Shin et al., 2004). In fascin bundled networks, a simi-
lar behavior was also observed (Lieleg et al., 2007), and the loading rate-dependence
of the rupture force of a single bond was observed for the maximum stress of the
whole network even though not all fascin-actin bonds were broken at the same time
(Lieleg and Bausch, 2007). The maximum stress of HMM crosslinked F-actin also
agrees well with the unbinding force of rigor HMM-actin, and it shows no stress-
rate dependencies, which is also consistent with the single bond behaviors (Lieleg
et al., 2008). However, curved bundles crosslinked by filamin show no dependence
of on (Schmoller et al., 2008), and rupture stress is linearly proportional to
cA and independent of (Gardel etal., 2004b). In the nonlinear regime, the differ-
ential shear modulus K ( ) = d d may also be used to characterize the actin
network sensitivity to stress or strain. K ( ) is a function of crosslinking density but
independent of cA when the stress is over a certain threshold, and K ( ) 3/2
for scruin bundled F-actin (Gardel et al., 2004a). However, fascin-bundled F-actin
exhibits K 3/2 (Lieleg et al., 2007). Another possible reason for stress soften-
ing is the buckling of actin filaments, though it would primarily occur at very high
stresses (Chaudhuri et al., 2007).
The actin cytoskeleton of intact cells has multiple ACLPs, and F-actin filaments
are crosslinked into both bundles and branched networks. Competitive binding
and cooperative binding between ACLPs potentially exists. In some developmental
systems, two or more ACLPs are needed to work in concert to build necessary cel-
lular structural elements. In terms of cellular mechanics, different crosslinkers also
show a diverse array of interactions, ranging from additive to non-additive effects
(Girard et al., 2004; Reichl et al., 2008). However, there is limited experimental
data on the cross-talk between ACLPs in purified systems. Conceptually, synergis-
tic enhancement of mechanical properties can be theoretically generated just by the
welding of two structurally complementary sub-networks crosslinked by two dif-
ferent types of ACLPs. Two examples illustrate the diversity of possibilities. First,
fascin and filamin crosslinked actin network displays both bundled and branched
42 T. Luo and D.N. Robinson

microstructures; yet these networks display little cross-talk between the two proteins
since the corresponding mechanical properties appear to be determined by the
dominant ACLP as shown in Fig. 2.7 (Schmoller et al., 2008). By contrast, actin
networks crosslinked by -actinin and fascin displayed synergistic enhancement of
elasticity (Tseng et al., 2005). The underlying mechanism may be the complement
between bundled and orthogonal branched networks. Actin networks crosslinked
by -actinin and filamin exhibit more solid-like behaviors than those crosslinked
by the individual crosslinkers, specifically displaying increased G ( ) at low
regime with little enhancement of G () as shown in Fig. 2.9 (Esue et al., 2009).
Thus, further experiments are needed to fully elucidate the crosstalk between other
ACLPs and their synergistic effects on the mechanical properties of purified actin
networks.
Crosslinked networks can also be described as affine or nonaffine. In general,
in the high strain regime, the deformation of the whole network remains affine and
microscopically every filament has almost the same strain, whereas in the low strain
regime the deformation may be nonaffine. The transition from nonaffine to affine is
controlled by three length parameters: filament contour length Lc , distance between
 
crosslinkers lc (mesh size ) and material length lb that is defined as lb = b ,
where is the stretch modulus of the filaments (Wilhelm and Frey, 2003; Head et al.,
2003; Das et  al., 2007; Buxton and Clarke, 2007). The transition can be measured

by = Lc / lc3 lb . Independent of the strain magnitude, nonaffine deformation
3

and affine deformation occur when < 2 and > 20, respectively, and transient
behaviors exist in the range of 2 < < 20. For a fixed contour length (actin dynam-
ics reaches steady state), elasticity is dominated by the stretch modulus under high
crosslinker density or small lc conditions (affine); the bending modulus dominates
under low crosslinker density or large lc conditions (nonaffine). The apparent strain-
stiffening during deformation is basically a nonaffine to affine transition (Onck et al.,
2005; Gardel et al., 2004b). The nonaffine-affine transition theory also successfully

Fig. 2.9 Mechanical properties of actin networks crosslinked by -actinin and fascin. The syn-
ergistic effect on the elasticity of -actinin and fascin is shown in a. The elasticity displays
[fascin]/[-actinin] dependence in b. The exponent of the elasticity as a function of frequency
is shown in c. The total concentration of ACLPs is 0.96 M in b and c and the actin concentration
is 24 M for all panels. Reproduced from Tseng et al. (2005) with permission of Elsevier
2 The Role of the Actin Cytoskeleton in Mechanosensation 43

interprets the scalings between G (or K ) and cA , and the pre-stress observed
in these experimental systems. The corresponding microstructural transition was
observed in scruin-bundled actin network by confocal microscopy (Liu et al., 2007).

2.2.2.3 Effects of Myosin II on the Mechanical Properties of the Actin


Network
In the absence of ACLPs, myosin II motors pull on actin filaments along their axial
directions as shown in Fig. 2.10b. Over time, actin filaments aggregate, resulting in
a heterogeneous distribution of actin filaments (super-precipitation). In the presence
of saturating ATP, active myosin II does not change the shear modulus of an actin
filament solution, whereas in the absence of ATP, inactive myosin II crosslinked F-
actin, leading to a >10-fold enhancement of the shear modulus (Humphrey et al.,
2002). Additionally, the decreasing of G G and the relaxation time associated
with active myosin II indicates that myosin II increases the fluidity of the actin net-
work. This apparent fluidization likely arises from cycles of pulling and releasing,
which generate local fluctuations inside the actin network.

Fig. 2.10 Microstructures and mechanical properties of actin meshwork with muscle myosin II
and human filamin A. Electron micrographs of pure actin, actin+myosin ([myosin]/[actin] = 0.02
and 5 mM ATP) and filamin A +myosin+actin ([filamin]/[actin] = 0.005, [myosin]/[actin] = 0.02
and 5 mM ATP) are shown in a, b and c, respectively. The corresponding fluorescent images are
found in the inserts. The illustration of an active stiffening mechanism is shown in d. Stiffening
behaviors of active networks (with myosin to actin ratio of 0.02 (blue squares), 0.005 (green
squares), and 0.001 (red squares)) and a passive network at fixed [filamin]/[actin] of 0.005 (white
triangles) are shown in e. The filamin concentration dependence of rupture force is shown in f.
Critical strain decreases as a function of the internal stress generated by myosin II. Scale bars are
10 m in all panels. Reproduced from Koenderink et al. (2009) with permission by Proc Natl Acad
Sci USA
44 T. Luo and D.N. Robinson

In the presence of ACLPs, internal tensile stresses associated with the myosin
II motor stiffen the actin network, leading to stress-stiffening such that G ()
G () 1/2 (Mizuno et al., 2007). In assembled networks, myosin II and filamin
A work together to enhance the network stiffness as shown in Fig. 2.10 (Koenderink
et al., 2009). Increasing the myosin concentration leads to higher differential mod-
ulus and increasing the filamin A concentration makes the network able to sustain
higher stresses. The critical strain displays a power-law decay of the internal stress
that is dependent on the myosin II concentration. Because ACLP binding to actin is
dynamic, myosin may break the binding between ACLPs and actin when the myosin
concentrations become high enough, resulting in the power-law decay of the critical
strain with increasing myosin concentration.

2.2.3 In Vivo Measurements of Cell Mechanics

In comparison to reconstituted actin cytoskeletons, the shear moduli of intact cells


are several orders higher (Hoffman et al., 2006; Girard et al., 2004; Reichl et al.,
2008). One reason for this difference is that there are tens of ACLPs with concen-
trations on the order of 1 M in intact cells compared to just one or two ACLPs
as in the in vitro experiments. Another reason is that myosin II motors generate
contractile forces that not only stiffen the actin network but also enhance the bind-
ing between some ACLPs and actin filaments that further increases the stiffness of
the actin cytoskeleton (Reichl et al., 2008). In Dictyostelium, myosin II null cells
have decreased cortical tension and elastic moduli compared to wild type cells.
Similarly, mutant cells depleted with various ACLPs also have softer cortices as
shown in Fig. 2.11 (Girard et al., 2004; Reichl et al., 2008). In the myosin II null
background, a complex relationship between the ACLPs and myosin II is observed.
Some ACLPs (for example, dynacortin) have large effects upon their depletion from
a wild type background but smaller effects when depleted from a myoII mutant
background. By contrast, the depletion of fimbrin has an even more complicated
effect being both time-scale sensitive and myosin II dependent: fimbrin contributes
only to the viscoelastic moduli on fast time-scales in a wild type background, but
also impacts the cortical tension of myoII null cells. More generally, similar to
the reconstituted actin cytoskeletons, the cytoskeleton of intact cells also display
G () G () 3/4 in the high frequency regime, indicating that the mechani-
cal properties of cells are dominated by the entropic vibrations of actin filaments at
these frequencies. However, in the low frequency range, cells exhibit elastic moduli,
G () G () , where 0 < < 0.3 (Hoffman et al., 2006; Deng et al.,
2006; Girard et al., 2004; Reichl et al., 2008). The difference in the low frequency
regimes between intact cells and artificial actin cytoskeletons has been primarily
attributed to the forces generated by motors that push the system out of thermal
dynamic equilibrium (Lau et al., 2003), which is consistent with the in vitro obser-
vations (Humphrey et al., 2002; Mizuno et al., 2007). Indeed, myosin II null cells
have a fundamentally different character in the low frequency range that is more
consistent with a passive network (Girard et al., 2006).
2 The Role of the Actin Cytoskeleton in Mechanosensation 45

Fig. 2.11 Mechanical properties of Dictyostelium cells. a and c show the frequency dependence
of complex moduli of different cell-lines. b and d show the effective cortical tension for these cells.
wt and myoll represent wild-type and myosin II-null cells, respectively. dynhp and fimhp
refer to dynacortin-hairpin and fimbrin-hairpin, respectively. Dynacortin and fimbrin are ACLPs,
and hairpin constructs are used to silence gene expression through RNA interference. Reproduced
from Reichl et al. (2008) with permission of Elsevier

Many of the studies of living cells draw upon laser-based tracking of single or
multiple particles embedded in the living network. Because the particles may fluctu-
ate due to thermal or active forces that act on the particles, the fluctuation-dissipation
theorem (FDT) is used to extract viscoelastic parameters. However, the FDT should
only be applied to systems at equilibrium, not out of equilibrium. Therefore, by
measuring the mean square displacement (MSD) of the particles as a function of
correlation time, it was found that the FDT cannot describe the particle behaviors in
the low frequency regime (Lau et al., 2003; Bursac et al., 2005; Girard et al., 2006;
Mizuno et al., 2007; Wilhelm, 2008; Reichl et al., 2008). Furthermore, in some
contexts, the FDT is violated because the apparent diffusive behavior has a much
larger magnitude than expected, considering the viscous damping for the particle
size. This suggests that local active processes can essentially facilitate the stirring
of the cytoplasm (Brangwynne et al., 2009). It was further demonstrated that the
mechanochemistry of myosin II motors in combination with ACLPs regulates the
46 T. Luo and D.N. Robinson

MSDs in the low frequency regime (Girard et al., 2006). ACLP mutant cells also dis-
play significant effects on the complex modulus over a wide frequency range (Girard
et al., 2004). The coupled effect of myosin and ACLPs was also investigated (Girard
et al., 2006; Reichl et al., 2008). The frequency-dependent mechanical behaviors
of cells imply that microscopic processes, such as unbinding events and conforma-
tional changes of ACLPs and motors with a broad distribution of characteristic times
play important roles in regulating cell mechanics.
Based on these types of experimental observations, three major cell mechan-
ics models have been proposed: tensegrity (Ingber, 2003), soft glassy rheology
(Fabry et al., 2001; Trepat et al., 2007; Zhou et al., 2009) and the sol-gel hypothesis
(Janmey et al., 1990). In the tensegrity model, the actin network, including myosin II
and ACLPs, is considered to be primary sources of pre-stress (Wang et al., 2001),
and cell stiffness is proportional to the pre-stress that cells experience. Tensegrity
can help explain the stress-stiffening, but it does not predict the power-law behaviors
of cells. The soft glassy rheology (SGR) model considers the cells glassy mate-
rials that are microstructurally disordered and thermodynamically close to a glass
transition. The deformations in glassy materials are microscopically inelastic, local-
ized and time-dependent. The SGR model successfully accounts for the power-law
behavior in the low frequency regime but it does not capture the stress-stiffening.
The sol-gel model assumes that the cell is a gel of filamentary polymers embed-
ded within a fluid cytosol, which predicts a weaker frequency dependency in the
low frequency regime than is observed experimentally. Thus, all models capture
some aspects of cell mechanics, but a single model that accounts for all of cellular
behaviors has yet to emerge.

2.3 Functions of the Actin Cytoskeleton in Mechanosensation

Mechanosensing, the sensing of mechanical force, is crucial for a range of processes


that extend over a wide range of length- and time-scales and from the molecular to
organismal levels. At the cellular level, stretch receptors in the plasma membrane
and many components of the cytoskeletal network are obvious targets of external
forces. However, internally generated forces are also felt by the same machin-
ery, allowing intrinsic regulation and cross-talk to occur through the heterogeneous
cytoskeletal network. Here, we will focus on two mechanisms for mechanosensing
in the actin network: the crosslinked actin network complete with myosin II motors,
which governs the cell shape changes particularly during cytokinesis (Effler et al.,
2006; Ren et al., 2009) and the actin-associated proteins found in focal adhesions
(Vogel and Sheetz, 2006).

2.3.1 Mechanosensing Through Myosin II and Actin Crosslinking


Proteins

Dividing cells have a mechanosensory system that they use to monitor their shape
as they cleave into two daughter cells. The system could be activated in a controlled
2 The Role of the Actin Cytoskeleton in Mechanosensation 47

Fig. 2.12 Mechansensing system in Dictyostelium. a and b show the accumulation of fluores-
cently labeled cortexillin I and myosin II, respectively. Cortexillin I and myosin II are green. The
microtubules are labeled red with RFP-tubulin. The lever arm length of different myosin II mutants
is shown in c. The percentage of cells displaying mechanosensing increases with elevated pressure
as shown in d. Reproduced from Ren et al. (2009) with permission of Elsevier

fashion, using micropipette aspiration (Effler et al., 2006). Here, myosin II and the
actin crosslinker cortexillin I accumulate cooperatively in highly deformed regions
in dividing wild type cells as shown in Fig. 2.12. Both myosin-II and cortexillin-I
are necessary for this function since cell-lines devoid of either protein are unable
to respond to cellular deformations (Ren et al., 2009). Furthermore, only fully wild
type myosin-II and wild type cortexillin are able to fully restore mechanosensing
while many of the functions of each protein are expendable for cytokinesis contrac-
tility (Ren et al., 2009). Except for cortexillin-I, other ACLPs that play a major role
in cytokinesis and the microtubules may be dispensed for mechanosensing in divid-
ing Dictyostelium cells (Effler et al., 2006). In sum, these observations indicate that
the cooperative interaction between myosin II and cortexillin I provide the core of
the mechanosensor.
Myosin-II mechanochemistry appears to be the essential active component of the
mechanosensory module. Regulatory light chain phosphorylation leads to the activa-
tion of Dictyostelium myosin-II. Interestingly, this phosphorylation step is essential
for mechanosensing (Ren et al., 2009) but is not required for cytokinesis (Beach
and Egelhoff, 2009). The lack of a requirement for light chain phosphorylation in
cytokinesis is likely due to the fact that RLC phosphorylation only leads to a 3-5-
fold activation of the actin-activated ATPase activity in Dictyostelium myosin II, and
that the myosin II mechanochemistry is not rate limiting for cytokinesis over at least
a 30-fold range (Zhang and Robinson, 2005; Chen et al., 1995). By contrast, many
48 T. Luo and D.N. Robinson

other myosin II isoforms are activated 30-fold by RLC phosphorylation. However,


with respect to mechanosensing, this observation suggested that the motor domain
of myosin-II was the critical active component of cellular-scale mechanosensing.
To test this, the maximum force (Fmax ) of the myosin-II was shifted by altering the
lever arm length (Fmax lever arm length1 ), which shifted the applied pressure-
dependency of the mechanosensory response (Uyeda et al., 1996; Ren et al., 2009).
Because myosin-II must undergo a full lever arm swing before it can release its
ADP, this result strongly indicates that the local accumulation of myosin-II is regu-
lated by the kinetics of its binding/unbinding to actin filaments. It is also likely that
the extent of accumulation during mechanosensing is proportional to the magnitude
of the applied force as shown in Fig. 2.12 (Ren et al., 2009), which is consistent
with catch-bond behaviors of myosin II (Guo and Guilford, 2006).
The regulated assembly and disassembly of myosin II into bipolar thick filaments
(BTFs) is also required for the mechanosensory system. Without assembling into
BTFs, myosin II cannot generate contractile force and therefore experience tension.
Because myosin II accumulates during the mechanosensory response, unassembled
monomeric myosin II must be able to diffuse to the site, requiring these myosins
to be disassembled. Therefore, the full assembly/disassembly dynamic is required
for this process. It should be noted that myosin-II BTF assembly in Dictyostelium
is independent of myosin-II light chain phosphorylation but is fully dependent on
the heavy chain phosphorylation. In contrast, mammalian myosin II assembly is
regulated by both heavy chain and regulatory light chain phosphorylation (Beach
and Egelhoff, 2009 and references therein). Regulatory light chain phosphoryla-
tion results in a conformational change from a thick filament assembly incompetent
state to an assembly competent state (the so-called 10S 6S transition) (Craig
et al., 1983). Because Dictyostelium myosin-II does not undergo this transition, it
does not require the RhoA-ROCK kinases pathway for regulation of contractility.
Consistently, the Dictyostelium genome is devoid of ROCK kinase.
Dividing cells have distinctive mechanical properties (the cells soften from
anaphase through cytokinesis completion) and the global/polar actin crosslinkers
(dynacortin, fimbrin, and enlazin) become more cytoplasmic as compared to inter-
phase cells (Robinson and Spudich, 2000; Reichl et al., 2008). Similarly, wild type
cells show a much stronger mechanosensory response during cell division than dur-
ing interphase. However, the mechanosensitive localization occurs very strongly in
interphase RacE null cells (Ren et al., 2009). Unlike RhoA that regulates myosin-II
functions, RacE, a Rac-family small GTPase, is known to be upstream of global
ACLPs, such as dynacortin, enlazin and fimbrin (Robinson and Spudich, 2000;
Zhang and Robinson, 2005). Experimental data shows that the cortical stiffness of
RacE null cells is much lower (70% lower) than that of wild type cells, indicat-
ing RacE helps to maintain the mechanical integrity of the actin cortex. It is not
known yet whether RacE directly inhibits the mechanosensory pathway or if the
mechanosensory response only occurs within a certain range of cortical stiffness.
The molecular mechanisms for the cooperative accumulation of myosin II and
cortexillin I remain to be fully defined. However, there are four processes that
may contribute to their accumulation. The first is that forces stabilize the bipolar
2 The Role of the Actin Cytoskeleton in Mechanosensation 49

thick filaments in highly deformed region by increasing the binding life time of
myosin-actin. This tight binding then promotes additional myosin accumulation
through cooperative interactions between motor domains of assembled (in BTF
form) and unassembled myosin monomers. The second mechanism is that the
binding of cortexillin and the binding of myosin to actin filaments facilitate each
other, i.e. cortexillin and myosin II may bind actin cooperatively. A third possible
mechanism is that the curvature sensitivity of phosphatidylinositol 4,5-diphosphate
(PIP2 ) lipid molecules might lead to PIP2 accumulation in the pipette, result-
ing in cortexillin accumulation since cortexillin binds PIP2 (Stock et al., 1999).
Consequently, the cortexillin accumulation increases the local stiffness of the actin
network and enhances the force propagation, which can potentially lead to myosin
II accumulation through the first mechanism. Finally, the fourth possible mech-
anism is that BTF assembly regulatory enzymes, including MHCK and myosin
heavy chain phosphatase (which is less well characterized), may be force sensitive.
The reduced phosphorylation (by inhibiting MHCK or activating the myosin heavy
chain phosphatase) of myosin heavy chains with increasing force could promote
local accumulation of the BTFs. However, force-sensitive activation or inhibition
of these enzymes may not be essential; rather the enzymes may only be required
to maintain the available free pool of myosin monomers, which is essential for
the mechanosensory response. These four mechanisms are not mutually exclu-
sive. In the next sections, we will show how force-dependent promotion of BTF
assembly and cooperativity between myosin II and cortexillin might lead to the
inter-dependent, mechanical stress-induced accumulation of these proteins.

2.3.1.1 How Force Might Modulate Myosin II Bipolar Thick Filament


Assembly
To consider where force might act in myosin BTF assembly, a sensitivity analysis
of the BTF assembly pathway was assessed. The reactions of myosin II assem-
bly and the corresponding reaction rates are listed in Table 2.1. M0 , M, D and
T represent phosphorylated (assembly incompetent) monomer, unphosphorylated
(assembly competent) monomer, dimer and tetramer, respectively. BTF3 , BTFn and

Table 2.1 Kinetics of


myosin II assembly based on k1 k1 = 0.0008 s1
M0  M,
the dimer addition model. k1 k1 = 0.1 s1
Reproduced from Ren et al. k2 k2 = 0.37 M1 s1
(2009) with permission of M+M  D,
k2 k2 = 0.01 s1
Elsevier
k3 k3 = 0.0395 M1 s1
D+D  T,
k3 k3 = 0.0045 s1
k4 k4 = 1.25 M1 s1
T +D  BTF3 ,
k4 k4 = 0.025 s1
k5 k5 = 10 M1 s1
BTFn + D  BTFn+1 ,
k5 k5 = 0.2 s1
50 T. Luo and D.N. Robinson

BTFn+1 are the bipolar filaments having 6, 2n and 2(n+1) monomers, respectively.
The formation of the anti-parallel tetramer T is the nucleation step, and the subse-
quent steps are the growth phase of BTF assembly where dimers D are added to the
BTF. The reaction rates are derived from a combination of in vitro kinetic studies of
BTF assembly as well as in vivo fluorescence recovery after photobleaching exper-
iments (Mahajan and Pardee, 1996; Moores and Spudich, 1998; Reichl et al., 2008;
Ren et al., 2009). From this analysis, the most likely process that may be affected
is the ratio of k1 to k1 . Figure 2.13 shows the kinetics of assembly after this ratio
is shifted ten-fold, which could mimic inhibition of MHCK, activation of myosin
phosphatase, or a lowering of the energy barrier required for incorporating M0 into

Fig. 2.13 Accumulation of myosin II in response to force in Dictyostelium cells during anaphase:
(a) spatial distribution of myosin II intensity in pipette region normalized by the intensity at the
opposite pole of the cell; (b) averaged transient curve of myosin II accumulation where intensity
is normalized by the intensity in the cytoplasm; (c) simulation result of the kinetics of myosin II
accumulation when k1 in Table 2.1 is decreased by 10 fold to mimic the force effect; (d) the BTFs
distribution before (without force) and 60 s after 10-fold change in k1 . Reproduced from Ren et al.
(2009) with permission from Elsevier
2 The Role of the Actin Cytoskeleton in Mechanosensation 51

a pre-existing BTF. The force dependence of these steps generally can have the form
similar to Eq. (2.1).
Overall, this analysis suggests that the largest impact on the assembly mechanism
might be in the transition from assembly-incompetent to assembly-competent states.
Although force might act on MHCK or a myosin heavy chain phosphatase, this sce-
nario requires additional enzymes and steps. A very appealing mechanism is one
where motor domains found in pre-existing mini-BTFs are stabilized in the mechan-
ical transition state by force, which then leads to the local accumulation of myosin
monomers, putting them in close proximity to the BTF where they can be directly
inserted. The motors found in the HMM form of myosin II (dimeric but unassem-
bled myosin monomers; see Fig. 2.2) binds actin in a highly cooperative fashion
but only during the transition state of the actomyosin-ADP+Pi complex (Tokuraku
et al., 2009) or if the actin structure has been altered such as by being assembled
with Ca2+ cations (Ca2+ ATP-actin as opposed to Mg2+ ATP-actin found in cells)
(Orlova and Egelman, 1997). This mechanism has the appeal that no additional
enzymes are required, and that all of the necessary parts are included directly in
the myosin motor and thick filament assembly region. In this model, the MHCK
and myosin heavy chain phosphatase are still required to maintain the pool of avail-
able myosin monomers M0 and to ensure that the system relaxes back once the
mechanical signal subsides. However, more experiments are required to see if this
mechanism accounts for mechanosensitive BTF assembly.

2.3.1.2 Cooperativity Between Myosin II and Cortexillin


Since myosin II-binding to actin can be highly cooperative depending on the actin
conformation and the transition state of the motor-actin complex (as discussed in
the previous section), it is very tempting to consider that this cooperativity may be
extended to interactions between myosin and actin-associated proteins. For muscle
myosin II, the motors bind cooperatively to actin filaments, but only in the pres-
ence of tropomyosin-troponin (Geeves and Halsall, 1987; Hill et al., 1980; Chen
et al., 2001). On the other hand, the binding of actin-binding proteins (ABPs),
such as scruin (Owen and DeRosier, 1994) and formins, induces noticeable struc-
tural changes in F-actin. Since F-actin is the common binding substrate of myosin
II and ACLPs, it is possible that the binding of myosin and ACLPs to actin are
cooperative, i.e. the structural changes in F-actin due to one kind of binding facili-
tate another kind of binding (Williamson, 2008). Mathematically, the cooperativity
between myosin II and cortexillin can be written as

dCM  M  dCC  
= g (CC ) kon CM koff
M
and = h (CM ) kon
C
CC koff
C
. (2.11)
dt dt

where C is the concentration. The superscripts/subscripts M and C represent myosin


and cortexillin, respectively. g and h are functions characterizing the cooperativ-
ity between the two proteins. Here, only positive cooperativity is considered and
52 T. Luo and D.N. Robinson

both g and h always have non-negative values. Furthermore, because of the force-
dependence of myosin-actin M is assumed to be a function of force, i.e.
binding, koff
 
M (f ) = k0 exp f x
koff off kB T .

2.3.2 Mechanosensation Through Focal Adhesion Complexes


Mechanosensitive behaviors of focal adhesions (FAs) are important in many cellu-
lar processes such has cell growth, differentiation and motility. FAs are mechanical
linkages between the cytoskeleton and the extracellular matrix (ECM). FAs are
large multi-molecular complexes that can extend several-micrometers and consist
of a large number of different proteins, including integrins and ABPs such as talin,
vinculin, paxillin, and tensin (Zamir and Geiger, 2001; Geiger et al., 2009). In
FAs, integrins form heterodimers consisting of and subunits non-covalently
bound and each consisting of an extracellular domain, a single-pass transmem-
brane helix, and a short cytoplasmic tail (Puklin-Faucher and Sheetz, 2009). The
tail of -integrin binds to the talin head domain. Talin may then anchor directly
to actin or indirectly through vinculin. FAs can be stationary or mobile while dis-
playing a continuous exchange of components with the cytoplasmic pool. FAs grow
with increasing local force (Tan et al., 2003) and tend to orient in the direction of
applied force (Riveline et al., 2001), which has been attributed to stretching forces,
which may enhance the binding affinity of integrins to the ECM (Katsumi et al.,
2005).
FA formation is initiated with the activation of integrins. Inactive integrins adopt
a bent shape whereas the active forms have an extended shape (Hynes, 2002 and
references therein). Integrins may be activated either by their head binding to the
ECM (so-called outside-in signaling) or by the tail binding to talin (so-called inside-
out signaling). The activation of the head and the resulting binding to the ECM
are thought to occur through long-range conformational changes that propagate
through the integrin extracellular domain. Activation of integrin 5 1 in cells can
be switched on mechanically, and the corresponding strength of FAs increases with
the rigidity of the extracellular matrix, indicating that the integrin-ECM interac-
tion fits a catch-bond model (Friedland et al., 2009). Single molecule measurements
have also shown that the catch-bond behavior of integrin 5 1 may be attributed to
the mechanical activation of the headpiece, but not integrin extension, over a force
range of 430 pN (Kong et al., 2009).
The next mechanosensitive protein in the FA is talin, a large protein consisting
of an N-terminal head region and a long rod region. Near talins amino-terminus
is a FERM (band 4.1, ezrin, radixin, moesin) domain through which talin binds to
integrin, focal adhesion kinases (FAKs) and other receptors. Talin-binding to the
integrin tail disrupts an intracellular salt bridge between the - and -integrin sub-
units, increasing the integrin affinity for ECM (Tadokoro et al., 2003). Additionally,
talin has eleven vinculin binding sites (VBSs) in its rod region. Single molecule
measurements discovered that only one VBS is active in the absence of force and
2 The Role of the Actin Cytoskeleton in Mechanosensation 53

two more VBSs appear when the talin rod is stretched by relatively low (12 pN)
forces (del Rio et al., 2009). In the absence of force, these two force-sensitive VBSs
are thought to remain buried in adjacent amphipathic helices through hydrophobic
interactions.
Talin then links to vinculin, a 116 kDa actin-binding protein, which links the core
FA proteins to the actin cytoskeleton. The vinculin head domain consists of seven
-helices arranged as two four-helical bundles (eight -helices), and its tail domain
has five -helices that form an anti-parallel bundle. The strong interaction between
the head and the tail masks the binding sites for other proteins such as talin, F-actin,
-actinin and paxillin and keeps vinculin in its inactive states. Upon talin-binding
to -actinin, the tail domain of vinculin is displaced away from the head domain,
which activates vinculin (Izard et al., 2004) and enables its binding to F-actin and
other molecules.
The mechanical stretching of FAs triggers many downstream signaling pathways
by activating the SH2 domain-containing phosphatase SHP-2 and non-receptor pro-
tein tyrosine kinases, such as Src and FAK (Tamada et al., 2004; Giannone and
Sheetz, 2006). These enzymes regulate the assembly/disassembly of FAs by con-
trolling the actin stress fiber (actin bundles with myosin II thick filaments) formation
(Vicente-Manzanares et al., 2009). Activity of some of these kinases, such as FAK
kinase, requires actin and myosin II-dependent tension (Tilghman and Parsons,
2008). Furthermore, stretching of p120Cas in cells or using an in vitro reconsti-
tution system exposes more Src kinase binding sites and leads to its local activation
(Sawada et al., 2006). This stretch-induced activation can be very fast as Src at
remote sites may be activated within 0.3 s, demonstrating just how fast signals can
propagate through the elastic cytoskeleton (Na et al., 2008).

2.3.3 The Actin Cytoskeleton Works as a Force-Transmission


Highway

Both chemical and mechanical signals can be transmitted over long distances.
Propagation of chemical signals occurs mainly through the diffusion of molecules
in the cytosol and its speed is limited by the chemical reaction (such as phospho-
rylation) rates, unbinding/binding rates and diffusion rates. Diffusion is usually the
limiting step since the diffusion coefficient of molecules in cells is in the range of
0.01100 m2 s1 , depending on the molecular size and shape and the viscosity of
cytoplasm on the length-scale of the diffusing particle (Howard, 2001). For exam-
ple, it can take one molecule 1100 s to travel 10-m using diffusion alone. On
the other hand, mechanical signals may be transmitted through the deformation of
cytoskeleton along actin filaments, microtubules and intermediate filaments. The
speed of transmission depends on the elastic modulus of the cytoskeleton where
signals may propagate over a 10-m distance on sub-second time-scales, indicating
that proteins in cells can sense mechanical stimuli much more rapidly than chem-
ical signals (Forgacs, 1995). This propagation of mechanical deformations likely
54 T. Luo and D.N. Robinson

depends on the pre-stress as well as the elastic modulus of the cytoskeleton, and
the magnitude of deformation decays exponentially in space with a characteristic
length that is comparable to or larger than the size of the cell (Wang and Suo,
2005).
One consequence of signal propagation through the integrated elastic actin
cytoskeleton is that signals can be transmitted over long distances and broad areas
and to a range of organelles. For example, actin filaments are connected to the
nuclear envelope through a complex of SUN and nesprin proteins (Wang et al.,
2009), to the plasma membrane through proteins such as ezrin, radixin, and moesin
(ERM proteins) (Sato et al., 1992), to stretch-activated channels (SACs) by myosin
I motor proteins (Fettiplace and Hackney, 2006), and to mitochondria by mitochon-
drial ABPs (Boldogh et al., 1998). Forces transmitted by the actin cytoskeleton
to the nucleus alter gene expression, which may in turn regulate actin remodel-
ing. The SACs can also be activated or deactivated by cytoskeleton stretching,
resulting in ion flux, regulating stress fiber formation and orientation and myosin
II bipolar thick filament assembly. Mechanical stimuli propagated to membrane-
bound or associated proteins through the actin-membrane connections can lead
to changes in activity of membrane-bound signaling molecules and other ion
channels.

2.4 Remodeling of the Actin Cytoskeleton During


Mechanosensation

The actin cytoskeleton is composed of highly dynamic structures. Besides


mechanosensing and transmitting mechanical signals, the cytoskeleton can rear-
range its structures in response to the mechanical stimuli this is referred to as
remodeling. Actin remodeling is determined by ABPs that control linear elon-
gation, shortening and organization of actin filaments in response to signaling
cascades (Stossel et al., 2006). However, superimposed over these mechanosensi-
tive actin-binding proteins are signaling molecules, such as kinases, Rho-family
GTPases (e.g. RhoA, Cdc42 and Rac), phosphoinositides and WASP-family pro-
teins, which are also spatially and temporally coordinated biochemically and,
indirectly, mechanically.
Most Rho proteins switch between active (GTP-bound) and inactive (GDP-
bound) conformations. The activities of Rho proteins are regulated by Rho guanine
nucleotide exchange factors (Rho-GEFs) and Rho GTPase-activating proteins
(Rho-GAPs). The Rho-GEF promotes the exchange of GDP for GTP while the
Rho-GAPs enhance GTP hydrolysis. Rho guanine-nucleotide dissociation inhibitors
(GDIs) also bind to prenylated GDP-bound Rho proteins and allow their translo-
cation between membrane and the cytosol (Buchsbaum, 2007). RhoA and its
effector Rho-kinase elevate myosin II light chain phosphorylation and thereby
promote myosin II activation. Cdc42 activates WASP, which subsequently medi-
ates the branched actin-network formation by activating Arp2/3 (Pollard, 2007).
PIP2 can induce G-actin dissociation from actin-monomer-binding proteins and
2 The Role of the Actin Cytoskeleton in Mechanosensation 55

uncapping of the actin filament barbed ends, and can enhance the linkages between
the actin cytoskeleton and the plasma membrane by activating ERM proteins
(Nebl et al., 2000). PIP2 can also activate vinculin, promoting FA assembly
(Sechi and Wehland, 2000), and WASP, increasing actin polymerization (Pollard,
2007).

2.4.1 How Mechanically Activated Kinases Regulate the Actin


Cytoskeleton
Mechanically induced FAs trigger the activation and recruitment of many down-
stream kinases (Brakebusch and Fssler, 2003), and these kinases affect actin
remodeling by regulating small GTPases and ABPs. Focal adhesion kinase binds
to integrin, talin and paxillin, and this binding enhances FAK activities, which pro-
motes stress fiber formation by increasing the recruitment of talin and paxillin.
Integrin-linked kinase (ILK) binds the tails of integrin subunits, paxillin and phos-
pholipids, which induces the phosphorylation of PKB/AKT protein kinases that are
upstream of actin polymerization. ILK also forms a complex with other proteins
to recruit F-actin to FAs. The FAK-Src complex stimulates Rac1 activity, recruits
the GEF for Cdc42 and Rac1, and mediates the suppression of Rho-GTP by reg-
ulating Rho-GEFs and Rho-GAPs (Huveneers and Danen, 2009 and references
therein).

2.4.2 Crosstalk Between Microtubules and Actin Cytoskeleton

Increasing evidence shows crosstalk between microtubules and actin cytoskeleton.


For example, centrosome separation and positioning during mitosis depend on the
integrity of the actin cytoskeleton and F-actin cortical flow (Rosenblatt et al., 2004).
By contrast, actin nucleation near the plasma membrane is coordinated by micro-
tubules and microtubule-associated proteins (Martin et al., 2005; Siegrist and Doe,
2007; Rosales-Nieves et al., 2006). Similar to the concept of the tensegrity model,
cortical motors may pull on astral microtubules and conversely, astral microtubule
polymerization exerts a pushing force against the actin cortex, promoting centro-
some separation and positioning. The activation of Src was observed at cortical sites
where microtubules appear to deform the cortex (Na et al., 2008). Microtubules
also affect the spatial distribution of active small GTPases, thereby regulating the
organization of the actin cortex (Siegrist and Doe, 2007 and references therein),
and many unproven mechanisms have been proposed for this crosstalk. However,
the list of structural linkages between microtubules and F-actin continues to grow.
Among the first identified linkages was the splice-variant of the mitotic kinesin-like
protein (MKLP1) called CHO1. CHO1 has a kinesin-family motor domain, which
can move on microtubules, and an additional microtubule-binding domain and an
F-actin binding domain in its tail (Robinson and Spudich, 2004). Indeed, MKLP1
proteins help organize the central spindle microtubules, and CHO1 can integrate this
56 T. Luo and D.N. Robinson

system with the cortical actin network. In Drosophila melanogaster, cappuchino (an
FH2 containing protein) and spire (a WH2 containing protein) can crosslink micro-
tubules and actin filaments (Rosales-Nieves et al., 2006). In Schizosaccharomyces
pombe, tea4p and tea1p also localize to the plus ends of microtubules, and a com-
plex of tea1p, tea4p and the formin for3p is necessary for the establishment of cell
polarity and actin nucleation at new cell ends (Martin et al., 2005). Thus, several
linkages between the actin and microtubule networks promote their integration and
may facilitate force-propagation, and therefore signal-propagation, through these
systems.

2.5 Experimental Techniques for Measuring


Mechanosensation In Vitro and In Vivo Methods

In addition to traditional micropipette aspiration (MPA), the past few decades have
witnessed the development of various new techniques using the combinations of
nanomanipulation, microfabrication, magnetic techniques, and optical techniques
(Bao and Suresh, 2003; Addae-Mensah and Wikswo, 2008). These methods include
atomic force microscopy (AFM), magnetic tweezers, optical tweezers, magnetic
twisting cytometry (MTC), particle-tracking microrheology (PTM), microfluidic
devices, stretching devices, traction force microscopy (TFM) and MEMS-based
devices. Classic MPA still offers a number of advantages in that it is relatively easy
to implement and can be readily adapted to a broad array of cell-types, particularly
those which are not highly adherent. MPA can be used to measure effective tension
and elastic and viscous properties of the cell. Perhaps more significantly, MPA is
very useful for imposing deformations to cells so that the cells response may be
monitored using an array of fluorescence methods. However, the major limitation of
MPA is that it is difficult to measure properties occurring on fast sub-second time-
scales or to measure frequency-dependent features. For these sorts of measurements,
many of the other methods, for example AFM, MTC and PTM, are more suitable
(Girard et al., 2004; Hoffman et al., 2006). PTM can be categorized into single-
bead, two-bead and multiple-bead modes (Wirtz, 2009). Depending on the driving
force, PTM has two working modes: passive and active. To measure the mechanical
properties of single molecules, AFM is commonly used for the high force ranges
(>10 pN) whereas optical tweezers and magnetic tweezers are commonly used
for relatively low forces (Neuman and Nagy, 2008; Finer et al., 1994). AFM and
optical tweezers also allow three-dimensional manipulation of molecules. AFM is
often used to study protein folding/unfolding and protein-protein interactions while
tweezers are usually used to study biological motors, including cytoskeletal motors
and DNA and RNA polymerases. Additionally, many of these methods have been
combined with fluorescence microscopy techniques, such as total internal reflection
fluorescence (TIRF) imaging and fluorescence resonance energy transfer (FRET)
(Sarkar et al., 2004; Moffitt et al., 2008; del Rio et al., 2009).
Microfluidic devices and flow chambers are used to study the cell responses to
shear flow (del lamo et al., 2008; Wang and Levchenko, 2009). Stretching devices
2 The Role of the Actin Cytoskeleton in Mechanosensation 57

are usually coupled with fluorescence imaging methods to quantify the effects of
stretch on actin cytoskeleton rearrangement, activation of kinases, gene expression
and cell differentiation (Sawada et al., 2006; Kurpinski et al., 2006). TFM was
invented to investigate the traction force that cells apply to the substrates (Pelham
and Wang, 1997). Initially, TFM methods utilized fluorescent beads embedded in
polymeric substrates so that traction forces exerted by adherent cells on the substrate
could be calculated from the displacements of the beads. Recently, micropatterned
substrates by soft photolithography have been used to control the cell adhesion areas
and cell shapes (Balaban et al., 2001; Tan et al., 2003; Thry and Bornens, 2006).
Therefore, a broad range of mechanical measurements and manipulations are now
possible across a broad array of length- and time-scales and from the molecule to
cellular levels.

2.6 Conclusion and Perspectives

Mechanical inputs must have been among the first signals that cells received and had
to respond to, and the ability of cells to sense and react to these inputs likely evolved
at a very early time point. Thus, it is not surprising that there are numerous overlaps
between the mechanotransduction and traditional chemical signal transduction
pathways. Because of the overlap between these pathways, mechanical-chemical
coupling and feedback loops are a natural consequence of this system integra-
tion. Because the actin cytoskeleton is structurally integrated with nearly every
aspect of the cell, mechanical inputs can be transmitted quickly throughout the cell.
Furthermore, individual proteins may be involved in multiple pathways and con-
tributing multiple functions. Therefore, to fully understand the roles of individual
proteins and the cooperativity among them in the actin cytoskeleton, in vitro exper-
iments involving single molecule measurements, reconstituted actin networks, and
computational simulations of protein folding/unfolding and protein-protein inter-
actions have to be combined with quantitative in vivo observations. Challenges
for understanding mechanosensation through the actin cytoskeleton include reveal-
ing how proteins function cooperatively over short nanometer-length-scales and
fast sub-second time-scales. Direct observations of force propagation in cells and
eventually between cells within tissues will be essential. Novel designs of mechan-
ical strain sensors using fluorescence readouts such as FRET pairs and inventive
imaging setups will be needed to fulfill these demands. Applying a repertoire of
these approaches to a genetically tractable organism, such as Dictyostelium cells
as they perform physiologically and medically important processes like cell divi-
sion, will continue to provide unique insights into cellular mechanosensing and
mechanotransduction.

Acknowledgements We are grateful to the insightful discussions and comments on the


manuscript from Alexandra Surcel and Sheil Kee. We acknowledge the support of the National
Institute of Health (Grant #GM066817) and the American Cancer Society (Grant #RSG CCG-
114122).
58 T. Luo and D.N. Robinson

Glossary List
Affine Describes an actin network, which is co-linear during stretching and
shearing
Non-affine Describes an actin network in which co-linearity is absent during
stretching and shearing
Stress The force applied on a unit area
Strain The ratio between the length-change associated with deformation and
the original length (no deformation)
ABD Actin-binding domain
ABP Actin-binding protein
ACLP Actin-crosslinking protein
BTF Bipolar thick filament
ELC Essential light chain
FJC Free-joint-chain
HMM Heavy meromyosin
RLC Regulatory light chain
WLC Worm-like-chain
D Bundle size
E Youngs modulus: the proportionality between stress and the resulting
strain
fb Bending modulus: the proportionality between bending momentum
and the resulting curvature
G Complex modulus
G Shear modulus the real part of G : the proportionality between shear
stress and the shear strain
G Loss modulus the imaginary part of G
K Differential shear modulus: the differential proportionality between
shear stress and the shear strain
Lc Contour length: the integrated length along the polymer chain
Lp Persistence length: the length over which correlations in the direction
of the tangent are lost
Le Distance between entanglements
Mesh size
Crosslinking density: the ratio between the cross-linker concentration
and actin concentration

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Chapter 3
Effect of Cytoskeleton on the Mechanosensitivity
of Genes in Osteoblasts

Qiang Fu, Yiping Zhang, Yajuan Xu, Yourui Li, Ling Guo, and Minfeng Shao

Abstract Mechanosensitivity is the ability of tissues and cells to detect and make
response to mechanical stimuli. Osteoblast is a kind of important mechanosen-
sitive cell in bone tissue, while cytoskeleton plays an important role in the
mechanotransduction in osteoblasts. This article reviewed the roles of cytoskeleton
on the mechanotransduction of genes in osteoblasts, summarized that cytoskele-
ton integrity is essential for the expression of bone formationrelated genes in
osteoblasts, and concluded that cytoskeleton reorganization inhibition can enhance
the mechanosensitivity of some genes in osteoblasts. Further researches on the
specific mechanisms of cytoskeleton will shed new light on the transmission mech-
anisms of mechanical stress in osteoblasts, and will lay the foundation for the
further investigations on the biomechanical behaviors of bone cells and even bone
tissues.

Keywords Cytoskeleton Osteoblast Mechanotransduction Mechanosensitivity

3.1 Introduction

Cytoskeleton is the protein fiber network system in eukaryotic cells. The general
concept of cytoskeleton includes nucleus cytoskeleton (intranuclear cytoskele-
ton, nuclear lamina, and chromosome cytoskeleton in mitotic state), cytoplasm
cytoskeleton (microfilament, microtubule, intermediate fibre, and microtrabecular
lattice), cell membrane cytoskeleton and extracellular matrix. The narrow sense
of cytoskeleton only refers to the cytoplasm cytoskeleton. Cytoplasm cytoskele-
ton, which is a fibrous structure system mainly distributed in the eukaryotic cells,
consists of three basic filaments: microtubule, microfilament and intermediate fibre.

Q. Fu (B)
Department of Prosthodontics, Guanghua School & Hospital of Stomatology, Sun Yat-Sen
University, Guangzhou 510055, Guangdong, China
e-mail: fuqiangds@yahoo.com.cn; fuqiangds@gmail.com

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 67


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_3,

C Springer Science+Business Media B.V. 2011
68 Q. Fu et al.

These three filaments are polymerized from different protein subunits (skelemin)
in different specific ways (Qian et al., 2009). The cytoskeleton we reported in this
review is mainly about the cytoplasm cytoskeleton.

3.2 Characteristics and Roles of Cytoskeleton

3.2.1 Characteristics of Cytoskeleton

One of the greatest characteristics of cytoskeleton is its dynamics variance which


occurs in a short time by accommodating to the structure and function of cells.
Dynamics variance is based on the continual polymerization of skelemin, which
prolongs the filaments; or on the continual depolymerization of skelemin, which
decurtates the filaments, and even clears it away. So, the cytoskeleton in cells is in a
condition of continual dynamic reorganization.
The other characteristic of cytoskeleton is its connection with cell nucleus, cell
membrane and some other organelles, and such the connection is reversible due
to the dynamics variance of cytoskeleton itself. In this connected network, the
cytoskeleton system is the main fiber network, around which some other cellular
structures and cytoplasmic matrix of biomacromolecules are cohered and embed-
ded. The dynamic variance of cytoskeleton also endows the cytoplasmic matrix with
the same characteristic.

3.2.2 Roles of Cytoskeleton

The cytoskeleton plays a crucial role not only in maintaining the cellular morphol-
ogy, bearing loads and keeping the intracellular structures in order, but also in lots of
life activities, for examples: guiding the chromosome segregation during cell divi-
sion, transporting kinds of vesicles and organelles along it in a directional transport.
Recently, the increasing evidences indicate that several cellular activities, includ-
ing activation of several signal transduction pathways and transcriptional activity of
many genes, are based on the reorganization of the cytoskeleton. The cytoskeleton
has the function of transferring and dispersing stress, as well as responding to the
mechanical signals (Higuchi et al., 2009).

3.2.2.1 Tensegrity Model


The cytoskeleton, as the core of the ECM-Integrin-CSK-Nucleus network system,
transmits extracellular signals into cells in two different ways: One is that mechan-
ical stress induces the redistribution of the tension in cytoskeleton, and then the
rearrangement of cytoskeleton and morphological change of cells. The role of
the cytoskeleton in this way is mainly the physical conduction. The other is that
the cytoskeleton, entirety or partly as a translator of mechanical-chemical signals,
3 Effect of Cytoskeleton on the Mechanosensitivity of Genes in Osteoblasts 69

makes come true the transformation from mechanical signals to biological signals
and chemical signals. The transmission and transduction of mechanical signals can
be completed by the cooperation of these two ways (Charras and Horton, 2002).
The function of cytoskeleton, directly as the conductive track to transmit mechan-
ical signals into cells, can be explained by the tensegrity model. In this model, actin
microfilaments are the major part to bear the tension, meanwhile, microtubules can
be viewed as the load-bearing element for compression, and intermediate fibres,
as the potent integrated components, connect the microtubules with the inotropic
microfilaments and fix them to the membrane surface and the nucleus. If the con-
ducting system is based on the tensegrity, it must be instant for the mechanical
signals to be transmitted into cells, and faster than any signal conducting system
based on diffusion, which enhances the conducting efficiency of the mechanical
signals (Ingber, 2003). Mechanical stimulus can be transmitted to cytoskeleton
through integrin-microfilament pathway and cadherins-microtubule pathway, which
can change the distribution and polymerization of cytoskeleton, increase the perme-
ability of ion channels coupled with cytoskeleton and the activity of some surface
receptors, and alter the function of the proteins and functional enzymes in many
signal conducting systems coupled with cytoskeleton, then produces the second
messengers and transmits the signals to nucleus, and finally promotes the metabolic
activities of the proliferation and differentiation of cells and the synthesis and
secretion of proteins (Sawada and Sheetz, 2002). The extracellular signals can be
transmitted to cells through this continuum structure, vice versa. Such is the major
way for microfilaments to participate in signal transduction (Chou et al., 2001).

3.2.2.2 Response of Cytoskeleton to Mechanical Forces


The mechanical signals transmitted to cytoskeleton could cause the rearrangement
of microfilaments and microtubules, as well as the change of orientation of cells. The
reorganization of cytoskeleton is good for the extracellular signals to be transmitted
into cells, and then the signals spread along the cytoskeleton, which induces the
gene transcription and the change in cell cycle and cell shape. The response of the
cells to mechanical signals is mainly based on the F-actin cytoskeleton. Studies have
indicated that the cytoskeleton integrity plays a crucial role in the expression of
c-fos and cox-2 genes in osteoblasts induced by fluid shear stress (Chen and Fu,
2008; Wu and Fu, 2009).

3.2.2.3 Cytoskeleton and Genes Transcription and Expression


The cytoskeleton not only participates in the mechanical signal-chemical signals
transduction, but also affects the genes expression in nucleus. Chen et al. (2000)
have confirmed that the increasing of the expression and synthesis of c-fos and cox-2
genes induced by fluid shear force needs the recombination of actin into stress fibers.
The structural change of cytoskeleton could directly activate NF-KB, which greatly
enhances the binding activity of NF-KB with DNA, and then affects the expression
70 Q. Fu et al.

of nuclear genes. Now this function is thought to be determined and performed


mainly by the state of microtubules.
Concerning the mechanism of how the mechanical agents affect the gene expres-
sion through cytoskeleton, Ingber (2003) reported that the distortion of cytoskeleton
could expose some genes around the nucleus, which made them much easier to
be identified and activated. Stein et al. (1999) thought that the rearrangement
of cytoskeleton induced by mechanical stress may stretch the promoter through
karyoskeleton, change its spatial conformation, and then regulate its transcription
activity, which may be one of the pathways for microgravity to affect the genes
expression. Lammerding et al. (2004) have observed that gene mutations of nuclear
lamina would change the mechanical properties of nuclear lamina and the gene
expression induced by tension force.
When some intranuclear genes are activated by the mechanical signals, the imme-
diate early genes will respond rapidly, and the mRNA localization of these immedi-
ate early genes is related to the cytoskeleton. The c-fos mRNAs of most interphase
cells are located in cytoskeleton, but when the microfilaments are depolymerized by
Cytochalasin D, most mRNAs will be transferred into the endochylema.
The actin microfilaments and microtubules in cytoskeleton also participate in the
RNA transfer; the former in short distance transportation, while the latter in long
distance. And both of them have their own corresponding molecular motors. While
the intermediate fibres are tied up with nuclear matrix, their roles in intranuclear
signal transduction and genetic transcription should not be ignored (Guzman et al.,
2006).

3.3 Effect of Cytoskeleton on the Genes Expression


in Osteoblasts

Bone formation-related genes, which could promote the proliferation and activa-
tion of osteoblasts, include c-fos, early growth response gene-1 (egr-1), osteopontin
(OPN), cyclooxygenase-2 (COX-2), extracellular signal-regulated kinase (ERK),
and so on. When these genes are activated, some bone formation-related proteins
are expressed, and then the proliferation and differentiation of osteoblasts and new
bone formation are promoted (Kapur et al., 2004; Lee et al., 2008).

3.3.1 C-fos Gene


C-fos gene, as an important member of the immediate-early gene (IEG) family, can
respond to external stimuli in a few minutes (Lau and Nathans, 1987). C-Fos protein,
as the product of c-fos gene, can combine with c-Jun through leucine zipper to form
the activator protein-1 (AP-1) heterodimer. AP-1, as an important transcription fac-
tor, which has the DNA-binding activity, can combine with the AP-1 binding sites of
the DNA sequences of downstream target genes, and then activate the transcription
3 Effect of Cytoskeleton on the Mechanosensitivity of Genes in Osteoblasts 71

activity of these genes. AP-1 plays a significant role both in the proliferation and
differentiation of osteoblasts and the promotion of bone fracture healing (Cowles
et al., 2000; Zayzafoon et al., 2005). The DNA sequences of the important bone
formation-related genes that are known so far, such as OPN (Nomura and Takano-
Yamamoto, 2000), COX-2 (Ogasawara et al., 2001) and so on, have AP-1 binding
sites. That means, the expression of these genes would be regulated by c-fos gene.
Studies have found that tensile stress and compressive stress could activate
c-fos gene expression of osteoblasts, but when the cytoskeleton is destructed by
Cytochalasin D, the cytoskeleton would depolymerize, which could interrupt the
c-fos gene expression of osteoblasts induced by tensile stress and compressive stress
(Nomura and Takano-Yamamoto, 2000; Ogasawara et al., 2001). Our previous work
has showed that Cytochalasin D used to destruct the cytoskeleton could also inter-
rupt the induction of c-fos gene in osteoblasts under fluid shear stress (Chen and Fu,
2008).

3.3.2 Egr-1

Egr-1, also a member of the immediate-early gene family, can be activated by a


kind of stimulus such as mechanical stress and then express the corresponding pro-
tein quickly. Egr-1, which is fairly conservative in evolution, generally exists in
the eukaryocytes from yeast to human. Egr-1 protein, with 533 amino acids, is a
karyo-phosphorylated protein of 80 kD and a transcription factor. Egr-1 has three
functional areas in structure (Thiel and Cibelli, 2002): the N-terminal end as the
transcriptional activation domain, the structural domain with three duplicate zinc
fingers as the central zone, which has the characteristics of combination with DNA,
and the area between the N-terminal end and the zinc fingers as the transcription
inhibition zone. McMahon et al. (1990) indicated that egr-1 protein regulates the
transcription of the target gene by combining with the specific sequence of the
gene, and then promotes the cell proliferation. High level expression of egr-1 could
be detected in places including periosteum during the bone formation (Chen and
Fu, 2008). Moalli et al. (2000) showed that mechanical stress could induce the
gene expressions of egr-1 and c-fos, meanwhile, could enhance the cytoactive of
osteoblasts, which comes to the conclusion that egr-1 and c-fos play an important
role in enhancing the cytoactive of osteoblasts.
Ogata (1997) have reported that fluid shear stress could induce the expression
of egr-1, but Cytochalasin D could interrupt this induction (the expression of egr-1
was reduced 40%).

3.3.3 OPN

OPN, a non-collagenous bone matrix protein, is one of the phenotypes of


osteoblasts. In different species, the sizes and forms of OPN are not exactly the
same, but they all have the same specific conserved sequences: signal peptide
72 Q. Fu et al.

sequences, seven to ten consecutive Asp sequences, RGD sequences and tyro-
sine kinase-phosphorylation recognition sequences. The RGD (arginine-glycine-
aspartic acid) sequence, a special structure of OPN, could combine with integrin,
and then facilitate the bone cells to adhere to the extracellular mineralized matrix.
So, it mediates lots of significant physiological functions.
Many studies showed that mechanical stress could induce the expression of OPN
in osteoblasts (Toma et al., 1997). Morinobu et al. (2003) have established an osteo-
genesis model induced by stress to observe the role of OPN in bone formation
with stress. They found that the osteogenesis under stress was obvious in wild-type
mouse, and the expression of OPN protein was significant in new bone formation
area, but not in OPN knock-out mice. Fujihara et al. (2006) found that bone remod-
eling induced by mechanical stress was significantly inhibited in OPN knock-out
mice, which suggested that OPN also plays an important role in bone remodeling.
But other studies showed that OPN is also an important regulatory factor in bone
loss (disuse atrophy) (Ishijima et al., 2006). So, OPN may play important roles in
both bone formation and bone resorption.
After studying the OPN signaling pathway induced by mechanical stress, both
Toma et al. (1997) and Carvalho et al. (2002) have found that the cytoskele-
ton destruction with Cytochalasin D could interrupt the induction of OPN under
mechanical stress.

3.3.4 ERK

ERK, as a member of the mitogen-activated protein kinases (MAPKs) family, with


a wide catalytic activity, could phosphorylate a series of plasmosins, membrane
proteins, intranuclear transcription factors and transcription regulons. ERK is also
considered as the upstream molecule that cells make response to external signals,
and as the junction of many kinds of important signal conduction pathways.
Present studies suggested that ERK can be quickly activated by mechanical stress
(Liu et al., 2006; Li et al., 2007; Wadhwa et al., 2002; Kapur et al., 2003). Many
bone formation-related genes as we know, such as c-fos, COX-2, OPN and egr-1,
are regulated by ERK, because the genes expression could be interrupted when the
activation of ERK is inhibited (Kapur et al., 2004, 2003). Meanwhile, ERK plays
an important role in the proliferation and differentiation of osteoblasts promoted by
mechanical forces (Lee et al., 2008; Kapur et al., 2003).
Liu et al. (2006) indicated that tensile stress and compressive stress can activate
ERK, but when the cytoskeleton was destructed by Cytochalasin D, the activation
would be interrupted.

3.3.5 COX-2

Cyclooxygenase, also named prostaglandin synthetase, could oxidize the arachi-


donic acid into prostaglandin E2 (PGE2). PGE2 could increase the contents of
3 Effect of Cytoskeleton on the Mechanosensitivity of Genes in Osteoblasts 73

calcified tissue in bone, accelerate fracture healing, enhance the proliferation and
differentiation of osteoblasts (Norrdin and Shih, 1988), and also mobilize the pre-
cursor cells of osteoblasts to differentiate into osteoblasts (Suponitzky and Weinreb,
1998). Cyclooxygenase includes two isoenzyme types: Conservative COX (COX-1)
and induction COX (COX-2). COX-1 is widely distributed in various tissues and is
maintained at a stable level under physiological conditions to regulate the physio-
logical function of organisms, while COX-2, as an inducible enzyme, which can be
induced by mechanical stress, is a rate-limiting enzyme for PGE2 synthesis under
mechanical stress. Studies showed that COX-2 plays a significant role in the PGE2
expression induced by fluid shear stress (Bakker et al., 2003).
Fluid shear stress could induce the expression of COX-2 and the release of PGE2
in osteoblasts, and both of them are necessary for mechanical stress to stimulate
bone formation (Bakker et al., 2001; Klein-Nulend et al., 1997; Reich et al., 1997;
Smalt et al., 1997; Chow and Chambers, 1994). The selective inhibition of COX-2
gene expression could interrupt the bone formation in vivo induced by mechanical
stress (Chow and Chambers, 1994; Forwood, 1996).
FU et al. had indicated that cytoskeleton integrity plays an important role in the
expression of cox-2 gene in osteoblasts induced by fluid shear stress (Wu and Fu,
2009).
To sum up, cytoskeleton integrity is necessary for the expressions of bone
formation-related genes (such as c-fos, egr-1, OPN, ERK, COX-2, and so on)
induced by fluid shear stress.

3.4 Cytoskeleton Reorganization Inhibition Enhances the


Mechanosensitivity of Some Genes in Osteoblasts

Mechanosensitivity refers to the ability that tissues or cells detect and make response
to mechanical stimuli.
Many studies showed that the response of bone cells to external stress could be
changed (Turner, 1999; Schriefer et al., 2005; Jaasma et al., 2007). Cytoskeleton
plays an important role in the biotransformation of mechanical signals transmitted
to bone tissues. The previous work of Fu et al. has proved that LIMK2 gene plays
a crucial role in cytoskeleton reorganization of osteoblasts induced by fluid shear
stress (Fu et al., 2008; Fig. 3.1).
Presently, Fu et al. have studied the effects of cytoskeleton reorganization inhi-
bition on c-fos and cox-2 genes in osteoblasts induced by fluid shear stress. They
found that cytoskeleton reorganization inhibition with RNAi (RNA interference)
could enhance the expression of c-fos and cox-2 genes induced by fluid shear stress
(Chen and Fu, 2008; Wu and Fu, 2009). Thus, Fu et al. concluded that on condition
of maintaining the cytoskeleton integrity, cytoskeleton reorganization inhibition can
promote the expression of c-fos and cox-2 genes in osteoblasts induced by fluid
shear stress. In other words, cytoskeleton reorganization inhibition can enhance the
mechanosensitivity of c-fos and cox-2 genes in osteoblasts.
74 Q. Fu et al.

Fig. 3.1 Cytoskeleton immunofluorescence staining of 14 groups of osteoblasts (200). F-actin


was stained using FITC-phalloidine (green fluorescence), and the nucleus was stained using DAPI
(blue fluorescence) (Fu Q et al., 2008). The cells were taken photographs in the same condition.
As shown in the pictures, compared with the negative control (Group1), when LIMK2 gene was
interfered with siRNA, cytoskeleton reorganization of osteoblasts was significantly inhibited in
loading groups, and the F-actin became disordered and was in filamentary arrangement (Group 2)

3.5 Conclusion and Perspectives


About the mechanisms that mechanical signals can be transformed into biologi-
cal signals and chemical signals, and then induce the expression of some bone
formation-related genes when osteoblasts are loaded by mechanical stress, it is not
clear until now. For the c-fos and cox-2 genes, Fu et al. have indicated that cytoskele-
ton reorganization inhibition could enhance their mechanosensitivity. But for the
other genes of osteoblasts, similar investigations should be conducted in the future.
Further researches on the specific mechanisms of cytoskeleton will shed new light
on the transmission mechanisms of mechanical stress in osteoblasts, and will lay
the foundation for the further investigations on the biomechanical behaviors of bone
cells and even bone tissues.
Acknowledgments This work was supported by the Science and Technology Projects of
Guangdong Province (No. 2008B030301121 and 2009B050700027), China.

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Chapter 4
Involvement of the Cytoskeletal Elements
in Articular Cartilage Mechanotransduction

Emma J. Blain

Abstract The cytoskeleton of all cells is a three-dimensional network comprising


actin microfilaments, tubulin microtubules and intermediate filaments. The
cytoskeletal networks are highly organised in structure enabling them to fulfil their
biological functions. They are involved in many diverse cellular processes including
alteration of cell shape, migration, cell division, movement of organelles, endocy-
tosis, secretion and extracellular matrix assembly. This review will primarily focus
on the organisation and function of the three major cytoskeletal networks in artic-
ular cartilage chondrocytes. Articular cartilage is a major load-bearing tissue of
the synovial joint; it is well known that the cytoskeleton acts as a physical inter-
face between the chondrocytes and the extracellular matrix in sensing mechanical
stimuli. The effect of physiological and abnormal mechanical load on cytoskeletal
element expression and organisation will also be reviewed.

Keywords Cytoskeleton Actin microfilaments Tubulin microtubules Vimentin


intermediate filaments Articular cartilage Chondrocyte Mechanotransduction

4.1 Introduction

4.1.1 Structure and Function of the Three Major Cytoskeletal


Elements

The cytoskeleton is a three-dimensional network which provides a physical inter-


face between the cell and the extracellular matrix in sensing a mechanical
stimulus. Converting a mechanical stimulus into a biochemical signal to initi-
ate downstream biosynthetic responses is primarily mediated by the three major

E.J. Blain (B)


Connective Tissue Biology Laboratories, Biomedical Sciences Building, School of Biosciences,
Cardiff University, Museum Avenue, Cardiff, CF10 3AX, UK
e-mail: Blain@cardiff.ac.uk

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 77


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_4,

C Springer Science+Business Media B.V. 2011
78 E.J. Blain

cytoskeletal elements comprising actin microfilaments, intermediate filaments and


tubulin microtubules (Benjamin et al., 1994). The cytoskeletal proteins have distinct
roles in many diverse cellular processes as discussed below.

4.1.1.1 Actin
Actin, a 43-kDa globular protein, is reported to be the most abundant protein in
eukaryotic cells (Disanza et al., 2005) and comprises three different monomers (,
and ). Under physiological conditions, the actin monomers (globular/G-actin)
assemble into long, highly organised filaments (filamentous/F-actin) (Fig. 4.1), and
it is the formation of these F-actin filaments which are critical to the activities of
many fundamental cellular events including cell migration (Heath and Holifield,
1991) and adhesion (Turner and Burridge, 1991), movement of organelles (Simon
and Pon, 1996), secretion (Sontag et al., 1988; Koukouritaki et al., 1996), alteration
of cell shape (Sims et al., 1992) and extracellular matrix assembly (Hayes et al.,
1999).
Actin assembly/disassembly is coupled with continuous ATP hydrolysis; a cleft
containing the nucleotide ATP (or ADP) and the cations Mg2+ and Ca2+ exists
within the G-actin moiety to meet the energy demands of actin polymerisation.
This ATP-dependent process of actin filament dis/assembly is referred to as tread-
milling, and it is the mechanism controlling cell locomotion, morphogenetic
movements and intracellular transport (Pollard and Borisy, 2003). Actin filaments
are polarised structures comprising a fast-growing (plus or barbed) end and a
slow-growing (minus or pointed) end (Ono, 2007). Under physiological conditions,
Mg2+ -ATP-bound G-actin is incorporated into growing filaments at the barbed end
(Cooper and Schafer, 2000; Ono, 2007). ATP is hydrolysed during F-actin poly-
merisation and a phosphate moiety is released. The resulting ADPactin filaments
are disassembled through the loss of ADPG-actin from the pointed end (Cooper
and Schafer, 2000; Ono, 2007). The released ADPG-actin undergoes nucleotide
exchange to produce ATPG-actin enabling continuation of F-actin polymerisation
at the barbed end.

Monomer Subunits Polymerised Actin Microfilament

Globular Actin
59nm
(G-actin)

Filamentous Actin
(F-actin)

Fig. 4.1 Schematic representation of the assembly of the F-actin microfilaments


4 Involvement of the Cytoskeletal Elements 79

4.1.1.2 Actin-Binding Proteins


Actin treadmilling and its organisation into functional cytoplasmic networks is
regulated by a variety of actin-binding proteins (Pollard and Borisy, 2003) which
function by:

(1) promoting nucleation of actin e.g. Arp2/3 complex (Goley and Welch, 2006),
(2) inducing actin filament depolymerisation e.g. the actin-depolymerising factor
family including ADF and cofilin (Ono, 2007),
(3) associating with G-actin moieties e.g. profilin and thymosin 4 (Yarmola and
Bubb, 2004),
(4) capping the F-actin filament end e.g. gelsolin (Cooper and Schafer, 2000).

The actin-binding proteins are therefore an essential prerequisite for regulating


actin treadmilling, and this is fulfilled by a series of tightly controlled cell signalling
pathways which co-ordinate the activities of these proteins (Disanza et al., 2005;
Pullikuth and Catling, 2007).

4.1.2 Intermediate Filaments

The intermediate filaments (1012 nm: intermediate in size between the micro-
filaments and microtubules) are highly organised fibrous protein structures which
connect the plasma with the nuclear membrane. Although intermediate filament
function(s) has not been completely characterised to date, they, in addition to the
other cytoskeletal elements, have been implicated in intracellular mRNA transport
(Glotzer and Ephrussi, 1996; Lopez de Heredia and Jansen, 2004) and distribution of
organelles and proteins (Herrmann and Aebi, 2000). The intermediate filaments are
involved in the localisation of organelles including the Golgi apparatus, endosomes,
lysosomes and nuclei in the cell cytoplasm (Styers et al., 2005).
However, compelling evidence exists to demonstrate their involvement in signal
transduction (Traub, 1995), and in particular, they fulfil a critical role in mechan-
otransduction i.e. the propagation of a mechanical stimulus into a downstream
biochemical response(s) (Lazarides, 1980; Wang et al., 1993). The role of interme-
diate filaments in mechanotransduction has been verified in several human genetic
diseases (Omary et al., 2004) and in knockout studies (Eckes et al., 1998; Broers
et al., 2004) where cells lacking their usual complement of intermediate filaments
have been demonstrated to be mechanically fragile. These studies infer that the
intermediate filaments confer mechanical integrity to the cells and tissues.
Five classes of intermediate filaments exist including: class I cytokeratins
(epithelia), class II desmin (muscle cells), class III vimentin (mesenchymal
cells), class IV glial fibrillar acidic protein (glial cells) and neurofilaments (neu-
rons), and the class V nuclear lamins which are present in the nuclei of all
cell types. Unlike the other two major cytoskeletal elements, intermediate fila-
ment expression can be both developmentally regulated and cell-type specific i.e.
80 E.J. Blain

only vimentin class III intermediate filaments are observed in chondrocytes, and
may reflect their diversity in biological function. Intermediate filaments all con-
tain common highly-conserved structural features including a central -helical
rod domain flanked by non--helical N-(head) and C-terminal (tail) domains. The
highly-conserved central -helical rod primarily consists of seven-residue repeats
which configures a coiled-coil structure (Parry et al., 2007). Intermediate filaments
assemble through the association of two monomeric subunits aligned in parallel and
in register to form a homodimer. Two homodimers associate to form anti-parallel,
half-staggered tetramers, and eight of these tetramers associate laterally to form a
supercoiled sheet which has the characteristic appearance of a rope-like filamen-
tous structure (Fuchs and Weber, 1994; Sokolova et al., 2006) (Fig. 4.2). However,
it is the degree of heterogeneity in the non--helical head and tail domains (size
and sequence can differ) which provides the array of intermediate filament classes
indicated above.
The intracellular organisation of intermediate filament networks i.e. the dynam-
ics of assembly and disassembly is under the control of specific protein kinases
and phosphatases (Izawa and Inagaki, 2006). Phosphorylation of the intermedi-
ate filament protein occurs on specific serine and threonine residues, resulting
in filament disassembly; most of these phosphorylation sites are located in the
N-terminal non--helical head domain (Beuttenmuller et al., 1994; Izawa and
Inagaki, 2006) (Fig. 4.3). As a consequence of phosphorylation, filament disas-
sembly generates a pool of soluble subunits in preparation for the next phase of
polymerisation. Conversely, dephosphorylation of the intermediate filament insti-
gates filament assembly, and the architecture undergoes a rearrangement (Herrmann
and Aebi, 2000).
The direct involvement of intermediate filaments in signal transduction has been
demonstrated in many cell types, as many of the regulatory proteins appear to be
dependent upon their interaction with the filaments. Several studies have demon-
strated that protein kinases phosphorylate intermediate filaments at specific sites in
vitro (reviewed in (Izawa and Inagaki, 2006; Minin and Moldaver, 2008)). These
phosphorylation sites are located mainly on the N-terminal protein domains, which
are imperative for intermediate filament assembly. In vivo, vimentin is one of
the predominant phosphoproteins in the cytoplasm, and in vitro provides a good
substrate for second messenger protein kinases (Fig. 4.3). The dynamic status of
vimentin de/phosphorylation is critical in numerous signal transduction pathways

812nm
Monomer Dimer Tetramer Rope-like filament
(8 tetramers)

Fig. 4.2 Schematic representation of the assembly of the vimentin intermediate filaments
4 Involvement of the Cytoskeletal Elements 81

N- L1 L1-2 L2 -C
1A 1B 2A 2B

Phosphorylated serine residues in vimentin head domain


6 8 9 20 24 25 28 33 38 41 46 50 55 64 65 71 72 82 86
PKA + + + + + + + +
PKC + + + + + + + + + + + +
PAK + + + + +
Rho-kinase + +
CaM kinase II + +
cdc2 kinase + +
Plk1 +
Aurora-B + + + + + + + +

Fig. 4.3 Schematic representation of the phosphorylation sites in the vimentin head domain.
Serine residues are phosphorylated by PKC (protein kinase C), PKA (protein kinase A), PAK
(p21-activated kinase), Rho-kinase, CaM kinase II (Ca2+ /calmodulin-dependent protein kinase II,
cdc2 kinase, Plk1 (polo-like kinase 1) and Aurora-B (adapted from Izawa and Inagaki, 2006; Minin
and Moldaver, 2008)

including the MAP kinases ERK 1/2 (Perlson et al., 2006); Blain, unpublished
observations), various 14-3-3 signalling protein isoforms and apoptotic factors (Kim
and Coulombe, 2007).

4.1.2.1 Nuclear Lamins


The ubiquitous nuclear lamins are the principal components of the nuclear lamina;
their primary role is to provide a structural framework to maintain interphase nuclear
shape, but the nuclear lamins are also essential for many aspects of normal nuclear
function including DNA replication, chromatin organisation and gene expression
(Dechat et al., 2008). The nuclear lamina, which resides beneath the inner nuclear
envelope, comprises four major lamin proteins: A, B1, B2 and C (Burke and Stewart,
2006), and as with the class III filaments, the nuclear lamins are also developmen-
tally regulated; all cells express at least one lamin B, however the A-type lamins are
absent from early embryonic development, embryonic stem cells and certain stem
cell populations in adults (Rober et al., 1989; Cohen et al., 2008).

4.1.3 Tubulin Microtubules

Tubulin microtubules are highly conserved / dimeric proteins, each monomer


being approximately 450 amino acids in length with a molecular mass of 55 kDa
82 E.J. Blain

(Valiron et al., 2001), that self-assemble to form the polymeric microtubular struc-
ture. Many functions have been attributed to the microtubules including cell motility,
mitosis, cell morphogenesis, organisation and transport of organelles. The principal
function of microtubules occurs during mitosis when they rearrange to form the
mitotic spindle to orientate the plane of cell cleavage (Mitchison et al., 1986).
The microtubules also operate as an intracellular supramolecular motor to segregate
the chromosomes to the cell poles during anaphase (Mitchison et al., 1986). The
ability of the microtubules to act as a supramolecular motor allows them to also act
as a highway for bi-directional transport: protein trafficking and secretion results
from the delineation of microtubule tracks along which the cargo are shuttled during
endo- and exocytosis (Vale, 1987; Thyberg and Moskalewski, 1999). Microtubules
organise the cell interior and are also involved in assembling intracellular organelles
e.g. endoplasmic reticulum and the Golgi apparatus (Rogalski and Singer, 1984).
Microtubules are also involved in sensing and responding to mechanical stimuli as
they are one of the principal constituents of the axonemal structures found in cilia
(Gibbons, 1981; Poole et al., 2001) and flagella (Woolley, 2000).
Tubulin assembly/disassembly is coupled with GTP hydrolysis; -tubulin binds
a GTP molecule in a non-exchangeable manner whereas GTP--tubulin binding is
exchangeable. GTP hydrolysis occurs upon the addition of a tubulin heterodimer
to the microtubule end, with the -tubulin subunit of the heterodimer exposed on
the faster growing end (plus end) and the -tubulin subunit exposed on the slow
growing end (minus end) (Valiron et al., 2001). Microtubules organise into linear
protofilaments that assemble to form 24 nm-wide cylindrical structures (Fig. 4.4).
The numbers of protofilaments which constitute a microtubule is generally consid-
ered to be 13 in vivo (Wade and Hyman, 1997), although the number may vary
depending on assembly conditions. As a result of the unique organisation of the
- and -tubulin heterodimers, the microtubule protofilament is polarised result-
ing in differing kinetic and structural properties. Microtubule polarity is critical
for the correct implementation of the microtubule motor proteins (comprehensively
reviewed in (Mallik and Gross, 2004; Wu et al., 2006)). Recognition of microtubule

Subunit

-tubulin GTP GDP -tubulin

Protofilament 24nm

8nm

Fig. 4.4 Schematic representation of the assembly of the tubulin microtubules


4 Involvement of the Cytoskeletal Elements 83

polarity by the motor proteins enables the specific motor employed to determine
the direction of transport along the microtubule tracks (Mallik and Gross, 2004).
Dynein shuttles cargo toward the microtubule minus-end (Hirokawa, 1998), sus-
taining microtubule-dependent trafficking of newly synthesised proteins from the
endoplasmic reticulum to the cis side of the golgi stacks, and providing vesicular
transport for endosomal clearance (Thyberg and Moskalewski, 1999). In contrast,
the motor protein kinesin moves toward the plus-end of the microtubules bringing
cargo toward the cell periphery (Hirokawa, 1998), e.g. by trafficking membrane and
secretory proteins (following post-translational modifications) from the trans side
of the golgi stacks. Kinesin is also involved in propagating the return transport of
endoplasmic reticulum components from the cis side of the golgi stacks (Thyberg
and Moskalewski, 1999).

4.2 Articular Cartilage Structure and Function

The major load-bearing tissue of the synovial joint is articular cartilage which pro-
vides a smooth and resilient surface for joint locomotion. It is unique in being
avascular, aneural and alymphatic and contains a sparse population of cells the
chondrocyte interspersed in a dense extracellular matrix. The ability of articu-
lar cartilage to withstand highly repetitive stresses, brought about by movement,
depends on the structure and composition of this extracellular matrix.

4.2.1 Tissue Composition

The unique mechanical properties of articular cartilage are dependent on the organ-
isation (Fig. 4.5a) and composition of the tissue (Fig. 4.5b). Morphologically, there
are four distinct zones including the superficial, transitional, deep, and calcified zone
which merges into the subchondral bone. Articular cartilage (wet weight) comprises
approximately 6580% water, with 80% being in the superficial zone and 65% in the
deep zone; the highly hydrated nature of the tissue allows for load-dependent defor-
mation, as well as providing a medium for lubrication to maintain a low-friction
bearing surface zone (Buckwalter and Mankin, 1998). Collagens comprise 1020%
of the wet weight of articular cartilage with type II forming the principal component
(9095%); parallel arrangement of the collagen fibrils in the surface zone is respon-
sible for providing the greatest tensile and shear strength (Duance et al., 1998).
The other principal constituent of articular cartilage is proteoglycan (1020%); two
major classes of proteoglycans are found in articular cartilage: large aggregating
monomers e.g. aggrecan and the small leucine-rich proteoglycans e.g. decorin and
biglycan (Hardingham et al., 1992). Aggrecan is most abundant in the transitional
and deep zone, where due to its viscoelastic nature (hydrated gel) provides cartilage
with its function of compressibility under load. Articular cartilage also contains
a multitude of other extracellular matrix macromolecules which contribute to its
unique structural/mechanical integrity, but this lies outside the scope of this review.
84 E.J. Blain

hyaluronate

link protein
A. B.
aggrecan
decorin

SZ HA receptor COMP
glycosaminoglycan
TZ

biglycan

DZ type IX collagen
chondrocyte
SB

type II collagen
fibromodulin
core type XI collagen

Fig. 4.5 Organisation and composition of articular cartilage. (a) Haematoxylin and Eosin staining
of articular cartilage from the carpal metacarpal joint of a 7-day-old bovine calf. (b) Schematic car-
toon illustrating the interactions between the major components of articular cartilage (adapted from
Silver and Glasgold, 1995). Collectively the composition and organisation of these matrix macro-
molecules provides the tissue with its unique biomechanical properties to withstand compressive
load

Chondrocytes envelop themselves in basket-like networks of fine fibrils of elabo-


rate structure termed a chondron (Muir, 1995). As individual units, chondrons
appear to be compression-resistant, fluid-filled elements that dampen mechanical,
osmotic and physicochemical changes induced by mechanical loading (Muir, 1995).
The elaborate structure of articular cartilage allows for many complex interactions
between the chondrocyte and its surrounding environment, and it acts as an ideal
intermediary for sensing and responding to alterations in mechanical load.

4.2.2 Tissue Function


Articular cartilage is a specialised connective tissue that functions in dissipating
mechanical loads across the joint. All components of the joint i.e. articular cartilage,
subchondral bone, meniscus, synovium, muscles, ligaments and tendons partici-
pate in load transmission but it is the articular cartilage itself, which provides a
resilient and compliant articulating surface to the bones in diarthrodial joints. In
simple terms, the articular cartilage prevents potentially damaging local stress con-
centrations from being experienced, whilst providing a low friction-bearing surface
to enable free movement of the joint for locomotion (Jeffrey et al., 1997). During
normal walking, articular cartilage is subjected to a complex state of dynamic
4 Involvement of the Cytoskeletal Elements 85

loading which is primarily applied perpendicular to the surface of the articular car-
tilage (Armstrong et al., 1984). Dynamic, compressive loading of articular cartilage
induces cell deformation, changes in hydrostatic or fluid pressure and deformation
of the charged extracellular matrix, resulting in alterations in osmolality and pH
(Urban, 1994; Hall et al., 1996a), as depicted (Fig. 4.6). Chondrocytes can detect
these physico-chemical signals, and respond by altering their metabolism via
the activation of specific intracellular signalling pathways (Palmoski et al., 1980;
Palmoski and Brandt, 1984; Kiviranta et al., 1988; Sah et al., 1989).

Mechanical synovial membrane


Load
synovial fluid

soluble factors
territorial matrix

fluid flow
hydrostatic pressure

fluid expression deformation

compression of matrix growth factor release


[cations] osmolality
pericellular matrix

51 integrin

IL-4
F-actin FAK
Ca2+

iNOS
NO vimentin

ERK
tubulin
transcriptional
ECM responses
macromolecules Stretch-activated
macromolecule synthesis channels

ECM assembly/turnover MMPs


Aggrecanases
TIMPs

Fig. 4.6 A schematic representation of how mechanical deformation, acting on articular cartilage,
can alter both the intra- and extracellular environment of the cells. Exposure of cells to mechan-
ical stimulation disseminates further effects across the extracellular matrix including opening of
stretch-activated channels, focal adhesion kinase signalling via the integrins, and rearrangement of
the cytoskeletal elements to induce changes in gene and protein expression (adapted from Urban,
1993)
86 E.J. Blain

Under compressive load, cartilage chondrocytes are subject to deformation which


induces an alteration in cell volume and/or surface area (Guilak et al., 1995; Knight
et al., 1998), and in native tissue this deformation is associated with an increase in
extracellular osmolality (Guilak et al., 1995; Buschmann et al., 1996). Compression-
induced adaptations in chondrocyte volume and/or surface area activate various
membrane ion transporters (Urban et al., 1993; Hall et al., 1996b), stretch sensi-
tive ion channels and induce alterations in membrane potential (Guilak et al., 1994;
Wright et al., 1996). Compressive load also results in nuclear deformation, concomi-
tant with a reduction in nucleus volume (Guilak, 1995; Buschmann et al., 1996).
Load-induced deformation of the intracellular organelles e.g. the nucleus is thought
to effect alterations in matrix synthesis (Buschmann et al., 1996; Janmey, 1998). The
mechanism by which compressive load is mediated through the cell to the nucleus
to effect a metabolic response is thought to involve the cytoskeleton (Ben-Zeev,
1991; Wang et al., 1993; Ingber et al., 1994; Maniotis et al., 1997; Janmey, 1998).

4.3 Cytoskeletal Element Composition in Articular


Chondrocytes

The chondrocyte cytoskeleton comprises actin microfilaments, vimentin interme-


diate filaments and tubulin microtubules (Fig. 4.7), in addition to the ubiquitous
nuclear lamins (Benjamin et al., 1994).

4.3.1 Organisation of the Cytoskeletal Elements in Articular


Chondrocytes

The architecture of the three major cytoskeletal networks in chondrocytes have been
characterised in articular cartilage explants (Durrant et al., 1999; Langelier et al.,
2000), in primary chondrocytes cultured in agarose (Idowu et al., 2000; Knight et al.,
2001; Trickey et al., 2004; Sasazaki et al., 2008) and in high-density monolayer
cultures (Blain et al., 2006). Key morphological features are described (Table 4.1A).
F-actin is predominantly cortical in localisation and distributed at the periph-
ery of the chondrocyte (Idowu et al., 2000; Langelier et al., 2000; Knight et al.,
2001; Trickey et al., 2004; Blain et al., 2006; Sasazaki et al., 2008). In contrast,
the vimentin intermediate filaments extend throughout the cytoplasm and form a
highly organised architecture in chondrocytes, traversing from the plasma to the
nuclear membrane (Idowu et al., 2000; Langelier et al., 2000; Holloway et al.,
2004; Trickey et al., 2004; Blain et al., 2006; Sasazaki et al., 2008). Tubulin micro-
tubules are uniformly distributed throughout the chondrocyte cytoplasm and exhibit
a loose mesh-like network (Jortikka et al., 2000; Langelier et al., 2000; Trickey
et al., 2004; Blain et al., 2006). Zonal differences are also evident in the organ-
isation of the cytoskeletal networks in articular cartilage chondrocytes (Langelier
et al., 2000). A comparison of cytoskeletal element distribution through the depth of
4 Involvement of the Cytoskeletal Elements 87

Table 4.1 Organisation of the three major cytoskeletal elements: actin, vimentin and tubulin in
[A] normal and [B] osteoarthritic articular chondrocytes. All three structures are highly organised
in normal chondrocytes and their localisation in the cytoplasm reflects their biological functions
within the cell. However, in chondrocytes from osteoarthritic cartilage there is a general loss of
actin, vimentin and tubulin organisation, and a reduction in protein amounts. The loss of vimentin
organisation in osteoarthritic cartilage may be attributed to an increase in vimentin degradation
products which are derived from N-terminal domain cleavage i.e. that which is responsible for
filament assembly

Cytoskeletal Organisation normal


element chondrocytes Reference

F-Actin cortical distribution Langelier et al. (2000), Idowu et al.


predominantly localised (2000), Knight et al. (2001),
to cell periphery Trickey et al. (2004), Blain et al.
(2006), Fioravanti et al. (2003),
and Sasazaki et al. (2008)
Vimentin highly organised structural Langelier et al. (2000), Idowu et al.
network (2000), Holloway et al. (2004),
traverses cytoplasm connecting Trickey et al. (2004), Blain et al.
nuclear membrane with cell (2006), Fioravanti et al. (2003),
periphery and Sasazaki et al. (2008)
Tubulin loose, basket-like meshwork Jortikka et al. (2000), Langelier
uniformly distributed et al. (2000), Trickey et al.
throughout cell cytoplasm (2004), Blain et al. (2006), and
Fioravanti et al. (2003)

Table 4.1B

Cytoskeletal
element Organisation osteoarthritic chondrocytes Reference

F-Actin less well-defined and localised diffusely in the Fioravanti et al. (2003)
cytoplasm or limited to cell periphery [Human]
Vimentin vimentin-rich multiple, elongated processes Holloway et al. (2004)
(correlating with greatest histological and
macroscopic signs of OA) [Human]
loss of expression 37.1% reduction. Capin-Gutierrez et al.
Predominantly localised to the superficial zone; (2004)
ranged from simple disorganisation to total
disruption of architecture [Rat partial
menisectomy model]
diffuse cytoplasmic distribution [Human] Lambrecht et al. (2008)
vimentin degradation products derived from Lambrecht et al. (2008)
cleavage of N-terminal domain (responsible for
filament assembly) [Human]
Tubulin often absent [Human] Fioravanti et al. (2003)
loss of expression 20.1% reduction [Rat partial Capin-Gutierrez et al.
menisectomy model] (2004)
88 E.J. Blain

Fig. 4.7 The cytoskeleton of


primary bovine chondrocytes
depicting F-actin
microfilaments, vimentin
intermediate filaments and
tubulin microtubules cultured
in high density monolayer for
7 days. Cells were visualised
using FITC-conjugated
antibodies in conjunction
with scanning confocal
microscopy
F-actin microfilaments

Vimentin intermediate filaments

Tubulin microtubules

skeletally mature bovine articular cartilage demonstrated that F-actin was uniformly
distributed; in contrast, both vimentin and tubulin were predominantly detected
in cells of the superficial zone (Langelier et al., 2000). Interestingly, there was
an increasing gradient in both vimentin and tubulin network organisation in car-
tilage situated in proximity to weight-bearing regions (Langelier et al., 2000). The
exhibition of specific structural characteristics in chondrocyte cytoskeletal network
organisation reflects their distinct functions within the cell.
4 Involvement of the Cytoskeletal Elements 89

4.3.2 Cytoskeletal Element Functions in Articular Chondrocytes


In addition to the generic functions already outlined above (Section 4.1), the
cytoskeletal elements provide further, more specific functions in articular chondro-
cytes.

4.3.2.1 Actin Microfilaments


The ability of a chondrocyte to withstand compressive loads is dependent on the
organisation of the F-actin cytoskeleton (Guilak, 1995). A chondrocytes response to
mechanical stimulation is thought to occur as a consequence of an alteration in cell
shape and volume which activates intracellular signalling cascades. Deformation
of the chondrocyte nucleus is involved in relaying intracellular signals via the
F-actin network. Disruption of the F-actin cytoskeleton with cytochalasin D affected
the relationship between matrix deformation and alterations in nucleus shape and
height (Guilak, 1995). In the presence of cytochalasin D, chondrocyte stiffness
decreased by 90% and viscosity by 80% (Trickey et al., 2004), demonstrating the
importance of the actin cytoskeleton in maintaining the mechanical integrity of the
cell (refer to Section 4.4 for more detail). Actin microfilaments are also crucial in
maintaining cell matrix interactions. Studies have demonstrated that cytoskele-
tal actin disruption within articular cartilage uncouples the chondrocytes from the
matrix, altering tissue synthesis and structure by inhibiting type II collagen and
sulphated glycosaminoglycan synthesis (Takigawa et al., 1984; Brown and Benya,
1988; Newman and Watt, 1988; Loty et al., 1995). Cytochalasin D induced disrup-
tion of F-actin reduced chondrocyte pericellular matrix assembly and the retention
of proteoglycans within the cartilage tissue (Nofal and Knudson, 2002). This loss
of function was attributed to a reduction in the anchorage of CD44 in the chon-
drocyte membrane which reduced the capacity of CD44 to bind to its extracellular
ligand.

4.3.2.2 Vimentin Intermediate Filaments


Vimentin intermediate filament organisation has been characterised in articular car-
tilage tissue (Durrant et al., 1999; Langelier et al., 2000; Holloway et al., 2004), in
isolated chondrocytes (Idowu et al., 2000; Langelier et al., 2000; Holloway et al.,
2004; Trickey et al., 2004; Sasazaki et al., 2008) and in high-density monolayer
cultures (Blain et al., 2006), but their function(s) are still being unravelled. The
vimentin knock-out mouse displays no obvious phenotype, being able to develop
and reproduce normally; the skeleton is apparently normal (Colucci-Guyon et al.,
1994), although the morphology of the articular cartilage was not fully assessed.
Interestingly, vimentin / fibroblasts cultured in vitro displayed reduced mechan-
ical stability when exposed to external stimuli (Eckes et al., 1998). Vimentin /
mesenteric resistance arteries decreased their response to blood flow (Henrion et al.,
1997), and in vimentin / carotid arteries flow-induced remodelling was reduced
90 E.J. Blain

(Schiffers et al., 2000), demonstrating a requirement of vimentin in the mechan-


otransduction of shear stress. These studies suggest that the absence of a vimentin
filament network does not impair basic cellular functions, but that cells are mechan-
ically compromised; therefore Eckes et al. proposed that cellular functions that are
dependent upon mechanical stability become impaired. This argues well with the
observation of a more prominent vimentin expression in weight-bearing areas of
articular cartilage in situ (Eggli et al., 1988). More recently, the contribution of
the vimentin filaments in withstanding mechanical load has been investigated in
chondrocytes (Lahiji et al., 2004; Trickey et al., 2004). In conjunction with actin
microfilaments, the vimentin cytoskeleton confers some mechanical integrity to
the cell, as a decrease in stiffness and viscosity was observed in chondrocytes
treated with 40 mM acrylamide to disrupt the vimentin filaments (Trickey et al.,
2004).
We have demonstrated a novel function of the vimentin network in contributing
to the maintenance of the chondrocyte phenotype (Blain et al., 2006). Chondrocyte
matrix synthesis is regulated by the state of vimentin network assembly/disassembly
(using 5 mM acrylamide to disrupt vimentin architecture), as evidenced by a signif-
icant reduction in de novo collagen and sGAG biosynthesis at both the mRNA and
protein level (Blain et al., 2006). Preliminary studies indicate that this response may
be due to sustained phosphorylation of ERK1/2 (unpublished observations), which
has previously been reported to inhibit the chondrocyte phenotype (Seghatoleslami
et al., 2003).

4.3.2.3 Tubulin Microtubules


The microtubule network does not directly confer any mechanical integrity to the
chondrocyte, as disruption of tubulin with colchicine did not alter the stiffness or
viscosity of the cell (Trickey et al., 2004). However, a sophisticated tubulin net-
work, in the form of acetylated -tubulin, has been observed in the primary cilia of
chondrocytes (Poole et al., 2001). The primary cilium is a highly conserved, sin-
gle cytoplasmic organelle which projects into the pericellular matrix and monitors
the mechanical environment of the cartilage tissue (McGlashan et al., 2008). The
primary cilium interacts with pericellular matrix components including collagen
types II and VI (Poole et al., 1997; McGlashan et al., 2006). Ultrastructural studies
have also identified a direct connection between extracellular collagen fibres and the
proteins which decorate the ciliary microtubules, suggesting a matrix-cilium-Golgi
continuum in chondrocytes to detect mechanical fluctuations in the surrounding
matrix (Poole et al., 2001).
However, as in all cell types, the microtubules have a critical function in protein
trafficking and secretion (Thyberg and Moskalewski, 1999). The integrity of the
tubulin network, manipulated using disrupting agents i.e. colchicine or nocodazole,
is also essential for the synthesis and secretion of both collagens and proteogly-
cans in chondrocytes (Jansen and Bornstein, 1974; Lohmander et al., 1976, 1979;
Madsen et al., 1979; Takigawa et al., 1984). Colchicine dissociates the Golgi appa-
ratus of chondrocytes (Moskalewski et al., 1975), therefore its negative impact on
4 Involvement of the Cytoskeletal Elements 91

matrix synthesis may reflect alterations to the normal functioning of the Golgi
apparatus, or its disruption of normal routes of intracellular transport.

4.4 Biomechanics and the Chondrocyte Cytoskeleton

4.4.1 Contribution of the Cytoskeletal Elements to the Mechanical


Properties of the Chondrocyte

Cytoskeletal element organisation is a highly dynamic process, as demonstrated in


isolated chondrocytes whereby F-actin, vimentin and tubulin assembly increased
markedly over a culture period of days (Lee et al., 2000a; Blain et al., 2006).
The biomechanical integrity of the chondrocyte is dependent on the organisation
of these major cytoskeletal elements, as each of the networks serve an important
function in the cells ability to resist and recover from mechanical forces. Ofek
et al. hypothesised that each cytoskeletal component would differentially contribute
to the compressive properties and behaviour of single chondrocytes, which would
have specific ramifications for mechanotransduction pathways (Ofek et al., 2009).
Chemical disruption of individual cytoskeletal elements prior to the application
of a range of compressive strains demonstrated that F-actin was the greatest con-
tributor to chondrocyte stiffness, vimentin and tubulin contributed significantly to
volumetric changes/compressibility and that tubulin was essential for cell recovery
post-deformation (Ofek et al., 2009).
These specific functions relate to the distribution of the cytoskeletal elements
within the chondrocyte (Fig. 4.7). F-actin is localised to the periphery of the cell
where it provides mechanical reinforcement for the cytoplasm (Guilak et al., 1995;
Trickey et al., 2004; Knight et al., 2006). Conversely, tubulin forms a loose mesh-
work throughout the cell cytoplasm, increasing the ability of the cell to revert to its
original morphology post-compression. Vimentin, comprising a coiled-coil struc-
ture with flexible regions at its head and tail (Fig. 4.2) acts primarily to resist
tensile forces and counteract compression-induced volumetric changes (Ofek et al.,
2009). Chondrocyte stiffness can play a critical role in the cells interpretation and
ability to function under an altered mechanical environment; the functional role
of the cytoskeleton in resisting compression is particularly important as cell vol-
ume alterations can influence matrix synthesis and tissue homeostasis, potentially
predisposing the tissue to pathological conditions e.g. osteoarthritis (OA).

4.4.2 Mechanical Load Influences Cytoskeletal Element


Organisation
The application of a physiological mechanical load is imperative for maintaining
articular cartilage integrity (Fig. 4.6); extracellular matrix synthesis, i.e. sGAG and
collagen production, is regulated by mechanical load, and sustained synthesis is
92 E.J. Blain

in itself required to maintain the ability of the cartilage to withstand the forces
that are associated with tissue deformation (Urban, 1994). However, abnormal non-
physiological loads including disuse (Palmoski et al., 1980; Palmoski and Brandt,
1984) and high, repetitive loads (Vasan, 1983; Radin et al., 1984) inhibit extracel-
lular matrix synthesis, and is detrimental to the properties of the tissue in vivo.
Altering the biomechanical properties of the chondrocyte influences cell-matrix
interactions (Guilak and Mow, 2000), supporting the role of the cytoskeleton in
mechanical signal transduction. Collectively, the three major cytoskeletal networks
play a fundamental role in maintaining the phenotype of chondrocytes and act-
ing as a physical interface between the chondrocyte and the extracellular matrix
in sensing mechanical stimuli (Table 4.2).

4.4.2.1 Actin Microfilaments


The F-actin cytoskeleton is intimately involved in the conversion of a mechanical
stimulus into a biochemical response during articular chondrocyte mechanotrans-
duction (Wang et al., 1993; Banes et al., 1995; Guilak, 1995; Grodzinsky et al.,

Table 4.2 The effect of mechanical load on the expression and organisation of cytoskeletal
elements in articular cartilage chondrocytes

Cytoskeletal
element Effect of load on chondrocyte cytoskeleton Reference

F-Actin Loss of actin stress fibres with increasing hydrostatic Parkkinen et al. (1995)
pressure (1530 MPa); reversible after 2 h
Compressive strain (1015%) induced punctuate Knight et al. (2006)
reorganisation of F-actin replacing uniform
cortical distribution in unloaded cells
Actin reorganisation in response to osmotic stress Erickson et al. (2003)
loss of cortical F-actin distribution due to severing and Chao et al.
and detachment from cell periphery (2006)
Actin- Increased thymosin 4 mRNA: 0.5 MPa, 1 Hz, Blain et al. (2002) and
binding 10 min reorganisation of thymosin 4 and F-actin Blain et al.(2003)
proteins architecturesa
Increased cofilin and destrin mRNA: 15%, 1 Hz, Campbell et al. (2007)
10 min temporal disassembly of cortical F-actin

Vimentin Increased content in weight-bearing regionsa Eggli et al. (1988)


Increased filament organisation: 04 MPa, 1 ha Durrant et al. (1999)
Reduction in vimentin mRNA after continuous or Karjalainen et al.
cyclic (0.5 Hz) stretch (5 MPa) (2003)
Increased vimentin mRNA in response to cyclic Lahiji et al. (2004)
strain (24 h)

Tubulin Loss of organisation: (24 MPa, 3 h) Fioravanti et al. (2005)


a Cartilage explants
4 Involvement of the Cytoskeletal Elements 93

2000). The application of hydrostatic pressure to isolated articular chondrocytes cul-


tured in monolayer (Parkkinen et al., 1995) and alginate (Fioravanti et al., 2005), or
dynamic compression of cells embedded in agarose (Knight et al., 2006) reorganises
the F-actin cytoskeleton. A polygonal arrangement of F-actin was observed through-
out the cytoplasm of non-loaded cells however, this organisation was lost under
increasing levels of hydrostatic pressure (Parkkinen et al., 1995; Fioravanti et al.,
2005). Also, the number of cells containing an intact F-actin network decreased
in cells subjected to a cyclic pressure of 15 MPa, with a near total disappearance
of the F-actin networks, concomitant with retraction of the cells, after exposure
to 30 MPa (Parkkinen et al., 1995). Interestingly, a recovery period of 2 h post-
load was sufficient to reverse the effect of hydrostatic pressure on the F-actin
network. In contrast, the application of compressive (10 or 15%, 1 Hz) or static
compression (15%) on chondrocytes resulted in F-actin remodelling, with a more
punctate organisation replacing the uniform cortical distribution observed in non-
loaded cells (Knight et al., 2006). However, a recovery period of 1 h was sufficient
to reorganise the actin filaments to that observed prior to loading. An increase in
punctate actin cytosolic features was also observed in cyclically compressed chon-
drocytes embedded in agarose (010% strain, 0.5 Hz, 20 min) (Haudenschild et al.,
2008a, b).
The actin cytoskeleton is also sensitive to mechanical distortions as a conse-
quence of osmotic loading (Erickson et al., 2003; Chao et al., 2006). Prolonged
mechanical compression or release of compression can alter the osmotic environ-
ment of the chondrocyte; manipulation of the osmotic pressure to 250 mOsm in
vitro resulted in F-actin rearrangement from a predominantly cortical distribution to
the severing and subsequent detachment from the chondrocyte periphery (Erickson
et al., 2003; Chao et al., 2006). Conversely, exposure to hypo-osmotic stress led
to a progressive disorganisation of F-actin followed by a gradual reorganisation
(Erickson et al., 2003; Chao et al., 2006).
Clearly, the F-actin cytoskeleton is highly dynamic in nature, responding to
mechanical stimulation by reversibly disassembling and reorganising the filaments
to maintain the structural integrity of the chondrocyte. Furthermore, mechanically
induced actin remodelling may provide a feedback mechanism through which
mechanical stimuli can modulate chondrocyte mechanosensitivity (Knight et al.,
2006). The mechano-responsiveness of the F-actin cytoskeleton is likely mediated
by the integrins, which form a link between the extracellular matrix and the cell.
Integrins are heteromeric transmembrane glycoproteins, consisting of a combina-
tion of - and -subunits, of which the 51 integrin is a major mechanoreceptor in
articular chondrocytes (Wright et al., 1997). The integrin receptor has an extracellu-
lar domain that acts as a ligand binding site, and a cytoplasmic tail which interacts
with intracellular molecules, including the actin cytoskeleton, enabling the integrin
receptor to transduce mechanical signals into intracellular biochemical responses
(Hynes, 1992). Integrin ligand binding, in response to mechanical stimulation (3700
strain, 0.33 Hz, 15 min), influences F-actin assembly concomitant with phos-
phorylation of actin accessory proteins e.g. FAK and paxillin (Lee et al., 2002)
and activation of downstream intracellular signalling cascades e.g. IL-4 signalling
94 E.J. Blain

(Millward-Sadler et al., 2000). Included in the downstream signalling cascades is the


Rho GTPase pathway which was activated in cyclically-compressed (010% strain,
0.5 Hz, 20 min) human articular chondrocytes embedded in agarose (Haudenschild
et al., 2008a, b). Rho activation was followed by Rho kinase (ROCK)-dependent
rearrangements of the actin cytoskeleton and by ROCK-dependent changes in
gene expression (Haudenschild et al., 2008a). The Rho GTPases promote F-
actin rearrangement and enhance the formation of focal adhesions, creating a
link between the cytoskeleton and the matrix. A major effector pathway down-
stream of Rho is the activation of ROCK, which phosphorylates and activates Lim
kinase, which in turn phosphorylates and inhibits the actin-depolymerising protein
cofilin.
Chondrocyte deformation is accompanied by hydrostatic pressure gradients, fluid
flow and streaming potentials generated by the displacement of water and ions
from the matrix (Urban et al., 1993). A local change in ion concentration is suf-
ficient to activate ion channels; stretch-activated Ca2+ -activated K+ channels were
shown to be active in compressed chondrocytes (Hall, 1999; Roberts et al., 2001).
The mechanical induction of intracellular Ca2+ release by the integrins is required
for the association of the actin accessory proteins (FAK, paxillin) with the F-actin
cytoskeleton. This effect may result from a requirement of Ca2+ ions for the activ-
ity of actin-binding proteins and other actin components in load-induced actin
reorganisation.

Actin-Binding Proteins
Actin remodelling in cartilage chondrocytes subjected to a mechanical stimulus has
been shown to involve the differential expression of the actin-binding proteins (Blain
et al., 2002, 2003; Knight et al., 2006; Campbell et al., 2007). We have previously
shown that thymosin 4, an actin-binding protein, is regulated by mechanical load
in articular chondrocytes (Blain et al., 2002, 2003). Thymosin 4 mRNA levels
were significantly elevated (20-fold) in articular cartilage explants subjected to a
dynamic low-physiological load (0.5 MPa, 1 Hz, 10 min); the response was tran-
sient as the thymosin 4 mRNA signal returned to basal levels after 60 min of load
(Blain et al., 2003). In unloaded explants chondrocytes, thymosin 4 protein was
cytoplasmically diffuse, but after 60 min of load, the protein was organised into
punctate loci throughout the cytoplasm; the distribution of thymosin 4 coincided
with a more abundant immunolabelling of F-actin in the loaded explant chondro-
cytes (Blain et al, unpublished data). Thymosin 4, an actin sequestering protein,
actively disassembles the actin filaments by binding in a 1:1 complex with G-
actin, thereby preventing its availability for polymerisation (Weber et al., 1992).
Other actin-disassembling proteins are also up-regulated by cyclic compression in
chondrocytes. mRNA levels of cofilin and destrin were significantly increased after
10 min of mechanical stimulation (15% strain, 1 Hz) in chondrocytes embedded in
agarose, concomitant with a temporal disassembly of cortical F-actin (Campbell
4 Involvement of the Cytoskeletal Elements 95

et al., 2007). Increased cofilin expression concomitant with F-actin disassembly


may be mediated by the regulation of the Rho kinase pathway (Haudenschild et al.,
2008a).

4.4.2.2 Vimentin Intermediate Filaments


Cartilage chondrocyte vimentin networks are also sensitive to mechanical pertur-
bations in the surrounding environment. Physiological loading conditions induce
vimentin filament assembly (Eggli et al., 1988; Durrant et al., 1999; Lahiji et al.,
2004), whereas the application of abnormal, non-physiological loads promote fila-
ment disassembly (Karjalainen et al., 2003; Henson and Vincent, 2008). In vivo,
increased vimentin content was observed in the more prominent weight-bearing
regions of young rabbit articular cartilage (Eggli et al., 1988). In vitro, reorgan-
isation of the vimentin network was observed in cartilage explant chondrocytes
subjected to increasing levels of physiological load (04 MPa, 1 h); with minimal
load, the explant chondrocytes behaved as free-swelling cultures as characterised
by vimentin network disassembly (Durrant et al., 1999). Interestingly, the vimentin
network in human osteoarthritic cartilage chondrocytes also reassembled when sub-
jected to cyclic strain (Lahiji et al., 2004). In contrast, when a non-physiological
single impact load (0.16 J) was applied to cartilage explants, vimentin disas-
sembly was observed in the injured chondrocytes as evidenced by peri-nuclear
collapse (Henson and Vincent, 2008). Vimentin mRNA levels were also signifi-
cantly decreased in human chondrosarcoma cells subjected to a non-physiological
continuous or cyclic (0.5 Hz) strain (5 MPa) (Karjalainen et al., 2003).

4.4.2.3 Tubulin Microtubules


Very few studies have been conducted on the effect of mechanical load on the chon-
drocyte tubulin cytoskeleton (Jortikka et al., 2000; Trickey et al., 2004; Fioravanti
et al., 2005). Tubulin network organisation was altered in chondrocytes exposed to a
continuous hydrostatic pressure (24 MPa, 3 h) (Fioravanti et al., 2005), but appeared
unaffected in response to a slightly lower hydrostatic pressure (> 15 MPa, 20 h)
(Jortikka et al., 2000). Chemical disassembly of the chondrocyte tubulin cytoskele-
ton (using colchicine) had no discernable effect on the mechanical properties of the
cell, suggesting that the F-actin and vimentin architectures provide the viscoelastic
properties of the chondrocyte (Trickey et al., 2004). However, -tubulin, the prin-
cipal cytoskeletal component of the mechano-sensing cilium is responsive to strain.
In a very recent study, it was demonstrated that the incidence and length of cilia
i.e. acetylated -tubulin increased in chondrocytes seeded in agarose subjected to
cyclic compressive strain (015%, 1 Hz) (McGlashan et al., 2010). It was previ-
ously determined that approximately 50% of chondrocyte cilia are decorated with
connexin 43 (Knight et al., 2009); further to this, the expression of a range of purine
receptors (P2X and P2Y receptor subtypes) were also identified through which
96 E.J. Blain

ATP activates downstream signalling events, suggesting a mechanism by which


mechanical load activates ATP release as part of a purinergic mechanotransduction
pathway involving the cilium (Knight et al., 2009).

4.5 Cytoskeletal Elements in Cartilage Chondrocyte Pathology

Abnormal mechanical load, be it highly repetitive, injurious or the absence of,


can result in degeneration of the articular cartilage; continued dissolution of the
tissue can ultimately lead to the development of osteoarthritis (OA). OA is a multi-
factorial disorder, but one of the primary risk factors involved in its progression is
inappropriate mechanical loading of the synovial joint (Cooper et al., 1994). OA
is characterised by a loss of cartilage tissue homeostasis where catabolic events
exceed synthesis culminating in a loss of matrix. OA is associated with altered chon-
drocyte gene expression (Reginato and Olsen, 2002) and metabolism i.e. reduced
proteoglycan and type II collagen synthesis, compounded by increased secretion of
pro-inflammatory cytokines and proteases (Goldring, 2000).

4.5.1 Cytoskeletal Element Organisation in Osteoarthritic


Cartilage Chondrocytes

Cell morphology, via cytoskeletal element organisation, influences cartilage chon-


drocyte metabolism (Ingber et al., 1994). Chondrocytes are normally devoid of actin
microfilament bundles with F-actin localising to the cell periphery (refer to Section
4.3 for detail); the predominantly cortical distribution of F-actin ensures that the cell
maintains a rounded morphology for preservation of the chondrocyte phenotype
(von der Mark et al., 1977; Benya and Shaffer, 1982). Morphological differences
between the cytoskeletal element(s) organisation in normal and OA chondrocytes
has been hypothesised to influence the cells biochemical responses to external stim-
uli e.g. mechanical load, pro-inflammatory cytokines. In recent years an association,
either directly or indirectly, between the presence of disorganised cytoskeletal net-
works in chondrocytes and cartilage pathology has been alluded to (Fioravanti et al.,
2003; Capin-Gutierrez et al., 2004; Holloway et al., 2004; Lambrecht et al., 2008).
Key morphological features of cytoskeletal element organisation in osteoarthritic
cartilage chondrocytes are described (Table 4.1B).
Characterisation of the cytoskeleton of human osteoarthritic cartilage chondro-
cytes, using transmission electron microscopy, demonstrated that the intensities and
distribution patterns of the F-actin, vimentin and tubulin elements contrasted with
observations in normal cells (Kouri et al., 1998). This early study was one of the first
to hypothesise that a modified cytoskeleton might contribute to the altered pheno-
type in OA (Kouri et al., 1998). It was subsequently demonstrated that F-actin was
apical in normal human chondrocytes, but was cytoplasmically diffuse or limited
to the cell periphery in human OA chondrocytes (Fioravanti et al., 2003). Although
tubulin was uniformly localised at the periphery of normal chondrocytes, it was
4 Involvement of the Cytoskeletal Elements 97

often absent in OA cells (Fioravanti et al., 2003). Fioravanti et al. postulated that
the loss of cytoskeletal element assembly in OA chondrocytes could compromise,
not only the metabolic activities of the cells, but also their biomechanical function
(Fioravanti et al., 2003). Disparities in cytoskeletal element expression and/or organ-
isation are not exclusive to human pathology but have also been reported in animal
models of OA. In a rat partial menisectomy model of OA, there was a reduction
in the number of OA chondrocytes immuno-positive for F-actin (4.7%) and tubulin
(20.1%) expression (Capin-Gutierrez et al., 2004).
Concomitant with this was the observed reduction in vimentin expression
(37.1%). Vimentin filaments which predominantly localised to the superficial zone,
ranged from a simple disorganisation to a total disruption of cytoplasmic archi-
tecture; this contrasted with the uniform distribution of vimentin filaments as
perinuclear bundles in normal rat chondrocytes (Capin-Gutierrez et al., 2004). The
vimentin cytoskeleton presented as a complex fibrous structure extending through-
out the cytoplasm in both normal and OA human chondrocytes (Holloway et al.,
2004). Interestingly, multiple elongated processes containing elaborate vimentin fil-
ament networks, extending up to 30 m into the surrounding matrix, were also
observed in the OA cartilage which corresponded to sites with the greatest histo-
logical and macroscopic signs of OA (Holloway et al., 2004). Thus, the aberrant
arrangement of the cytoskeletal networks in OA cells will impact on the mechano-
sensitivity of the cell, and the downstream signalling cascades activated as a
consequence.

4.5.2 Mechanotransduction in Osteoarthritic Cartilage: Effect(s)


on the Cytoskeleton

Chondrocyte stiffness i.e. the mechanical properties of the cell, is an important indi-
cator for how a cell will respond to mechanical perturbations in the surrounding
matrix environment (refer to Section 4.1). The observed aberration in cytoskeletal
element organisation in OA chondrocytes impacts on the mechanical properties of
the cell. Significant increases in elastic and viscous properties were observed in
OA chondrocytes from end-stage pathology (Trickey et al., 2000). Interestingly,
a recent study reported that the mechanical properties of cartilage chondrocytes
changed profoundly with ageing, and not OA pathology (Steklov et al., 2009).
Viscoelastic properties increased in aged cartilage (< 55 years of age), but there were
no apparent differences between the aged and age-matched OA cartilage. Clearly,
the biomechanical properties of the cartilage are dependent on cytoskeletal element
organisation irrespective of whether age and/or pathology are/is the confounding
influence.
An alteration to the biomechanical properties of the cell i.e. as a conse-
quence of cytoskeletal element aberrations, undoubtedly affects mechanotransduc-
tion responses. Cyclical pressure (15 MPa, 0.25 Hz, 3 h) did not affect F-actin
or tubulin distribution in OA chondrocytes, although the elements were subject to
reorganisation in normal chondrocytes (Fioravanti et al., 2005). It was suggested
98 E.J. Blain

that the cytoskeletal structures, which are not assembled appropriately in OA carti-
lage (Fioravanti et al., 2003), were unable to respond to similar loading conditions,
possibly becoming desensitised. This could compromise the metabolic activities
of the chondrocytes and hence the biomechanical integrity of the cartilage lead-
ing to pathology (Fioravanti et al., 2005). However, it was demonstrated in OA
tissue that the incidence and length of cilia, detected using an anti-acetylated tubu-
lin antibody, increased at the eroding articulating surface, resulting in an overall
increased proportion of ciliated cells with OA severity (McGlashan et al., 2008).
This study (McGlashan et al., 2008) would argue against the hypothesis that the
tubulin cytoskeleton becomes desensitised to mechanical stimulation in pathological
tissue (Fioravanti et al., 2003).
The mechanisms of cartilage chondrocyte mechanotransduction have also been
demonstrated to differ between normal and OA cells. As previously discussed
(Section 4.2.1), a mechanical stimulus is perceived by the chondrocyte and 5 1
integrin-mediated signalling is activated (Salter et al., 2001). This involves the
opening of stretch-activated channels, an alteration in membrane potential and
the rapid phosphorylation of focal adhesion proteins e.g. FAK125 , paxillin and
-catenin concomitant with F-actin treadmilling and the activation of downstream
signalling cascades e.g. PKC (Lee et al., 2000b). However, although the initial
response of OA chondrocytes to mechanical stimulation involved activation of 5 1
integrin, the mechanotransduction route was independent of the F-actin cytoskele-
ton (Millward-Sadler et al., 2000), which may imply an adaptation by the cell
to circumvent an aberrant F-actin network. Compressive stimulation activated an
integrin-dependent interleukin-4 (IL-4) autocrine/paracrine loop in normal chon-
drocytes, whereas mechanotransduction was mediated via the activation of an IL-1
autocrine/paracrine loop in OA cells (Salter et al., 2002). Interestingly, IL-1 has been
shown to increase F-actin amounts in chondrocytes (Pritchard and Guilak, 2006)
suggesting that a finely balanced interplay of soluble factors e.g. cytokines may exist
to regulate cytoskeletal element dynamics and hence organisation. Recently, a whole
genome array demonstrated that IL-1 inhibited the mRNA expression of both tubu-
lin and vimentin, as well as the LIM protein FHL2 which is associated with the actin
cytoskeleton (Joos et al., 2008). Therefore in an inflamed or pathological environ-
ment i.e. in an OA synovial joint, the organisation and expression levels of these
cytoskeletal proteins may be compromised which, in turn, will adversely influence
the biomechanical properties preventing normal modes of cartilage chondrocyte
mechano-signalling.

4.6 Conclusions and Perspectives

The cytoskeleton acts as a physical interface between the chondrocyte and the extra-
cellular matrix in sensing mechanical stimuli, and the cytoskeletal elements are
themselves subject to load-induced reorganisation. Although several mechanisms
of mechanotransduction involving the three major cytoskeletal elements have been
4 Involvement of the Cytoskeletal Elements 99

elucidated in articular cartilage chondrocytes there is undoubtedly much still to


discover. The reported loss of cytoskeletal element expression and/or organisa-
tion in OA chondrocytes has profound effects on cell morphology, negatively
impacting on both its biological and biomechanical functions within the cell. To
determine whether loss of cytoskeletal element architecture is a cause or effect of
abnormal mechanical load, we are currently assessing whether there is a thresh-
old above which load can disassemble the cytoskeleton. However, many more
questions remain to be answered to elucidate the signalling pathways that are acti-
vated downstream of load-induced cytoskeletal element reorganisation, and how
non-physiological loads may adversely impact on mechanotransduction pathways
leading to matrix dissolution and pathology.
Acknowledgements I wish to acknowledge the Arthritis Research Campaign (Grant No. 18221)
for funding.

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Part II
Molecular Mechanisms
of Mechanotransduction
and Ion Channels Modulation
Chapter 5
The Role of Nitric Oxide in the Regulation
of Mechanically Gated Channels in the Heart

Victor Kazanski, Andre Kamkin, Ekaterina Makarenko, Natalia Lysenko,


Natalia Lapina, and Irina Kiseleva

Abstract The article presents the effects of NO on myocardial functions including


its pronounced influence on myocardium contraction and heart rhythm. Attention is
given to cell signaling of nitric oxide in the heart. It is demonstrated that in general
the final effect of NO depends on the cellular source of NO, amount of NO release,
the prevailing redox balance and antioxidant status, stimuli such as coronary flow
rate and heart rate, the target tissue, interaction with neurohumoral and other stim-
uli, activity level of the immune system and activation of cGMP-dependent and
independent intracellular cascades. A number of experiments conducted on whole
hearts lets us suppose that NO and NO-synthases as NO origins, directly regulate the
conductivity of mechanically gated channels (MGCs). This study discusses exper-
imental data obtained from isolated ventricular myocytes of mouse, rat and guinea
pig by means of patch-clamp in the whole-cell configuration about the role of NO in
the regulation of MGCs. Presented data demonstrate that NO donors lead to MGCs
activation and appearance of MG-like currents in unstretched ventricular myocytes,
while in stretched cells with activated MGCs NO donors lead to inactivation and
inhibition of the conductivity of these channels. The NO scavenger PTIO causes
inactivation of all MGCs. In unstretched cells the conductance through MGCs is
blocked, which is present in control before deformation. PTIO causes complete inhi-
bition of stretch induced MG-current during presence of cellular stretch. Application
of non selective inhibitors of NO-synthases L-NAME or L-NMMA resulted in a
complete blockade of MGCs. The presented data are instituted on cells of trans-
genic mice. In ventricular myocytes of wild-type mice, NOS1/ and NOS2/
stretching of cells results in an activation of typical MG-currents. On the contrary,
in cells from NOS3/ mice stretch does not activate MG-currents. The results sug-
gest that NO plays an important role in the activation and inactivation of MGCs in
cardiomyocytes and demonstrate that NOS3 dominates as NO origin.

A. Kamkin (B)
Department of Fundamental and Applied Physiology, Russian State Medical University,
Ostrivitjanova 1, Moscow 117997, Russia
e-mail: Kamkin.A@g23.relcom.ru

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 109


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_5,

C Springer Science+Business Media B.V. 2011
110 V. Kazanski et al.

Keywords Heart Nitric oxide Myocardium contraction Heart


rhythm Isolated ventricular myocytes Mechanically gated channels PTIO
L-NAME L-NMMA NO donors NOS/mice

5.1 Introduction

Nitric oxide (NO) is a universal biological regulator contained in practically all


human body tissues. NO was first identified in 1987 as an endothelium-derived bio-
logical messenger causing dilatation of blood vessels (Ignarro et al., 1987). Since
then the amount of works devoted to NO has been growing fast. The Science
Journal proclaimed it in 1992 as the molecule of the year. The 1998 Nobel Prize in
physiology and medicine went to F. Murad, R. Furchgott and L. Ignarro, for dis-
covering the functional role of NO in the cardiovascular system. That was the first
boost to the gargantuan amount of publications devoted to research of the functional
qualities of this simple chemical compound.
NO possesses an obvious functional advantage over most biological regulators,
having a small molecular weight and no charge, enabling it to quickly diffuse and
freely penetrate through tight cellular layers and the intracellular space (Hughes,
2008). Going through the plasma membrane, NO acts not only as an intercellular
transmitter but also as a part of intracellular effectoral systems like other known
second messengers. NO has an unpaired electron, possesses high chemical activity
and easily reacts with numerous cell structures and chemical components (Hughes,
2008). For these reasons, NO insures exclusively diverse biological effects. This gas
is highly toxic in big concentrations, but in low or moderate concentrations it has
a wide range of regulatory effects (reviewed in Butler et al., 1995; Marletta et al.,
1994; Nathan and Xie, 1994; Schmidt et al. 1994; Stamler et al., 1994; Liaudet et al.,
2000; Bruckdorfer, 2005).
In human and animal organisms NO is produced as a result of enzymatic oxida-
tion of N-terminal L-arginine (Moncada et al., 1991; Mayer and Hemmens, 1997;
Hughes, 2008):

At the first stage, the hydroxylation of L-arginine occurs forming NG -hydroxy-


L-arginine, which oxidizes to NG -hydroxyarginine at the next stage. When the latter
splits, NO gets free and intermediate carbodiimide forms that hydrolyzes to cit-
rulline. At each of the two main stages of catalysis, one NADPH and one oxygen
molecules are used. The peroxidate of heme is the intermediate product of the cat-
alytic process (Marletta et al., 1994; Korth et al., 1994; Mayer and Hemmens, 1997;
Hughes, 2008).
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 111

The NO synthesis is carried out by a family of ferments NO-synthases (NOS)


that are presented in 3 different isoforms, named according to the place of their
discovery: (1) nNOS, subtype NOS1 (neuronal or brain), (2) iNOS, subtype NOS2
(inducible or macrophagal), (3) eNOS, subtype NOS3 (endothelial) (Moncada and
Higgs, 1993; Feron et al., 1996; Mayer and Hemmens, 1997; Xu et al., 1999). NOS
differ in their localization in the cell, activity regulation and substrate inhibitory
profile (Nathan and Xie, 1994; Moncada and Higgs, 1993). According to their
induction character and activity NOS are divided into 2 types: more active
calcium-independent inducible NO-synthase (NOS2); and less active calcium-
calmodulin-dependent constitutive NO-synthases (NOS1 and NOS3) (Moncada and
Higgs, 1993; Mayer and Hemmens, 1997).
In the heart the key role in NO level regulation plays the constitutive NO-
synthases. Thus, in mammals NOS1 is discovered in cardiomyocytes (Xu et al.,
1999; Papapetropoulos et al., 1999; Damy et al., 2003; Ziolo et al., 2008), as well
as in the preganglionic and postganglionic fibers innervating sinoatrial and atri-
oventricular nodes, subepicardial and endocardial neuronal cells. NOS3 is mainly
expressed in cardiac vessels and endocardial endotheliocytes, less in cardiomy-
ocytes and sinoatrial and atrioventricular node cells (Papapetropoulos et al., 1999;
Shah and MacCarthy, 2000; Ziolo et al., 2008). As opposed to NOS1 that is found in
the perimembrane area of cardiomyocite sarcoplasmic reticulum, NOS3 is located
in caveoles (Feron et al., 1996; Xu et al., 1999; Williams et al., 2006; Ziolo et al.,
2008), which play a significant role in limiting NO diffusion in heart cells with
high concentration of myoglobin (that binds NO with high affinity) and, what is
especially important in pathological processes, of superoxide-anions (that react with
NO and restrict its biological activity) (Casadei and Sears, 2003). Also, inducible
NOS2 was discovered in the heart which is expressed in infiltrating inflamma-
tory cells, microvessels, endocardial endotheliocytes, vascular smooth muscle cells,
fibroblasts and cardiomyocytes, often simultaneously with inflammatory cytokines
(Papapetropoulos et al., 1999; Shah and MacCarthy, 2000; Casadei and Sears,
2003).
The wide presence of NO-synthases in different types of cardiac cells proves
the necessity of nitric oxide for heart functioning. During the recent 20 years, the
role of NO as an important agent in myocardial function regulation has become
clear. It includes such effects like contractile activity, energy balance, substrate
metabolism, cellular growth and survival in physiological and pathophysiological
conditions (Shah and MacCarthy, 2000; Casadei and Sears, 2003). In this review,
rather than discussing the known effects of nitric oxide, we will discuss in detail its
effects on mechanically gated channels in heart.

5.2 Nitric Oxide and Cardiac Function

Since the first report on NO effects on myocardial contractility in 1991 (Smith et al.,
1991) a lot of data, often contradicting, have been accumulated on NO effects on
heart functions. In general it can be said that the final effect of this regulator depends
112 V. Kazanski et al.

on the following: (1) the cellular source of NO, (2) amount of NO release, (3) pre-
vailing redox balance and antioxidant status, (4) stimuli such as coronary flow rate
and heart rate, (5) the target tissue, (6) interaction with neurohumoral and other
stimuli, (7) activity level of immune system or disease, and (8) activation of intracel-
lular cGMP-dependent and independent subcellular cascades (Shah and MacCarthy,
2000).
NO takes part in regulating cardiac functions already at the embryogenesis stage.
NO affects the growth and development of cardiac cells in fetal and postnatal lives
(Strijdom et al., 2009). Moreover, several authors have considered NO as the key
signaling molecule in early embryonic development of the heart, at least in rodents
(Malan et al., 2004).
NO is cyclically secreted in the working heart with fast growing concentration
during early diastolic filling, which can be connected to the autoregulation during
the cardiac cycle (Pinsky et al. 1997). Endogenous NO causes different effects in
the heart, one of which is early relaxation of the left ventricle, that was shown on
isolated heart and isolated cardiomyocytes (Shah et al., 1994; Casadei and Sears,
2003). It was discovered that specific inhibition of NOS1 or destruction of NOS1
genes results in increased left ventricular contractility in vitro and in vivo (Ashley
et al., 2002; Casadei and Sears, 2003; Dawson et al., 2005). Clinical studies have
also shown that under NO there were changes in the onset time of left ventricle
relaxation, that manifests itself in an earlier onset of relaxation and decreased peak
and end-diastolic pressure of the left ventricle (Paulus et al., 1994). Stimulation of
NOS3 activity in healthy patients is also associated with changes in left ventricular
end-diastolic pressurevolume relationship. This suggests a sharp decrease of left
ventricular stiffness (Paulus et al., 1994).
NO also has a pronounced effect on myocardial contractility and heart rate,
and these NO effects have biphasic and concentration-dependent character. Low
concentrations of NO-donors (0.110 M) increase myocardial contractility and
heart rate, while high concentrations (over 100 M) produce negative inotropic and
chronotropic effects (Koja at al., 1996, 1997; Vila-Petroff et al., 1999; Brady et al.,
1993; Shah and MacCarthy, 2000; Casadei and Sears, 2003). It should be noted that
the positive inotropic effect is close to maximum, within the range of NO concentra-
tions corresponding to those in the normal heart with intact endothelium (e.g., 13
M, according to Pinsky et al., 1997) and therefore reflects the physiological role
of NO in healthy hearts (Casadei and Sears, 2003). Supporting this hypothesis is the
fact that coronary infusion of certain concentrations of NO-synthase inhibitors into
isolated rat hearts and healthy persons causes a small but reliable decrease of left
ventricle contractility (Kojda et al., 1999; Cotton et al., 2001).
NO plays an important role in regulating myocardium reaction to mechanical
stimuli, in particular to stretch. Myocardium response to stretch includes signif-
icant and fast increases of contractile force (Frank-Starling response), followed
by a slower and less pronounced increase of contractility (Anrep effect). NO can
be involved in the realization of both effects. Endogenous NO strengthens the
Frank-Starling response probably by increasing diastolic distensibility (Shah and
MacCarthy, 2000; Casadei and Sears, 2003; Zhang et al., 2009). It was shown that
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 113

the increases of NO production in the endothelium of coronary vessels or of intracar-


dial infusion of NO-donors causes fast left ventricle relaxation and reduction of the
end-diastolic resistance of left ventricle, that can affect the realization of the Frank-
Starling response by increasing end-diastolic volume of left ventricle and length of
myocardial myofibriles (Paulus et al., 1995). Vice versa, the inhibition of NO syn-
thesis blocks the increase of cardiac output in response to increased left ventricle
loading in isolated hearts (Prendergast et al., 1997), which attests that stretch-
mediated stimulation of constitutive NO production in myocardium plays a role in
regulation of cardiac output. An alternative autocrine mechanism for stretch induced
increase of contractive activity is the Anrep effect. NO release in myocardium cor-
responds to increased levels of intracellular Sa2+ , which results in positive inotropic
effect in response to stretch. This effect includes NOS3 activation as it is completely
blocked by NO-synthase inhibition or NOS3-gene destruction (Petroff et al., 2001).
Endogenous NO affects the expression of force-frequency relationship in car-
diomyocytes. Direction and expression of these effects depend on the experimental
conditions. Thus, positive force-frequency relation in myocardium is suppressed
by endogenous NO: this relation significantly rises after administering NOS1-
inhibitors in rat cardiomyocytes. The opposite effect is observed in vivo in
NOS1/mice. This testifies towards the ability of NOS1-produced NO to increase
the positive force-frequency relation (reviewed in Shah and MacCarthy, 2000;
Casadei and Sears, 2003).
NO is involved in the regulation of the response to adrenergic and cholinergic
stimulation in the heart. Thus, NO-donors modulate -adrenergic inotropic effects,
decreasing it in small doses and increasing it in high doses (Kelly et al., 1996;
Shah and MacCarthy, 2000; Massion and Balligand, 2003). It was also shown that
increased myocardial expression of NOS3 in pathophysiological conditions results
in inhibiting -adrenergic reactivity (Casadei and Sears, 2003). Moderate myocyte-
specific NOS3 overexpression results in inhibiting -adrenergic responses, but these
data are not confirmed in animals with high level of cardiomyocyte-specific NOS3
overexpression (Casadei and Sears, 2003). NO also mediates the effects of choliner-
gic stimulation of the heart: the first direct confirmation of the fact was given in the
work presented by Smith T.W. et al. (1995), similar data were received later using
NOS-inhibitors and NOS/mice (Shah and MacCarthy, 2000).
One more component of NO-mediated regulation of cardiac activity is its effect
on energy and metabolism qualities of myocardium. Paracrine NO release from the
endothelium of microvessels causes reversible inhibition of cardiac muscle oxy-
gen consumption (Shen et al., 1994; Trochu et al., 2000). Thus, stimulation of NO
release, by e.g., bradykinin, lowers mitochondrial respiration, and administration of
NO-synthase inhibitors results in increased total oxygen consumption in the heart
irrespective of changes of haemodynamics and contractile activity (Shen et al., 1994,
1995). Decreased oxygen consumption in the heart is connected with increased sub-
strate utilization of free fatty acids into glucose. Besides, there are data that NO
inhibits glycolysis (Zhang and Snyder, 1992).
One of the most important nitric oxide functions is modulation of cell survival
and death: it can both assist cell death and prevent it. The double role of NO in cell
114 V. Kazanski et al.

death depends on its concentration inside the cell and interaction with other bio-
logical molecules, such as oxygen and superoxide (Tsang et al., 2004; Calabrese
et al., 2009). In low concentrations NO produces cardioprotective, anti-apoptotic,
anti-hypertrophic effects protecting heart cells from damage caused by pathological
influences, like, for example, ischemia. In high concentrations NO is harmful when
it is presented in excessive amounts: pro-apoptotic, pro-necrotic effect on cardiomy-
ocytes (Casadei and Sears, 2003; Strijdom et al., 2009; Calabrese et al., 2009).
NO is involved in the regulation of cardiac activity in pathophysiological con-
ditions. Thus, expression and activity of NO-synthases increases in rat and mouse
myocardium under experimental infarction, as well as in humans with heart dis-
eases (Wildhirt et al., 1995; Casadei and Sears, 2003). NOS1 and NOS3 produced
NO influences contractility, energy balance and gene expression in myocardium
(e.g., with cardiomyopathies, infarctions), that can be an adaptive effect protecting
the injured heart from harmful influences of excessive catecholamine stimulation,
as well as from free radicals (Michel and Smith, 1993; Paulus, 2001). NOS2 par-
ticipates in developing immune-mediated heart diseases, e.g., infarction. NOS2
produced NO, on the contrary, causes apoptosis and is involved in inflammatory
processes under functional pathology of heart (Michel and Smith, 1993; Wildhirt
et al., 1995).

5.3 The Role of Nitric Oxide in the Regulation of Mechanically


Gated Channels in Isolated Ventricular Cardiomyocytes
from Guinea Pig, Rat and Mouse
Mechanical stretch is an important physiological and pathological stimulus in the
heart. It is well known that stretch increases myocardial contractility, but the mech-
anisms underlying this effect are not yet clear (Zhang et al., 2009). Thus, the
mechanical events can modify electric processes in the cardiomyocite membrane
by direct effects on mechanically gated channels (MGCs) (Kamkin et al., 2000).
It was shown in a number of works that mechanical stimuli (e.g., circumfer-
encial or longitudinal stretch) cause increased release of endocardial NO from
vascular endothelium and left ventricle cardiomyocites (Pinsky et al., 1997, Petroff
et al., 2001). Besides, in response to stretch in cardiac cells NO-synthase activ-
ity significantly increases, in particular NOS3 (reviewed in Shah and MacCarthy,
2000; Seddon et al., 2007). In Part 2 of this article we have described in brief the
participation of NO in heart response to stretch.
One of the possible mechanisms of realizing cardiomyocites reaction to mechan-
ical stress is the stretch stimulated NO effect on MGCs. Still, the NO input into
regulating the activity of mechanically gated currents (MG-currents) has been
hardly studied up till now due to serious methodical difficulties of such experiments,
although studies in this field have been conducted for a long time simultaneously by
two groups.
One group included G. Isenberg, V. Dyachenko and U. Rueckschloss and the
second group included A. Kamkin, V. Kazanski and I. Kiseleva. The authors
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 115

used similar methods of stretching cells and registering MG-currents. However


Dyachenko et al. (2009b) preincubated isolated ventricular mouse myocytes with
the PTIO, SNAP and L-NMMA (thus, not getting MG-currents control registration
from each cell under recording) after which they tried to stretch the cells and obtain
MG-currents. Preincubation of the cells means that the authors loaded drugs by stor-
ing the cells in drug containing KB-solution (see later) for the quoted period of time.
In the works presented by Kazanski et al. 2010a, b (See also this chapter) ventricu-
lar myocites of young guinea-pigs (3 months old), young Wistar rats (3 months old)
and mice were first perfused in physiological salt solution (PSS), and after regis-
tering the control MG-currents the perfusion with PSS containing the tested drugs
was started. With that, at each stage any and every drug was first tested without
deforming the cells, and secondly on the background of the previous deformation of
the cells. In both cases we tried to wash out the drug. The perfusion chamber had at
volume of 0.5 ml that was changed within 1520 s.
Ventricular myocytes were dispersed using the standard collagenase dissociation
technique. Briefly, the isolated hearts were perfused retrogradely (36 C). After an
initial 5-min period with Ca2+ -free PSS, a 15-min period followed where Ca2+ -free
PSS was supplemented with 0.1% collagenase type II (Worthington, Lakewood,
N.J., USA) and 20 M CaCl2 . Finally, the ventricles were chopped and gently trit-
urated to release the cells into Kraftbrhe (KB) medium (Isenberg and Klockner,
1982) containing (mmol/L) 30 KCl, 0.5 KH2 PO4 , 50 glutamic acid, 20 taurine,
10 glucose, 3 MgSO4 , 0.5 H4 EGTA, adjusted to pH 7.2 with KOH. The cell sus-
pension was filtered, resuspended and kept in KB medium at room temperature for
at least 2 h.
Ventricular myocytes were perfused with (37 C) PSS containing (in mM) 150
NaCl, 5.4 KCl, 1.8 CaCl2 , 1.2 MgCl2 , 20 glucose, 5 HEPES/NaOH, pH 7.4 (Kout ).
Patch pipette solution (Kin ) was composed of (in mM) 140 KCl, 5.5 MgCl2 , 5
Na2 ATP, 0.05 EGTA, 10 HEPES/KOH (pH 7.2). Patch pipettes had tip resistances
between 1.8 and 2.2 M (Kamkin et al., 2000, 2003).
The myocyte adhered with its bottom to the cover-slip. The patch pipette pressed
the cell to the glass bottom. After whole-cell access of the patch pipette, a fire-
polished glass stylus was attached to the membrane (Kamkin et al., 2000, 2003).
When the stylus was freshly polished and the surface membrane was clean, attach-
ment succeeded in approximately 70% of attempts. The stylus was then lifted 2 m
to prevent scratching of the lower cell surface on the cover-slip during stretch.
A motorized micromanipulator (MP 285, Sutter, Novato, Calif., USA, accuracy
0.2 m) increased the pipette stylus distance stepwise by up to 12 m, with
the pipette being the fixed point (Kamkin et al., 2000, 2003). Still, in most cases
we used a standard stretch equal to 10 m, as sufficiently soft for the cell.
Stretch and release of stretch could be repeated 35 times with the same cell, on
average.
Currents were recorded with an RK-300 amplifier (Biologic, Echirolle, France),
digitized (PowerCED, Cambridge Instruments, Cambridge, UK) and stored in a
computer. Cells were voltage-clamped to a holding potential of 45 mV in order
to inactivate voltage-dependent sodium currents. Changes in voltage-dependent
116 V. Kazanski et al.

membrane currents were studied with ramp-like repolarizations. To exclude a


contribution of voltage-dependent calcium currents the membrane was first clamped
to +50 mV for 50 ms and than repolarized from +50 to 100 mV at a rate of
100 mV/s. The resulting currents were plotted versus the potential as I-V-curves.
Since the amplitude of the currents depends on cell length and diameter, cells of
similar geometry were selected. The effect of a different size of the stretched mem-
brane was minimized by adjusting the glass tools to the same 40 m glass stylus
patch pipette distance prior to application of stretch. Since mechanical stretching
of the cell was restricted to a small unknown area between the stylus and the patch
pipette, we did not divide the mechanically gated currents (MG-currents) by whole
membrane capacitance. Currents in response to trains of short 5 mV pulses (applied
at 45 mV) were evaluated in terms of membrane capacitance (time integral) and
the access resistance (time constant divided by time integral (Gillis, 2000)). Results
are presented as mean S.D., n is the number of cells studied. Significant differ-
ences were detected by analysis of variance (ANOVA) with the Bonferroni test as
post-hoc test. This data demonstrates that the result of measurements in the three
animal groups do not differ significantly. There were no differences between exper-
imental series conducted on ventricular myocytes of different animals but with the
same drug.
Before stretch, the I-V curve was N-shaped (Fig. 5.1a: IC ). The intercept of the
I-V curve with the zero-current axis (Dyachenko et al., 2008) is the zero-current
potential (V0 ) which is equivalent to the diastolic membrane potential under current
clamp (V0 = 85 mV: meanS.D. V0 = 85 5 mV, n = 11). The data demon-
strated that under ramp-like repolarizations of ventricular myocytes (in Kin /Kout
solution) the hump of outward current at 60 mV was attributed to the inwardly

Fig. 5.1 Voltage dependence of membrane currents in a ventricular myocyte from young guinea-
pig in control and under cell stretch. a I-V curves recorded with repolarizing ramp commands
before (IC ) and during 10-m stretch (IS ). The main changes in the I-V curve during cell stretch
include reduction of V0 (intersection of the curve with zero-current-axis), induction of a negative
current at EK (equilibrium potential of potassium). The arrows show the direction of the I-V curve
shift. Compared to the control, 10-m stretch significantly reduces V0 , activates Ins and deactivates
IK1 . b Stretch-induced difference current (ISC : the difference of IS minus IC ). Modified from
Kazanski et al., 2010a with permission
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 117

rectifying K+ -current IK1 . At potentials positive to 40 mV, curve fit required the
superimposition of outwardly rectifying current components.
As we have shown, the 10 m stretch (1 min) shifted the I-V curve down-
wardly (Fig. 5.1a: IS ), reduced the hump of IK1 and depolarized V0 to 42 mV
(415 mV, n = 11). At the potassium equilibrium potential the potassium current
IK1 is zero. Therefore, the stretch-induced negative current I(EK ) should be inter-
preted as stretch-induced non-selective cation current Ins (Dyachenko et al., 2008),
otherwise called current through stretch activated channels ISAC (Hu and Sachs,
1997; Kamkin et al., 2000), or current through mechanically gated channels IMGC .
(Zhang Y and Hamill OP, 2000; Hamill and Martinac, 2001; White, 2006; Kamkin
and Kiseleva, 2008). I(EK ), the current at EK = 89 mV (61 mV log[5.4/155] =
89 mV), yielded a first estimate of the stretch-activated current Ins . In absence of
mechanical deformation, Ins (89 mV) was in the order of 0,03 nA (Dyachenko
et al., 2009a).
The stretch-induced difference current Isc (Fig. 5.1b) reversed polarity at
15 mV (11 2 mV, n = 11). Fit of the net and difference current according
to Ins = Gns (V Ens ) and Ins = Gns (V Ens ) with a voltage-independent Gns
yielded a straight line. At potentials negative to 20 mV mechanosensitivity is com-
posed of both Gns and GK1 . Gns has been reported to be voltage-independent
and to operate with a reversal potential Ens =10 mV (measurements with blocked
K+ -currents (Kamkin et al., 2000, 2003). The deviation of the difference current
from the straight line was attributed to the stretch-deactivation of GK1 because it
was abolished by substituting extracellular K+ by Cs+ ions (Kamkin et al., 2000,
2003). At potentials positive to 20 mV, mechanosensitive currents are carried by
K+ ions through both (Dyachenko et al., 2009a) TRPC6 outwardly rectifying chan-
nels (Hofmann et al., 1999; Spassova et al., 2006; Onohara et al., 2006) and TREK
(1 and 2) K2P 2.1 and K2P 10.1 leak channels (Honor et al., 2006; Li et al.,
2006; Patel and Honor, 2005) which are mechanosensitive.
Dyachenko et al. (2009a) proposed, that together with stretch-activated Gns
(Kamkin et al., 2000, 2003), stretch-induced deactivation of Kir2.3 and activation
of TRPC6 can destabilize the diastolic membrane, eventually leading to pacemaker-
like depolarizations and extra systoles (Kamkin et al., 2000, 2003; Lozinsky and
Kamkin, 2010; Zeng et al., 2000). After block of TRPC6 channels, stretch-induced
depolarizations remained small suggesting that stretch-activation of TRPC6 (and
not deactivation of Kir2.3 channels) is the key event Dyachenko et al. (2009a).
Thus, due to mechanosensitive currents carried by K+ ions, the difference cur-
rent Isc can only conditionally be called the MG-curent. Due to the simultaneous
activation of Gns , deactivation of Kir2.3, activation of TRPC6 and mechanosen-
sitivity of TREK Dyachenko et al. (2008) have introduced the term stretch
modulation of ion currents (SMIC: ISM ). But later these authors used the term
MG-current (Dyachenko et al., 2009b). Not to complicate the terminology here
and further on, in Figures and legends, we stick to the standard definition of
a difference current as Isc , and in presenting the data we use the term MG-
current, as MGC make the main contribution to control I-V curve modulation
under cell stretch (Dyachenko et al., 2009a). In our experiments the changes in
118 V. Kazanski et al.

MG-current increased with the amplitude of stretch and were reversible, i.e. they
have disappeared upon relaxation from stretch. These data coincide with the data
obtained under ramp-like repolarizations of ventricular myocyte Dyachenko et al.
(2008).
Since during cellular stretch (for example 10 m) both Ins (Ins(Vo) = 0.50 nA:
Fig. 5.1) and mechanosensitive currents carried by K+ ions contribute to the stretch-
induced difference current Isc , we calculated the value of stretch-induced currents
at the level of the holding potential, referring to it as IMGC(45 mV) (IMGC(45 mV) =
40 nA: meanS.D. IMGC(45 mV) = 0.410.02. Fig. 5.1). It seemed to represent
both MG-current and MG-like current.

5.3.1 NO Scavenger PTIO

The NO scavenger PTIO or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-


1-oxy-3-oxide was used for testing the involvement of NO in the regulation of MG-
current activity. Dyachenko et al. (2009b) preincubated mouse ventricular myocytes
for the period of 30 min in a solution of PTIO (100 mol/L). Then they recorded the
I-V relationship, altered by PTIO preincubation, and tried to stimulate MG-currents
by application of 10 m cellular stretch. Figure 5.2 shows the I-V curve registered
after the PTIO preincubation (IPTIO ) and the I-V curve of the same cell registered
after it was stretched by 10 m (IS,PTIO ). The absence of any changes caused by
stretch signifies the block of MG-currents on the background of PTIO. According
to the authors data the I-V curve crossed the voltage axis (zero current potential
V0 ) at 89 mV. It is a large enough resting potential for an isolated mouse cell. We
presupposed that incubation of cells with PTIO can result in closing a number of
MGCs that for certain reasons are activated in control, i.e. without artificial stretch
of the cell.

Fig. 5.2 Preincubation of


mouse ventricular myocytes
with the nitric oxide
scavenger PTIO
(100 mol/L, 30 min
preincubation) blocks the
induction of MG-currents by
10-m stretch. (From
Dyachenko et al., 2009b, with
permission from Elsevier)
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 119

First of all, we studied the reaction of the cell to PTIO. After whole-cell access of
the patch pipette, a glass stylus was attached to the membrane but no stretch of the
cell was conducted. After registering the control I-V curve we started perfusing the
cell with PTIO solution (500 mol/L). Such concentration of PTIO was used, for
example, for studying the regulation of cardiac calcium current by NO (Gallo et al.,
2001). Figure 5.3 shows the control I-V curves (IC ) as compared to I-V curves under
PTIO (IPTIO ) without stretching the ventricular myocyte from guinea-pig after 1 min
(Fig. 5.3a) and 3 min (Fig. 5.3b) of perfusion, and besides that the cells reaction
to washing out PTIO (W IPTIO ) for the period of 5 min (Fig. 5.3c). As the crossing
point of the I-V curve characterizes the diastolic membrane potential, it is obvious
that under the PTIO effect V0 increased by approximately 10 mV from 84 mV
(845 mV; n = 10) to 94 mV (942 mV; n = 10). PTIO shifted the I-V
curve downwardly, reduced the hump of outward current and shifted the hump from

Fig. 5.3 Voltage dependence of membrane currents in ventricular myocytes from guinea-pig under
the perfusion of unstretched cell by PTIO solution. a I-V curve measured in control (IC ) and after
1 min PTIO perfusion (IPTIO ). b After 3 min PTIO perfusion (IPTIO ) as compared to the control
(IC ). c After 5 min of PTIO wash out (W IPTIO ) as compared to control (IC ). Note: In panels (a)
and (v) the arrows show the direction of the I-V curve shift under PTIO perfusion of the cell after
1 and 3 min after the beginning of the perfusion accordingly. Modified from Kazanski et al., 2010b
with permission
120 V. Kazanski et al.

60 mV to 80 mV (Fig. 3a,b: IPTIO ). The value of IPTIOC is equal to 0.13


nA (IMGC(45 mV) = 0.15 0.02 nA, n = 10) for 3 min PTIO perfusion. As it
is known that NO increases cardiac IK1 (Gmez et al., 2009), it is understandable
that PTIO, reacting stoichiometrically with NO, decreases inwardly rectifying K+ -
currents IK1 . It is shown that after 3 min of PTIO perfusion the 5 min long wash
out did not result in any shifts of the curve towards the control, i.e. the unstretched
cell did not show even any partial release from PTIO effect during the registered
time. Similar data were registered from ventricular myocytes of mouse and rat.
In the following series of experiments after registering the control I-V curve
we first stretched the cell and registered the I-V curve. Then we perfused it
with the PTIO solution (500 mol/L) and registered the I-V curves during the
perfusion. Figure 5.4 shows I-V curves registered from ventricular myocytes of

Fig. 5.4 Voltage dependence of membrane currents in ventricular myocyte from young guinea-
pig under PTIO perfusion on the background of cell stretch. a I-V curve measured before stretch
(IC ) and during 10 m stretch (IS ). The arrows show the direction of the I-V curve shift. b , c
I-V curve modifications under PTIO perfusion with continued stretch for 2 and 8 min accordingly
(IS,PTIO ). The arrows show the direction of I-V curve shift at the given cell stretch under PTIO
perfusion. For better perception the panel D combines the control I-V curve (IC ) and the I-V curve
under 8 m cell stretch after 8 min of PTIO perfusion (IS,PTIO ). It is obvious that during the PTIO
perfusion the activity of MGCs is inhibited. Modified from Kazanski et al., 2010b with permission
and the authors data
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 121

young guinea-pig in control, under cell stretch and under further PTIO perfusion.
The voltage dependency of IC and its modulation by stretch IS is shown in Fig. 5.4a.
Before stretch (IC ), the I-V curve was N-shaped and crossed the voltage axis at
85 mV (86 5 mV, n = 10). The 10-m stretch shifted the I-V curve down-
wardly (Fig. 5.4a: IS ), reduced the hump of inwardly rectifying K+ -current IK1 and
depolarized V0 to 42 mV (39 4 mV, n = 10). Close to 10 mV, the I-V
curves recorded before and during stretch crossed each other, and at positive poten-
tials IS was increased. The stretch-induced difference current (ISC ) the difference
of IS minus IC (Fig. 5.5a corresponding to Fig. 5.4a) reversed polarity at 15 mV
(123 mV, n=10) and equaled 0.41 nA (IMGC(45 mV) = 0.40 0.02 nA, n =
10). At potentials negative to 20 mV mechanosensitivity is composed of both Gns
and GK1 . The deviation of the difference current from the straight line was attributed
to the stretch-deactivation of GK1 . Thus, stretch increases the MG-currents and
depolarizes the cell (Fig. 5.4a). Figure 5.4b shows the change of IS on the back-
ground of 10 m cell stretch 2 min after beginning the PTIO perfusion (IS,PTIO ), and
Fig. 5.4c demonstrates IS,PTIO under the same stretch but after 8 min of PTIO perfu-
sion. Disregarding the continuous cell stretch PTIO perfusion returns the I-V curve
to the original state, at which the curve got back to N-shaped and crossed the volt-
age axis at 84 mV (864 mV, n=10). The stretch-induced difference current
current during stretch minus current during stretch with PTIO ISS,PTIO (Fig. 5.5b
corresponding to Fig. 5.4c) reversed polarity at 10 mV and showed deviation from
the straight line and equals (+)0.33 nA IMGC(45 mV) = (+)0.35 0.03 nA, Erev =

Fig. 5.5 Stretch-induced difference currents from ventricular myocyte of young guinea-pig. a
Different current activated by 10 m of stretch (the difference of IS minus IC ), corresponding to
Fig. 5.4a (reversal potential Erev = 15 mV, IMGC(45 mV) = 0.41 nA. b Different current
under PTIO perfusion on the background of myocyte stretch: the difference of the current during
stretch (IS ) by 10 m minus same stretch with PTIO (ISS,PTIO ). Panel (b) corresponds to Fig. 5.4c
(reversal potential Erev = 5 mV, IMGC(45 mV) = (+)0.33 nA). Note: two curves for stretch of
10 m (a) and stretch of 10 m plus PTIO (b) show a nearly linear voltage dependence. Modified
from Kazanski et al., 2010b with permission
122 V. Kazanski et al.

2 2 mV, n = 10)1 . Thus, PTIO perfusion on the background of cell stretch


resulted in inhibiting MG-currents, and in partial restoration of IK1 . The I-V curve
registered under those conditions is practically the same as the I-V curve under
control (Fig. 5.4d). Only the hump of outward current at 60 mV was slightly
decreased and smoothed. Similar data were obtained from ventricular myocites of
mouse and rat. It is of interest, that under preceding cell stretch the PTIO perfusion
did not result in the shift of the hump of outward current and V0 towards negative
potentials.
We considered a possibility of PTIO wash out at least at the first stages of the
effect development. Figure 5.6 shows the experimental results obtained from mouse
ventricular myocytes. The 10-m stretch shifted the I-V curve downwardly, reduced
the hump of IK1 , depolarized V0 and caused the MG-current (Fig. 5.6a: IS ). PTIO
perfusion under up to 10 m cell stretch resulted in progressed inhibition of MG-
currents (Fig. 5.6b: IS,PTIO during 2 min and Fig. 5.6c: IS,PTIO during 3 min). To
check the possibility of PTIO wash out, it was started after 3 min, i.e. when the PTIO
effect came into force. Interestingly, the wash out immediately prevented further
inhibition of MG-currents but even after 5 min of wash out the curve remained
unchanged (W IS,PTIO Fig. 5.6d as compared to IS,PTIO Fig. 5.6c). Thus, the
treatment of PTIO for 3 min blocked a part of MSCs irreversibly, which manifested
itself in a decrease of MG currents, while the other part continued to function under
continuous stretch.

5.3.2 NO Donors
5.3.2.1 NO-Donor SNAP
The NO-donor SNAP or S-Nitroso-N-acetylpenicillamine was used for testing the
involvement of NO in regulating MG-current activities. First of all, we demonstrate
the reaction of an undeformed cell to SNAP. After registering the control I-V curve
we started perfusing the cell with SNAP (100 mol/L). Figure 5.7a demonstrates
the control I-V curve (IC ) registered in mouse ventricular myocyte and the I-V curve
received 1 min after the SNAP perfusion started (ISNAP ) without any cell stretch.
Figure 5.7b shows, as compared to the control curve, the I-V curve received 2 min
after SNAP perfusion started (ISNAP ). Before perfusion of SNAP (IC ), the I-V curve
was N-shaped and crossed the voltage axis (zero current potential V0 ) at 74 mV.
It is obvious that SNAP perfusion shifts V0 from 74 mV (85 4 mV, n = 8)

1 Here we must make the following note. The differential current that occurs under cell stretch as
a result of I increase (e.g., the difference of IS minus IC ) or the differential current occurring as
compared to the control under the drugs effect (the difference of IDrug minus IC ) was marked with
a minus sign. To make it easier for the reader and to avoid confusion with the differential current
under cell relaxation after stretch or the differential current resulting from I return registered in a
stretched cell to the control under the drugs effect, the latter we mark conventionally with a plus
sign in brackets (+).
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 123

Fig. 5.6 Voltage dependence of MG currents in ventricular myocyte from mouse under PTIO
perfusion of the cell and its wash out. a I-V curve measured before stretch (IC ) and during 10 m
stretch (IS ). b-, c- modified I-V curve under PTIO perfusion with continuous stretch for 2 and 3 min
accordingly (IS,PTIO ). In both panels IS characterizes MG-currents during 10 m stretch. d 5 min
of PTIO wash out under stretch (W IS,PTIO ). Note that the I-V curve W IS,PTIO did not change as
compared to I-V curve IS,PTIO in panel C. The authors data

to 50 mV (52 3 mV, n = 8) in 1 min and to 30 mV (28 4, n = 8) in


2 min. Besides, you can see a reduction of the slope of ISNAP at potentials negative
to V0 which could be attributed to stretch-deactivation of GK1 (Dyachenko et al.,
2009a). The appearing MG-like current equals to 0.38 nA and corresponds to the
stretch induced current appearing at 10 m cell stretch. The SNAP-induced differ-
ence current (ISNAPC : during perfusion of SNAP minus control) plotted in the I-V
relations in Fig. 5.7c, d (for 1 and 2 min, respectively) reversed polarity at 2 mV,
and at 2 mV (near zero potential: Erev = 1 + 1 mV). The deviation of the difference
current from a straight line was attributed to the stretch-deactivation of GK1 . The
value of ISNAPC is equal to 0.38 nA (IMGC(45 mV) = 0.39 0.02 nA, n = 8)
for 2 min SNAP perfusion vs. IMGC(45 mV) = 0.40 0.02 nA, n = 11 during
10 m cell stretch in control (P=NS). Thus, SNAP perfusion resulted in progressed
activation of MG-currents.
124 V. Kazanski et al.

Fig. 5.7 Voltage dependence of membrane currents in ventricular cardiomyocytes from mouse
under SNAP perfusion of unstretched cells. a I-V curve measured in control (IC ) and after 1 min
SNAP perfusion (ISNAP ). b After 2 min SNAP perfusion (ISNAP ) as compared to the control
(IC ). c Difference current activated by SNAP ISNAP-C (the difference of ISNAP minus IC ), cor-
responding to Panel A 1 min after the beginning of perfusion (reversal potential Erev = 2 mV,
IMGC(45 mV) = 0.13 nA). d ISNAP-C , corresponding to Panel B 2 min after the beginning of
perfusion (reversal potential Erev = 2 mV, IMGC(45 mV) = 0.38 nA). Note 1: In panels (a) and
(b) the arrows show the direction of the I-V curve shift under SNAP perfusion of the cell. Note 2:
After 2 min the different MG-like current (Panel d) corresponds to 10 m cell stretch. Modified
from Kazanski et al., 2010a with permission and the authors data

After 2 min of SNAP perfusion the wash out for 2 min results in an I-V curve
shift towards the control, i.e. the unstretched cell shows removal of SNAP effect
during registration time (Fig. 5.8a). After 5 min of wash out the control I-V curve
and the I-V curve received after SNAP wash out coincide completely, and the latter
crosses the voltage axis at 72 mV (82 5 mV, n = 8) (Fig. 5.8b). The value
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 125

Fig. 5.8 Voltage dependence of membrane currents in ventricular cardiomyocytes from mouse
after SNAP wash out. a After 2 min of SNAP wash out (W ISNAP ) as compared to the control (IC )
and to the perfusion of SNAP during 2 min (ISNAP ). b After 5 min SNAP wash out (W ISNAP ) as
compared to the control (IC ). The authors data

of W ISNAP is equal to (+)0.39 nA (IMGC(45 mV) = (+)0.40 0.03 nA, n = 8) for


5 min SNAP perfusion (not shown).
It is interesting that earlier Dyachenko et al. (2009b), who preincubated cells with
SNAP (200 mol/L), has shown that superfusion of cardiomyocytes with SNAP did
not significantly activate Gns or deactivate GK1 . We demonstrate that MG-like cur-
rents (Gns ) in cells isolated from all the animals change quite significantly, as well
as GK1 deactivation is observed. SNAP perfusion for approximately 23 min caused
the same changes as 10 m stretch. Since application of the NO-donor SNAP in the
absence of mechanical stimulation failed to induce MG-like currents we concluded
that availability of NO is crucial for MSC activation, and an increase in NO alone is
sufficient for its activation.
Further on we conducted a series of experiments in which we first stretched
cells by 10 m, and then perfused them in this stretched state with SNAP solution
(100 mol/L). Figure 5.9 shows the results of experiments carried out on ventricular
myocytes from mouse. The voltage dependency of IC and its modulation by stretch
(IS ) is shown in the I-V curves in Fig. 5.9a. Before stretch (IC ), the I-V curve was
N-shaped and crossed the voltage axis at V0 = 70 mV (85 4 mV, m = 8). The
10-m stretch shifted the net currents to more negative values (IS ), and V0 changed
to 35 mV (40 5 mV, n = 8). Close to 10 mV, the I-V curves recorded before
and during stretch crossed each other. The stretch-induced difference current (ISC )
is plotted in the I-V relations in Fig. 5.9c and equals 0.46 nA (IMGC(45 mV) =
0.42 0.02 nA, Erev = 8 2 mV, n = 8). The deviation of the difference cur-
rent from a straight line was attributed to the stretch-deactivation of GK1 , because at
126 V. Kazanski et al.

Fig. 5.9 Voltage dependence of membrane currents in ventricular myocyte from mouse under
SNAP perfusion on the background of the cell stretch. a Current-voltage relation (I-V curve)
measured before stretch (IC ) and during 10 m stretch (IS ). The arrows show the direction of
the I-V curve shift at the given cell stretch. b modified I-V curves on the background of
SNAP perfusion at continuous stretch during 2 min (IS,SNAP )and 3 min accordingly (IS,SNAP ).
The arrows show the direction of the I-V curve shift at the given cell stretch under SNAP
perfusion. It is obvious that during SNAP perfusion the activity of MGCs is inhibited. c
Difference current activated by 10 m of stretch (ISC ), corresponding to panel A (reversal
potential Erev = 10 mV, IMGC(45 mV) = 0.46 nA). d Difference current (IS-S,SNAP ): the
difference of the IS minus same stretch with SNAP IS,SNAP , corresponding to Fig. 9B (reversal
potential Erev = 3 mV, IMGC(53 mV) = (+)0.22 nA). Modified from Kazanski et al., 2010a with
permission and the authors data

potentials negative to 20 mV mechanosensitivity is composed of both Gns and


GK1 . Thus, stretch increases MG-currents and depolarizes the cell. Figure 5.9b
shows the change of IS on the background of 10 m stretch at the beginning of
SNAP perfusion (2 min) and then after 3 min. Despite the continuing cell stretch
the SNAP perfusion returned the I-V curve in 2 min to the state close to the original,
and the curve again turned N-shaped and crossed the voltage axis at 68 mV, in
3 min V0 = 78 mV (87 4 mV, n = 8). The stretch-induced difference current
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 127

(ISS,SNAP ) with SNAP 3 min after perfusion of SNAP is plotted in the I-V relations
in Fig. 5.9d and equals (+)0.53 nA (IMGC(45 mV) = (+)0.48 0.05 nA, n = 8 vs
control, P < 0.001; Erev = 3 1 mV). Thus, SNAP perfusion under 10 m cell
stretch resulted in inhibited MG-currents, probably due to the blocking of MGCs.
The I-V curve received in these conditions practically coincided after 2 min with
the I-V curve registered in the control. But the following SNAP perfusion already
at 3 min shifts the membrane potential value to 78 mV, which happens, in our
opinion, as a result of inhibiting MGCs that have background activity. Similar data
were obtained on ventricular cardiomyocytes from guinea-pig and rat.

5.3.2.2 NO-Donor DEA-NO


The NO-donor DEA-NO or 2-(N,N-Diethylamino)-diazenolate-2-oxide.diethy-
lammonium salt is a nitric oxide donor, useful for reliable generation of nitric oxide
(NO) in vitro or in vivo. We perfused isolated ventricular cardiomyocytes with
DEA-NO (250 mol/L) solution without deforming the cell. After registering the
control I-V curve we started perfusing the cell with DEA-NO. Figure 5.10a shows
the control I-V curve (IC ) registered in a ventricular myocyte from rat and the I-V
curve received 2 min after the beginning of DEA-NO perfusion (IDEA-NO ) without
stretching the cell. Before perfusion with DEA-NO control I-V curve was N-shaped
and crossed the voltage axis at 80 mV (86 5 mV, n = 9). It is obvious that
DEA-NO induced MG-like currents, and already after 2 min the I-V curve crossed
the voltage axis at 48 mV (45 3 mV, n = 9). The originating MG-like cur-
rent corresponds to the stretch-induced current originating to 10 m cell stretch.
The difference current activated by DEA-NO (IDEA-NOC ) is plotted in the I-V rela-
tions in Fig. 5.10c and equals 0.46 nA (IMGC(45 mV) = 0.43 0.03 nA, Erev =
3 2 mV, n = 9) for 2 min DEA-NO perfusion. It is shown that after 2 min per-
fusion with DEA-NO the wash out for 2 min results in I-V curve shifts towards the
control, i.e. the unstretched cell showing the loss of DEA-NO effect during registra-
tion time (Fig. 5.10b). After 2 min of wash out the control I-V curve (Fig. 5.10a) and
the I-V curve registered after DEA-NO wash out (Fig. 5.10b) coincided completely,
at which the latter crossed the voltage axis at 79 mV (864 mV, n = 9). The dif-
ference of the currents activated by DEA-NO without stretch minus currents under
wash out (IDEA-NOW ) is plotted in the I-V relations in Fig. 5.10d. IDEA NOW =
(+)0.47 nA (IMGC(45 mV) = (+)0.45 0.03 nA, Erev = 3 2 mV, n = 9)
Our data, accumulated on cells from different animals have demonstrated a pro-
nounced DEA-NO effect as an activator of MG-like currents. Since application of
the NO-donor DEA-NO in the absence of mechanical stimulation failed to induce
MG-like currents we have concluded that, although availability of NO was crucial
for MSC, an increase in NO alone was insufficient.
Further on, we conducted a series of experiments in which we first stretched
cells by 10 m and then perfused them with DEA-NO (250 mol/L). Figure 5.11
shows the results of the experiments conducted on ventricular myocyte from mouse.
The voltage dependency of IC and its modulation by stretch (IS ) is shown in the
I-V curves in Fig. 5.11a. Before stretch (IC ), the I-V curve was N-shaped and
128 V. Kazanski et al.

Fig. 5.10 Voltage dependence of membrane currents in ventricular cardiomyocytes from rat under
DEA-NO perfusion of unstretched cell. a I-V curve measured in control (IC ) and after 2 min
DEA-NO perfusion (IDEA-NO ). b after 1 and 2 min of DEA-NO wash out (W IDEA-NO ) as com-
pared to IDEA-NO . Note: In panels (a) and (b) the arrows show the direction of the I-V curve
shift under DEA-NO perfusion and under cell wash out. c Difference current activated by
DEA-NO (IDEA-NOC : the difference of IDEA-NO minus IC ), corresponding to Panel a 2 min after
the beginning of perfusion (reversal potential Erev = 5 mV, IMGC(45 mV) = 0.46 nA). At
2 min IDEA-NOC corresponds to 10 m cell stretch. d IDEO-NOC activated by DEA-NO without
stretch, 1 min after the beginning of wash out (reversal potential Erev = 4 mV, IMGC(45 mV) =
(+)0.22 nA. Modified from Kazanski et al., 2010a with permission and the authors data

crossed the voltage axis at V0 = 69 mV (83 4 mV, n = 9). The 10-m


stretch shifted the net currents to more negative values (IS ), and V0 changed to
35 mV (383 mV, n = 9). Close to 20 mV, the I-V curves recorded before and
during stretch crossed each other. The difference of currents during stretch minus
before stretch (ISC ) is plotted in the I-V relations in Fig. 5.11c and equals 0.31
nA (IMGC(45 mV) = 0.38 0.04 nA, Erev = 15 3 mV, n = 9). Thus, stretch
increases MG-currents and depolarizes the cell. Figure 5.11b shows the change of
IS on the background of 10 m stretch under DEA-NO perfusion of the cell (3 min).
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 129

Fig. 5.11 Voltage dependence of membrane currents in ventricular myocyte from mouse under
DEA-NO perfusion of the cell on the background of its stretch. a I-V curve measured before
stretch (IC ) and during 10 m stretch (IS ). The arrows show the direction of the I-V curve shift
at the given cell stretch. b modified I-V curves on the background of DEA-NO perfusion under
continuous stretch during 3 min accordingly (IS,DEA-NO ). The arrows show the direction toward
which the I-V curve shifts in response to DEA-NO application to prestretched cell. It is obvi-
ous that in the process of DEA-NO perfusion the activity of MGCs is inhibited. c Difference
current (ISC ) activated by 10 m of stretch corresponding to Fig. 5.11a (reversal potential
Erev = 20 mV, IMGC(45 mV) = 0.31 nA). d Difference current (ISS,DEA-NO ): the differ-
ence of IS minus same stretch with DEA-NO IS,DEA-NO , corresponding to Fig. 5.11b (reversal
potential Erev = 20 mV, IMGC(45 mV) = (+)0.34 nA). Modified from Kazanski et al., 2010a
with permission and the authors data

Despite the continuing cell stretch, the DEA-NO perfusion returns the I-V curve to
the state close to the original, at which the curve turns again N-shaped and crosses
the voltage axis at 75 mV (873 mV, n = 9). The difference of IS minus during
stretch with DEA-NO (IS,DEA-NO ) 3 min after perfusion is plotted in the I-V relations
in Fig. 5.11d and equals (+)0.34 nA (IMGC(45 mV) = (+)0.40 0.03 nA, n = 9 vs.
130 V. Kazanski et al.

control, P = NS; Erev = 14 4 mV). Thus, DEA-NO perfusion on the back-


ground of 10 m cell stretch resulted in inhibition of MG-currents, probably due to
blocking of MGCs. The I-V curve registered under these conditions shifted the value
of membrane potential after 3 min to 78 mV, which occurs, in our opinion, as a
result of inhibiting MSC that have background activity. Similar data were received
on ventricular cardiomyocytes from guinea-pig and rat.

5.3.3 Nitric Oxide Synthases Inhibitors NOS Inhibits


We used a non-selective inhibitor of nitric oxide synthase - L-NAME (hydrochlo-
ride) or L-NG -Nitroarginine methyl ester (hydrochloride). Ventricular myocytes
were preincubated in L-NAME solution (20 mol/L) for 2 h after which they were
put into the experiment. Figure 5.12 shows the results of the experiments conducted
on ventricular myocyte from guinea-pig. The 10 m stretch of the preincubated
cell does not cause any changes in the I-V curve, therefore MGCs are completely
blocked. An increase of L-NAME concentration to 100 mol/L with the same incu-
bation time does not cause any changes, there is no reaction of the cell to stretch.
But a concentration decrease to 2 mol/L with the same incubation time preserves
completely the cells reaction to stretch and relaxation returns the I-V curve to the
original state. Comparing Fig. 5.12 to Fig. 5.3 or Fig. 5.4 shows on guinea pig
cell samples that the cell preincubation in L-NAME solution shifted the I-V curve
downwardly and reduced the hump of outward current.
The use of nitric oxide synthase inhibitor L-NMMA or N -Methyl-L-arginine
acetate (200 mol/L, 2 h preincubation) showed results similar to the ones reported
above. In L-NMMA-pretreated cells MG currents were absent (Dyachenko et al.,
2009b). Thus, NO-synthase inhibitor L-NMMA completely blocked MGCs.

Fig. 5.12 Voltage dependence of stretch-induced membrane currents, K+ currents not suppressed.
Ventricular myocyte from guinea pig. Current-voltage relation (I-V curve) measured before stretch
(IC ) and during 10 m stretch (IS ) after preincubation of cells in L-NAME solution (20 mol/L,
2 h). Please, note that the cell preincubation in L-NAME solution changes the shape of the con-
trol I-V curve. See Fig. 5.3 compared to Fig. 5.4. Modified from Kazanski et al., 2010b with
permission
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 131

5.3.4 Cardiomyocytes Derived from NOS/Mice


Research of physiological effects of NO is complicated by the fact that it is a fast
inactivating molecule. Interpretation of the data is also complicated because in a
cell there are other compounds very similar to NO in their chemical qualities, e.g.,
free radicals that have analogical biological effects practically impossible to divide
pharmacologically. The solution is in using transgenetic animals, e.g. NOS/
mice.
MG currents were analyzed in cardiomyocytes derived from NOS/ mice
(Dyachenko et al., 2009b). In cardiomyocytes from NOS1/ mice, 10-m stretch
activated Gns similar as in cells from their wild-type littermates (10m Gns = 7.3
2.1 nS, n = 5 in NOS/ vs. 10m Gns = 6.2 2.5 nS, n = 8 in wild-type litter-
mates). In contrast, in cardiomyocytes from NOS3/ mice, 10-m stretch did not
significantly induce MG currents (0m Gns = 0.20.6 nS vs. 10m Gns = 2.03.4 nS
and 0m GK1 = 24 10 nS vs. 10m GK1 = 23 11 nS, n = 18; Fig. 5.12). This
result clearly points to NOS3 as the dominant source of NO involved in MGCs and
is in agreement with studies showing that NOS3 is activated by stretch (Petroff et al.,
2001; Kuebler et al., 2003; Fig. 5.13)

5.3.5 Possible Explanations of NO Involvement into Regulation


of MG-Currents

The data presented in Part 4 about the involvement of NO into regulating the activity
of MG-currents come down to several basic issues. In ventricular myocytes from
guinea pig, mouse and rat in all experimental conditions the NO scavenger PTIO
produces complete inhibition of stretch-activated MG currents and deactivation of
K1-currents. The extent of MG current block depends on incubation duration. The

Fig. 5.13 NOS3-derived NO


in MGCs. Cardiomyocyte
isolated from a NOS3/
mouse. 10-m stretch of the
myocyte does not induce
MSC (black: before stretch;
red: during 10-m stretch)
(From Dyachenko et al.
(2009b) with permission of
Elsevier)
132 V. Kazanski et al.

wash-out of PTIO does not reverse the blockade of MG current. Wash-out stops the
development of the blockade. We believe that application of PTIO scavengers NO,
thus blocking MG currents. However during the beginning of application of PTIO,
its removal prevents the development of the MG current blockade. PTIO blockade
of MG current cannot be reversed by its washout.
The NO-donors SNAP and DEO-NO in undeformed cell result in activation of
MG-like currents. Wash out removes these currents. On the contrary, SNAP and
DEO-NO inhibit MG currents in a previously stretched cell. This is an interest-
ing phenomenon that correlates with described bimodal effects of NO. It is known
that the effect of NO is bimodal, with a positive inotropic effect at low amounts
of NO exposure but a negative one at higher amounts (Massion et al., 2003).
Admittedly, defining what low or high amounts really mean is difficult, both in
terms of actual quantity of bioactive NO delivered (e.g., with different exogenous
NO donors) as well as the correspondence with amounts endogenously produced
in vivo. Similarly, Kojda et al. (1996) observed that low concentrations of the NO
donors SNAP and DEA/NO caused a moderate positively inotropic effect in adult
rat ventricular myocytes. Higher concentrations of either SNAP or DEA/NO sup-
pressed myocyte contractile function. Mohan et al. (1996) have reported a similar
biphasic inotropic response to NO donors in isolated feline papillary muscle strips
that appeared to be dependent on a GMP-mediated signaling pathway. Since the
direction of NO donors mediated effects can be dose dependent we suggest that
NO donors open MGCs in ventricular cardiomyocytes, which underlie MG-like
currents. We also register MG currents when stretching the cell, but with that we
increase the concentration of endogeneous NO. We speculate that in both first and
second cases we have that low concentration of exogenous or endogenous NO, that
activates MGCs. In case the cell, when stretched, shows higher endogenous NO
concentration, then an additional increase of exogenous concentration using NO
donors results in increased summary concentration, which probably is the reason for
inhibiting MGCs. As we have no data on MGCs ion channel structure in ventricular
myocytes, our speculation is based on analogues with voltage gated L-type calcium
channels which also show dose dependent activatory and inhibitory effects of NO
donors on ICaL . Thus, e.g., Mery et al. (1993) originally reported that SIN-1 (NO
donor 3-morpholino-sydnonimine), the active metabolite of molsidomine, has a
biphasic effect on ICaL in enzymatically dissociated frog ventricular myocytes. The
data of these authors suggest that the activatory and inhibitory effects of NO donors
on ICaL result from an inhibition of the cGMP-inhibited cAMP-phosphodiesterase
and an activation of the cGMP-stimulated cAMP-phosphodiesterase, respectively,
both linked to the activation of guanylyl cyclase, possibly a membrane form of the
enzyme.
The use of NOS inhibitors L-NAME and LNMMA results in MG currents that
do not appear as a response to cell stretch. In NOS1/ the reaction to stretch is
present, whereas in NOS3/ there is no reaction to stretch. This allows to consider
NOS3 as the dominant source of NO involved in MGCs and also define that NOS3
is activated by stretch.
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 133

5.4 Cell Signaling of Nitric Oxide in the Heart and Possible Role
in Regulation of MG-Currents

NO has a wide range of physiological effects, but the main intracellular target for
NO is considered soluble guanylyl cyclase that causes an increase of the concen-
tration of cGMP (Fig. 5.14), which can modulate the activity of cGMP-dependent
cyclic nucleotide phosphodiesterases and, respectively cAMP level, as well as
cGMP-dependent protein kinase G, that causes phosphorylation of a number of
proteins (Moncada et al., 1991; Murad, 1998; Lane and Gross, 1999; Shah and
MacCarthy, 2000; Bryan, 2009). The cGMP-mediated NO effect also includes influ-
ence on such effectors as cyclooxygenases, phosphatases, phospholipase S (Murad,
1998; Lane and Gross, 1999; Bryan, 2009).
The effect of the cGMP-dependent pathway on the function of the myocardium
consists of modulating Sa2+ influx from the sarcolemma, lowering myofilaments
sensitivity to Sa2+ , changing the functional activity of sarcoplasmic reticulum,
changing the action potential, changing cell volume, and decreasing oxygen con-
sumption (Ji et al., 1999; Shah and MacCarthy, 2000). cGMP-dependent NO effects
in the heart are: (1) early onset of relaxation mediated by troponin I phosphory-
lation by protein kinase G, resulting in lower myofilament sensitivity to Sa2+ ; (2)
positive inotropic effect of NO at low concentrations by affecting cGMP-inhibited
cyclic nucleotide phosphodiesterase resulting in increased levels of intracellular
cAMP, but cGMP participation can not be excluded, (3) negative inotropic effect
of higher NO doses, realized probably by activating protein kinase G, (4) mod-
ulation of -adrenergic and cholinergic responses, and (5) protective effect on
cardiomyocites under myocardial hypoxia at the expense cGMP-mediated opening

Fig. 5.14 NO-cGMP-mediated signal transduction in myocardium (Reproduced from Shah and
MacCarthy, 2000 with permission from Elsevier.)
134 V. Kazanski et al.

of KATP -channels (Ji et al., 1999; Casadei and Sears, 2003; Shah and MacCarthy,
2000).
The cGMP-independent mechanisms for realizing NO effects include its reac-
tions with amino, thiol (SH), diazo and tyrosyl groups in proteins, as well as haem-,
iron- or sulphur centers of proteins (Mateo and De Artiano, 2000; Landar and
Darley-Usmar, 2003; Hughes, 2008; Bryan et al., 2009). The most important fact is
the direct interaction of NO with chemically active thiol protein groups causing their
posttranslational modifications, which results in significant functional changes.
S-nitrosylation of reactive thiol groups can affect the activity of ion channels,
transporters and Sa2+ -binding proteins participating in the regulation of Sa2+ -
cycling in myocytes. Possible targets are ryanodine receptor Ca2+ release channels
(RYR) of sarcoplasmic reticulum, SERCA 2a, phospholambam, Sa2+ -ATP-ase of
the sarcolemma (Shah and MacCarthy, 2000; Casadei and Sears, 2003; Lim et al.,
2008; Petroff et al., 2001; Jaffrey et al., 2001). A NO mediated increase of Sa2+
concentration in cardial myocytes is the main mechanism of NO modulation of
myocardial excitationcontraction coupling and realization of cardiac cell response
to mechanical impacts (Shah and MacCarthy, 2000; Seddon et al., 2007; Lim et al.,
2008; Fig. 5.15).
One more important mechanism for realizing NO effects is its interaction with
active peroxide radicals. NO, especially under pathophysiological conditions, can
interact with high reactivity superoxide, which in norm is tied by superoxide
dismutase. As a result a high-activity compound is formed, such as peroxynitrite

Fig. 5.15 Regulation of cardiomyocyte functions by nitric oxide in normal heart (Reproduced
from Seddon et al., 2007 with permission from Elsevier)
5 The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels 135

(ONOO ) that can modulate ion channel functions, oxidizing, like NO itself, the
regulatory thiol groups of channel proteins (Mateo and De Artian, 2000; Landar
and Darley-Usmar, 2003; Hughes, 2008). Also shown was the effect of peroxynitrite
on the physiological activity of a number of enzymes, such as phospholipases of dif-
ferent types (Yuen et al., 2000; Guidarelli et al., 2000). Protonation of peroxynitrite,
especially under low rN, observed in heart pathologies, results in formation of per-
oxynitrous acid. As a result, highly reactive hydroxyl-like species are formed that
can cause toxic effects related to nitration and oxidation of not only functional but
also structural proteins (reviewed in Shah and MacCarthy, 2000).

5.5 Conclusion and Perspectives

There is a wide discussion going on about, how mechanical energy is transferred


to MGC: through the lipid bilayer of the membrane or through the cytoskeleton
and which of these mechanisms prevail. Several groups reported that MGCs are
activated by the stretch of the lipid bilayer, while other papers are focused in the role
of the cytoskeleton in MGCs activation. Some authors brought up the role of extra-
cellular matrix, although the forces distribution in the extracellular matrix remain
unknown. In addition to those issues we would like to add another one the possibil-
ity of MGCs modulation by intracellular second messengers and/or pharmacological
compounds. We have already mentioned that in our experiments stretch or several
pharmacological compounds, besides changing current through cation-nonselective
MGCs, also change mechanosensitive currents carried by K+ ions through both
TRPC6 and TREK which are mechanosensitive, together with inwardly rectifying
K+ -current IK1 (possibly through Kir2.3). Especially hard to explain is the appear-
ance of Ins in absence of cellular stretch. Regarding nitric oxide, it is possible that
S-nitrosylation of reactive thiol groups can affect the activity of MGCs.
In general, different studies report that besides direct activation of MGCs by
mechanical forces (via cytoskeleton or bilayer) fast indirect modulation of MGCs by
pharmacological compounds is also possible. Moreover some of those compounds
are capable of activating MGCs in the absence of actual cellular stretch, while oth-
ers inactivate MGCs despite continuous presence of cellular stretch. If this is true
for compounds other then nitric oxide, this provides an excellent opportunity for
development of new drugs for treatment of mechano-induced arrhythmias as well.
Acknowledgments This work was supported by grants from RFBR (09-04-01277a), DFG (Tr
02-A3) and a travel grant from the Humboldt-University (Berlin, Germany). VK, AK and IK
thank Prof. G. Isenberg and Prof. P. Persson for providing the opportunity to perform some of
experiments and general support of this work.

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Chapter 6
Role of Signaling Pathways in the Myocardial
Response to Biomechanical Stress and in
Mechanotransduction in the Heart

Danny Guo, Zamaneh Kassiri, and Gavin Y. Oudit

Abstract The heart is a mechanosensitive organ that adapts its morphology to


changing hemodynamic conditions via a process named mechanotransduction,
which is the primary means of detecting mechanical stress in the extracellular envi-
ronment. In the heart, mechanical signals are propagated into the intracellular space
primarily via integrin-linked complexes, and are subsequently transmitted from cell
to cell via paracrine signaling. The biochemical signals derived from mechani-
cal stimuli activate both acute phosphorylation of signaling cascades, such as in
the PI3K, FAK, and ILK pathways, and long-term morphological modifications
via intracellular cytoskeletal reorganization and extracellular matrix remodelling.
Mechanotransduction plays a fundamental role in cardiac (and vascular) function
and involves interaction between extracellular matrix and intracellular cytoskele-
tal proteins via cell adhesion complexes, which are modulated by PI3Ks. Loss of
PI3K signaling enhances the susceptibility to biomechanical stress while the loss of
its negative regulator, PTEN, is associated with a wide variety of adaptive mech-
anisms necessary to resist the progression of maladaptive ventricular remodelling
and heart failure. In this chapter, we discuss several of the key players involved in
mechanotransduction in the heart.

Keywords Mechanotransduction ECM Integrin ILK FAK Dystrophin


PI3K PTEN Cadherin Remodelling

G.Y. Oudit (B)


Division of Cardiology, Department of Medicine, Mazankowski Alberta Heart Institute, University
of Alberta, Edmonton, AB T6G 2S2, Canada
e-mail: gavin.oudit@ualberta.ca

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 141


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_6,

C Springer Science+Business Media B.V. 2011
142 D. Guo et al.

6.1 Introduction
Biomechanical signaling involves complex interactions between intracellular and
extracellular components. While whole organisms respond to external stimuli,
organs, tissues, and cells respond to mechanical signals from the extracellular space.
Common cellular responses to such signals include alterations in morphology,
intracellular structure, and extracellular structure. This extracellular transmission
of mechanical force followed by the subsequent intracellular conversion into bio-
chemical signals is termed mechanotransduction (Fig. 6.1). This process describes
the dynamic process of a mutually dependent relationship between cells and their
surrounding extracellular matrix (ECM). Responding to mechanical stress allows
cells to acclimate and adapt to changing environments. However, the benefits from
this intricate process are reversed if the carefully balanced equilibrium is tilted,
often leading to abnormal signaling and pathophysiological remodelling of cells
and tissues.
By detecting and responding to hemodynamic changes in the environment via
cell-ECM interactions, the heart develops compensatory responses utilizing intracel-
lular signaling cascades to maintain adequate function (Frey et al., 2004; Heineke
and Molkentin, 2006; Ruwhof and van der Laarse, 2000). Even at rest, the heart
is constantly exposed to much biomechanical and biochemical signals, causing it
to continuously adapt various aspects of its morphology such as the infrastructure
of its cytoskeleton as well as the composition of cell-ECM adhesion complexes.
This chapter will discuss several sub-cellular processes and signaling pathways
involved in mechanotransduction and the interactions between cardiomyocytes and
fibroblasts with the ECM.

Fig. 6.1 A conceptual framework of the key players involved in the detection of and the translation
of mechanical signals in the heart
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 143

6.2 Cell-ECM Adhesion


The ECM is like a framework of springs maintaining cells in a state of physi-
cal equilibrium, which, if deformed, mechanically stimulates the cells (Sussman
et al., 2002). The structural component of the ECM is a complex lattice of macro-
molecular proteins such as fibronectin and collagen; these proteins facilitate even
distribution of exogenous mechanical signals to the recipient cells (Berrier and
Yamada, 2007). Cellular attachment to the ECM via adhesion complexes such as
integrin and dystrophin-glycoprotein complexes warrants effective transmission of
extracellular mechanical signals into intracellular domains, stimulating changes in
cellular processes such as the cell cycle (Berrier and Yamada, 2007; Sussman et al.,
2002). Depending on the type and the mode of stimulation, downstream signals may
vary from mediating acute responses, such as the phosphorylation and activation of
signaling cascades and the generation of second messengers, to long-term cellular
modifications, such as changes in gene expression, initiation of compensatory path-
ways, and/or modification of intracellular and extracellular structural compositions.
For instance, cyclic and static stretch, elongation and compression, and unilateral
and bilateral strain all generate unique changes in transcriptional profile and total
gene expression in cardiac fibroblasts (Lee et al., 1999; Simpson et al., 1999).
Similarly, in vitro studies of cardiomyocytes cultured on deformable membranes
suggest that different magnitudes and directional orientations of stretch and com-
pression yield distinct intracellular effects (Kumar et al., 2002; Ruwhof et al., 2000).
Mechanical stimulation of one cell can spread to its neighbouring cells via paracrine
release of chemical messengers such as angiotensin 2 (Ang II), endothelin-1 (ET-1),
and transforming growth factor (TGF- ) (van Wamel et al., 2001) (discussed in
Section 6.3.2).
In order for cardiac cells to maintain the intricate cell-ECM interactions neces-
sary for mechanotransduction, sensitive adhesion complexes are required. Though
more than 50 proteins have been reported to be associated with these complexes
(Zamir and Geiger, 2001), cell-ECM adhesions in cardiomyocytes can be catego-
rized into two major groups: integrin-linked and dystrophin-glycoprotein complexes
(Fig. 6.2).

6.2.1 Cell-ECM Adhesion: Integrin-Linked Complexes

An increased pressure and/or volume hemodynamic load on the heart typically


results in the development of pathological ventricular hypertrophy and systolic and
diastolic dysfunction. One of the key players in mediating this response is the
integrin-linked complex, which contains a heterodimeric pair of integrins ( and )
that spans across the cytoplasmic membrane, binding to structural proteins in the
ECM, such as collagen, laminin, and fibronectin (Fig. 6.2) (Berrier and Yamada,
2007; Carver et al., 1994; Hynes, 1992; Schwartz et al., 1995). Integrin-linked
complexes also bind to cytoskeletal actin and sarcomeric components primarily
144 D. Guo et al.

Fig. 6.2 A schematic of integrin based complexes and their associated signaling pathways.
Integrin complexes linked to the Z-disc are called costameres. The ones directly linked to actin
are called focal adhesions

through the -subunits intracellular domain (Calderwood et al., 2000; Schwartz


et al., 1995). This bridging of intracellular and extracellular structural polymers
allows integrins to propagate bidirectional biomechanical communication between
cytoskeletal components and the ECM (Hynes, 2002).
Mice with cardiomyocyte specific ablation of 1-integrin have reduced cardiac
function, intolerance to increased hemodynamic workload, and gradual develop-
ment of heart failure with age (Shai et al., 2002) while mice with overexpression of
1-integrin develop an augmented hypertrophic response to mechanical stress (Ross
et al., 1998). In concordance, studies involving transgenic and adenoviral models
of 1-integrin both show that integrins downstream signaling cascades directly
promote growth, proliferation, and survival (Ieda et al., 2009; Ross et al., 1998)
implicating that 1-integrin is critical to and is directly responsible for inducing
cardiac hypertrophy.
Integrin-linked complexes are divided into costameres and focal adhesions in
cardiomyocytes (Fig. 6.2), both of which form in response to stretch (Sharp et al.,
1997). Structurally, these two categories only differ in the proteins clustered at their
intracellular domains and their intracellular anchoring points. While costameres
connect to sarcomeres via Z-disc proteins, focal adhesion complexes associate
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 145

with cytoskeletal actin filaments (Ervasti, 2003; Laser et al., 2000; Samarel, 2005;
Sharp et al., 1997). To some degree, both types of integrin-linked complexes share
overlapping signaling cascades with maintaining some key differences in signaling.
Integrins moderate cellular behaviour partially through physically bridging the
ECM to intracellular components. Nevertheless, the formation of mutli-protein com-
plexes at their intracellular domains is necessary for mechanotransduction (Berrier
and Yamada, 2007; Hynes, 2002). Because integrins do not exhibit inherent enzy-
matic activity, they must recruit signaling proteins to their cytoplasmic tails to
convert mechanical stimuli into biochemical signals (Berrier and Yamada, 2007).
In general, the proteins recruited to integrin-linked complexes are categorized into
3 major groups (Berrier and Yamada, 2007). The first group is integrin regulating
proteins such as talin, which bind to integrins and regulate their signaling and activ-
ity (Anastasi et al., 2009; Calderwood, 2004; Chen et al., 1995; Ulmer et al., 2003).
The second group is adaptor proteins, which act as scaffolds and link integrins to
structural and enzymatic proteins (Berrier and Yamada, 2007). An example is pax-
illin, a -integrin binding protein that recruits enzymes to integrin-linked complexes
(Schlaepfer et al., 1999; Tachibana et al., 1995). Another example is vinculin, which
in response to mechanical stimulation relocates to costameres and connect integrin-
linked complexes to cytoskeletal actin (Sharp et al., 1997; Wood et al., 1994). The
third group is enzymatic proteins such as focal adhesion kinase (FAK), integrin-
linked kinase (ILK) and phosphoinositide-3-kinase (PI3K) and which can initiate
a broad spectrum of chemical signaling cascades (Berrier and Yamada, 2007; Ross
et al., 1998).

6.2.1.1 Cell-ECM Adhesion: Focal Adhesion Kinase


Focal adhesion kinase (FAK), a non-receptor tyrosine kinase (Peng et al., 2008),
can be associated with focal adhesion complexes but can also localize at costameres
depending on the stimulus (Fig. 6.2) (Sharp et al., 1997; Torsoni et al., 2003). In
response to stretch and compression, FAK is recruited to integrin-linked complexes
via paxillin (Domingos et al., 2002; Tachibana et al., 1995), strategically allowing
FAK to receive mechanical signals directly; moreover, multiple FAKs can cluster
at these complexes in a number proportional to the strength of stimulation, sus-
taining and enhancing downstream signals accordingly (Katz et al., 2002; Torsoni
et al., 2003). However, it is important to note that FAK translocates differentially in
response to mechanical and chemical stimuli. In stretched cardiomyocytes in vitro,
FAK associates with Src adaptor proteins at costameres and autophosphorylates at
Tyr-397 (Laser et al., 2000; Torsoni et al., 2003). Src subsequently phosphorylates
FAK at Tyr-576 and Tyr-577, activating its kinase activity. The disruption of the
FAK-Src complex via Src inhibitors prevents FAK from binding to costameres lead-
ing to the loss of FAK mediated signaling (Domingos et al., 2002; Torsoni et al.,
2003). One of the many roles of the FAK-Src complex in cardiomyocytes is to
mediate stretched induced up-regulation of gap junctions (Yamada et al., 2005).
Stretching cardiomyocytes causes the release of VEGF in a FAK dependent manner,
increasingly ERK phosphorylation, an effect which is prevented via neutralization
146 D. Guo et al.

of VEGF suggesting that VEGF increases ERK phosphorylation via paracrine sig-
naling (Seko et al., 1999). The FAK mediated release of VEGF also up-regulates
the gap junction protein connexin-43 in neighbouring cells via paracrine signal-
ing (Li et al., 1997; Yamada et al., 2005). Increased expression of connexin-43
improves overall electrical signaling in the heart (Yamada et al., 2005). FAK-Src can
also up-regulate the expression of cell-cell adhesion proteins, such as N-cadherin,
desmoplakin, and plakoglobin (Yamada et al., 2005). Because stretch induced local-
ization of FAK to integrin-linked complexes increases with the magnitude and the
duration of stretch (Katz et al., 2002; Simpson et al., 1999), up-regulation of the
aforementioned cell-cell adhesion proteins in cardiomyocytes is likely proportional
to increases in work load. Cardiomyocyte adhesion to fibronectin stimulates a higher
level of N-cadherin and connexin-43 expression than adhesion to collagen suggest-
ing that matrix composition may alter focal adhesion binding and signaling (Shanker
et al., 2005). Together, these demonstrate that mechanical stimulation of FAK is
involved in the maintenance of both electrical and mechanical connectivity in the
heart.
Contrary to mechanical stimulation, chemical stimulation in cardiomyocytes
recruits FAKs to focal adhesions instead (Fig. 6.2) (Torsoni et al., 2003). For
instance, application of Ang II stimulates FAK mediated hypertrophy (Salazar and
Rozengurt, 2001). In concordance, application of AT1 receptor antagonists impairs
Ang II stimulation of FAK signaling without inhibiting cyclic stretch stimulation
of FAK signaling (Torsoni et al., 2003). This bimodal activation of FAK is one
of many examples that demonstrate the specificity of mechanotransduction and its
associated signaling cascades. While costameric and focal adhesion FAKs are, on
the most part, distinct in their respective signaling cascades, it should be noted that
there are circumstances of overlap. For instance, studies have shown that FAK acti-
vates phosphoinositide-3-kinase (PI3K) and its subsequent effectors by binding to
PI3Ks p85 adaptor subunit regardless of FAKs location (Chen and Guan, 1994;
Thamilselvan et al., 2007). Similarly, when activated, both costameric and focal
adhesion FAKs can bind to the mitogen activated protein kinases, ERK, JNK, and
p38, via Grb2 and Sos (Schlaepfer et al., 1999; Torsoni et al., 2003).
Focal-adhesion kinase importance to cardiac development is illustrated by
the heart-specific FAK-deficient embryonic mice (Table 6.1). These mutant mice
develop right ventricular eccentric hypertrophy due to ineffective FAK mediated
stimulation of physiological hypertrophic programs, ultimately resulting in decom-
pensation and maladaptive right ventricular remodelling (Peng et al., 2008). On the
other hand, after birth, these mice gradually develop left ventricular eccentric hyper-
trophy overtime due to age (Peng et al., 2008). Similar pathologies are observed
acutely when these mice are treated with Ang II or pressure overload (Peng et al.,
2008). This shift from right to left ventricular eccentric hypertrophy is likely due to
differences in ventricular dependence and workload between embryonic and postna-
tal mice (Peng et al., 2008). This model implies that FAK signaling is necessary for
myocardial tolerance of mechanical stress, possibly through eliciting hypertrophy
via the regulation of various transcription factors such as NF-B (Crosara-Alberto
et al., 2009; Gupta et al., 2002).
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 147

Table 6.1 Mutations in various genes involved in mechanotransduction and their effects on
signalling

Gene mutation Phenotype Reference

1-integrin Whole body mutant: Embryonic lethality. Ross et al. (1998)


Cardiac specific mutant: Reduced cardiac Shai et al. (2002)
function. Intolerance to stress.
Overexpression leads to augmented
hypertrophic response.
FAK Whole body mutant: Embryonic lethality. Peng et al. (2008)
Cardiac specific mutant: Right ventricular
eccentric hypertrophy during embryonic
stage and left ventricular eccentric
hypertrophy postnatal.
ILK Whole body mutant: Embryonic lethality. White et al. (2006)
Cardiac specific mutant: Attenuation of Lu et al. (2006)
hypertrophic response. Weakened
FAK/PI3K signaling.
N-Cadherin Whole Body mutant: Embryonic lethality. Tran et al. (2002)
Cardiac Specific mutant: Reduce AKT
signaling.
Dystrophin Whole body knock out: Increased ERK1/2 Khairallah et al. (2007)
stimulation. Decreased AKT activation. Kumar et al. (2004)

6.2.1.2 Cell-ECM Adhesion: Integrin Linked Kinase


Integrin linked kinase (ILK) plays a key role in mediating hypertrophic programs at
both costameres and focal adhesion complexes (Fig. 6.2) (Li et al., 1999; Sakai
et al., 2003; White et al., 2006). When integrins in cardiomyocytes bind to the
ECM, ILKs are recruited to costameres via paxillin and are subsequently activated;
this allows ILKs to interact with the cytoplasmic domain of 1-integrin and phos-
phorylate several downstream targets such as Rac1, AKT, and GSK-3 (Hannigan
et al., 2005; Li et al., 1999; Lu et al., 2006; Qian et al., 2005; Ross et al., 1998),
which have all been associated with mechanically stimulated cardiomyocyte hyper-
trophy (DeBosch et al., 2006; Matsuda et al., 2008; Satoh et al., 2006; Sugden,
2003).
In addition to its enzymatic roles, ILKs also act as scaffolding proteins by form-
ing multi-protein complexes at focal adhesions (Hannigan et al., 2005, 2007). In
cardiomyocytes, ILK is recruited to focal adhesions via binding to PINCH1 (Bock-
Marquette et al., 2004; Chen et al., 2005; Li et al., 1999), which if inhibited, leads
to reduced phosphorylation of AKT (Delcommenne et al., 1998; White et al., 2006)
and GSK-3 (Delcommenne et al., 1998) implying that the formation of the ILK-
PINCH1 complex may be necessary for ILK signaling. On the other hand, one study
suggested that PINCH1 is only necessary during embryonic development and is
dispensable in postnatal cardiomyocytes (Liang et al., 2005).
Integrin-linked kinase interaction with two regulatory proteins, -parvin and
-parvin, can further illustrate its role as a scaffolding protein (Chen et al., 2005;
148 D. Guo et al.

Tu et al., 2001). The mutually exclusive binding of -parvin and -parvin respec-
tively increases and reduces ILKs prohypertrophic activity (Hannigan et al., 2005).
Interestingly, formation of either -parvin-ILK or -parvin-ILK complexes have
been suggested to increase cell-ECM adhesion complexes (Hannigan et al., 2005;
Mongroo et al., 2004; Tu et al., 2001; Yamaji et al., 2001) demonstrating ILKs
importance as a scaffold in mediating the formation of integrin-linked complexes.
This also puts forth the idea that the enzymatic and scaffolding functions of ILK
may be independent of each other (Hannigan et al., 2005).
Structural analysis of ILK reveals a highly conserved Pleckstrin homology
domain, which activates ILK by binding to phosphoinositide-3,4,5-phosphate (PIP3 )
(Delcommenne et al., 1998), the primary product of PI3K activity (Vanhaesebroeck
et al., 1997) positing that activation of PI3K can subsequently activate ILK. In
addition, inactivation of PTEN, which is a lipid phosphatase that dephosphorylates
PIP3 into PIP2 (Oudit and Penninger, 2009; Oudit et al., 2004; Sun et al., 1999a),
results in constitutive activation of ILK (Edwards et al., 2005). Previous studies
have also shown that both enhancing PI3K activity and impairing PTEN activity
can enhance intracellular PIP3 levels, leading to cardiac hypertrophy (Crackower
et al., 2002; Shioi et al., 2000; Sun et al., 1999a). Together, these suggest that
PI3K activity can directly modulate ILK activity. Finally, ILK also directly phos-
phorylates AKT and GSK-3 (Troussard et al., 1999), two well studied downstream
effectors of FAK/PI3K signaling, supporting the notion that ILK may contribute to
the myocardial compensatory response to mechanical stress.
Since PI3K is involved in mediating acute responses to both chemical stimuli
via RTK and GPCR receptors (Oudit and Penninger, 2009; Oudit et al., 2004) and
mechanical stimuli via FAK (Chen and Guan, 1994), it is possible that ILK may also
be activated by chemical agonist stimulation. For instance, the application of Ang
II induces concentric hypertrophy in wildtype mice (Sadoshima and Izumo, 1993;
Sadoshima et al., 1993), an effect that is mitigated in ILK kinase dead mutants (Lu
et al., 2006). This partial attenuation of hypertrophy in the mutant strain suggests
that ILK amplifies, but is not necessary for, PI3K signaling. On the other hand,
one study suggested that ILK activation may be entirely PI3K dependent, as phar-
macological inhibitors of PI3K fully inhibited mechanically induced ILK activity
(Khwaja et al., 1997). However, an in vivo study stated that during maladaptive
cardiac hypertrophy, increased ILK association with Rac1 was observed without
phosphorylation of the PI3K signaling cascade (Lu et al., 2006). The same study
also demonstrated that ILK kinase dead mutants are unable to develop ventricular
hypertrophy in response to mechanical stress (Lu et al., 2006). Similarly, another
study postulated that ILKs can directly respond to RTK stimulation irrespective of
PI3K, further implicating that ILK and PI3K pathways may be independent (Knoll
et al., 2007). Though the results from these studies appear to conflict, their discrep-
ancies could be attributed to differences in the hearts condition; while ILK activity
may be PI3K independent during pathological states, ILK is likely PI3K dependent
during physiological conditions.
As one might expect, ILK is necessary for survival as mice with cardiomyocyte-
specific ablation of ILK develop heart failure and sudden death within 6 weeks
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 149

of age and develop spontaneous dilated cardiomyopathy (DCM) without the need
for a secondary stressor such as pressure overload or Ang II (Table 6.1) (White
et al., 2006). Similarly, a single mutation in the human ILK gene (A262V) has
been correlated with contractile dysfunction, ventricular dysmorphism, and DCM
(Knoll et al., 2007). As mice with cardiomyocyte specific ablation of PINCH1 do
not deviate from the wildtype phenotype (Liang et al., 2005), it is likely that ILK
has other physiological targets of interaction in cardiomyocytes allowing it to sus-
tain its signaling even in the absence of PINCH1. Interestingly, mice lacking ILK
have impaired FAK signaling indicating that ILK either contributes to FAK activa-
tion or mediates a part of FAKs downstream signaling (White et al., 2006). One
possible mechanism is that FAK indirectly stimulates ILK activity via PI3K/PIP3 ,
which is not impossible due to their close spatial proximity at integrin-linked com-
plexes (Chen and Guan, 1994; Thamilselvan et al., 2007). This is further supported
by FAK and ILKs overlapping downstream signaling (Antos et al., 2002; Bueno
and Molkentin, 2002; Delcommenne et al., 1998; Shioi et al., 2002; Troussard et al.,
1999).

6.2.2 Cell-ECM Adhesion: Dystrophin-Glycoprotein Complex

The dystrophin-glycoprotein complex (DGC) is another major class of actin-linked


adhesion complex (Hanft et al., 2006; Rybakova et al., 2000). DGCs are only briefly
covered in this report but its role in disease and cardiac physiology and disease have
previously been discussed elsewhere (Danialou et al., 2001; Ervasti, 2007; Lapidos
et al., 2004; Rybakova et al., 2000). DGCs were traditionally regarded as being only
involved in mechanical linkage, but recent studies illustrate that the loss of DGCs
results in altered protein expression and cellular contractile dysfunction (Danialou
et al., 2001; Hanft et al., 2006; Lapidos et al., 2004). Specifically, mutants lacking
dystrophins develop various diseases such as Duchenne muscular dystrophy, Becker
muscular dystrophy, and X-linked DCM, all of which are accompanied with the
development of cardiomyopathy (Lapidos et al., 2004).
One well studied DGC mutant model is the mdx mouse, which has a point muta-
tion in the dystrophin gene (Quinlan et al., 2004). These mice show no signs of
cardiomyopathy at 1012 weeks of age, but inevitably develop DCM and myocar-
dial contractile dysfunction at 40 weeks of age (Table 6.1) (Quinlan et al., 2004;
Quinlivan et al., 1996). It should be noted that though these mice do not have car-
diac dysfunction at 1012 weeks, they are less tolerant of hemodynamic stress and
are more susceptible to work load induced myocardial injury and adverse ventricular
remodelling (Danialou et al., 2001).
Though DGCs primarily modulate cell-ECM adhesion, its role in coordinating
intracellular chemical signaling can also be illustrated in mdx mice, which have
increased ERK1/2 activation both at rest and in response to mechanical stress
as compared to wildtypes (Kumar et al., 2004). On the other hand, a metabolic
study demonstrated that mdx hearts have a shift in basal metabolic substrate
dependence from fatty acids to carbohydrates (Khairallah et al., 2007) suggesting
150 D. Guo et al.

that a weakened energy supply may be responsible for its intolerance to stress.
However, since the overall cellular AMP to ATP ratios in the mdx cardiomyocytes
are not different from wildtypes (Khairallah et al., 2007), it is possible that mdx
cardiomyocytes either utilize a less efficient pattern of ATP allocation to sustain
normal contractile function or require more ATP to sustain physiological conditions
resulting in energy deficiency.
It is interesting to note that mdx hearts have decreased activation of AKT in vivo
(Khairallah et al., 2007) indicating that DGCs, like integrins, may associate with the
PI3K signaling cascade. Consistent with the mdx phenotype, AKT has been shown
to be necessary for physiological growth and cellular survival in the heart (DeBosch
et al., 2006). Moreover, since DGCs are located closely to integrin-linked complexes
within the cell (Rybakova et al., 2000), they could very likely interact with ILKs or
FAKs, posing a potential cause for the decreased activation of AKT observed in mdx
mice.

6.3 PI3K and PTEN

6.3.1 PI3K

PI3K, as previously described, is a highly conserved lipid kinase and an important


mediator of acute cellular responses to mechanical and chemical stimuli (Figs. 6.2
and 6.3) (Shioi et al., 2000; Oudit and Penninger, 2009; Oudit et al., 2004).The
PI3K signaling cascade is both directly and indirectly involved in mechanically
stimulated pathways. The direct mode of PI3K activation is at integrin complexes
via interactions with enzymes such as FAK and ILK (refer to Sections 6.2.1.1 and
6.2.1.2.). On the other hand, PI3K is indirectly activated during mechanical stim-
ulation via autocrine and paracrine signaling (refer to Section 6.4.2). The primary
PI3K isoforms expressed in the heart are PI3K and PI3K, which bind to recep-
tor tyrosine kinases (RTKs) and are activated by growth factors, and PI3K, which
binds to GPCRs and are activated by, among others, Ang II stimulation (Oudit and
Penninger, 2009). Though PI3K has traditionally been associated with RTK sig-
naling, later studies have given controversy to this belief revealing that PI3K can
be stimulated by binding to G-proteins (Kurosu et al., 1997; Maier et al., 1999; Yart
et al., 2002).
The generation of PI3K knockout mice was unsuccessful as PI3K is necessary
for embryonic development (Shioi et al., 2000). Thus, studies made use of PI3K-
DN instead, which overexpress a dominant but inactive form of PI3K in their
cardiomyocytes after birth (Shioi et al., 2000). PI3K-DN hearts are smaller than
wildtypes but do not display any signs of ventricular dysfunction under resting con-
ditions. Furthermore, unlike wildtypes, PI3K-DN hearts do not hypertrophy when
exercised, suggesting that PI3K is necessary for cellular growth and mechanosen-
sitivity to physiological stress (McMullen et al., 2003). Moreover, PI3K may also
protect the heart against pathological stress as PI3K-DN hearts rapidly develop
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 151

Fig. 6.3 The PI3K signaling cascade. PI3K signals through phosphatidylinositol-3,4,5-
triphosphate (PIP3 )/Akt pathway and stimulates growth, proliferation, and survival. RTK = rece-
ptor tyrosine kinase, GPCR = G-protein coupled receptor

DCM in response to pressure overload (McMullen et al., 2007). PI3Ks cardio-


protective properties are also observed in mutants that overexpress PI3K, which
are protected from PKC induced pathological hypertrophy (Bowman et al., 1997;
Rigor et al., 2009). PI3Ks effects on the heart are perhaps due to changes
atrial natriuretic peptide (ANP) expression, which increases in PI3K-DN mutants
exposed to pressure overload but not exercise (McMullen et al., 2003). On the other
hand, in exercised but not pressure overloaded wildtype mice, brain natriuretic
peptide (BNP) expression increases (McMullen et al., 2003), which can attenuate
ERK activation and pathological hypertrophy (Takahashi et al., 2003). Together
these observations denote that PI3K regulates physiological and pathological
hypertrophy.
Similarly to PI3K-DN, PI3K-KO mutant hearts are also more susceptible to
pathological stress as they rapidly develop DCM and ventricular lesions within
1 week after aortic banding (Patrucco et al., 2004). However, the cardiomyopathies
observed in these two mutant strains likely arise from distinct cellular patholo-
gies due to isoform specific functional differences. PI3K-KO, but not PI3K-DN,
mutants develop an increase in intracellular levels of cAMP due to reduced PDE
activity(Crackower et al., 2002; Patrucco et al., 2004). Increases in cAMP have
been shown to up-regulate expression of matrix metalloproteinase (Melnikova
et al., 2006), proteolytic enzymes of that target the ECM and cell-cell adhesion
complexes (Covington et al., 2006; Dwivedi et al., 2009), illustrating a potential
152 D. Guo et al.

mode of maladaptive ECM remodelling and a likely cause of heart failure in pres-
sure overloaded PI3K-KO hearts. Despite their redundant kinase activity, PI3K
isoforms demonstrate unique effects on cardiac hypertrophy. For instance, while
PI3K activity was shown to mitigate maladaptive hypertrophy (McMullen et al.,
2007), PI3K activity may exacerbate it. Studies of PI3K-KO and PI3K-DN
cardiomyocytes demonstrated that PI3K and PI3K respectively activate and
inhibit ERK (McMullen et al., 2007; Patrucco et al., 2004), whose activation has
been associated with pathological hypertrophy (Ainscough et al., 2009; Lorenz
et al., 2009).

6.3.2 PTEN

PTEN is a lipid phosphatase which dephosphorylates PIP3 into PIP2 (Stambolic


et al., 1998), and hence is a negative regulator of PI3K activity (Fig. 6.3) (Oudit
and Penninger, 2009). PIP3 is the primary catalytic product of PI3K which activates
AKT, stimulating physiological growth (Vanhaesebroeck et al., 1997). AKT signal-
ing also protects the heart from pathological hypertrophy (Rigor et al., 2009). The
PTEN null and caPI3K mutants, which have excessive PI3K activities, are resis-
tant against pressure overload induced heart failure (McMullen et al., 2007; Oudit
et al., 2008). Though PTEN-KOs are resistant to pressure overload, their maladap-
tive response to GPCR agonists worsens (Oudit et al., 2008) suggesting that loss of
PTEN is specifically protected against biomechanical stress. Indeed, PTEN is a key
regulator of cell-matrix adhesion and migration (Gu et al., 1999, 1998; Larsen et al.,
2003; Oudit et al., 2004) and PTEN regulate several distinct pathways involved
in remodeling of cell adhesion complexes and cytoskeletal organization (Gu et al.,
1999, 1998; Larsen et al., 2003; Oudit et al., 2004). For example, PTEN directly
dephosphorylates Shc and FAK which in turn modulates cell adhesion complexes
and the intracellular actin cytoskeleton (Gu et al., 1999, 1998; Larsen et al., 2003),
independent of AKT/PKB activity.

6.4 Intercellular Propagation of Mechanical Signals

6.4.1 Cell-Cell Adhesion

Intercellular transmission of mechanical signals is propagated in two distinct mech-


anisms: by directly stretching and straining neighbouring cells, and via paracrine
signaling. The direct transmission of force between adjacent cardiomyocytes is
propagated by cell-cell adhesion complexes called adherens junctions, which are
composed of several linkage proteins, with the traditional components being cad-
herins (with N-cadherin being the primary isoform in the heart) and catenins
( and ) (Fig. 6.4a). Though it is often assumed that like many striated mus-
cles, cardiomyocytes only experience mechanical stress along its longitudinal axis
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 153

Fig. 6.4 A schematic of adherens junctions (a) and gelsolin activity (b). The intracellular domain
of the transmembrane protein N-cadherin is anchored to actin filaments by - and -catenin while
gelsolin cleaves actins and prevents repolymerization by capping the actin filament
154 D. Guo et al.

via intercalated discs, it seems unlikely that cardiomyocytes do not experience lat-
eral strain from the surrounding ECM or from neighbouring cells, or exert lateral
force during systole (Schoenberg, 1980). In fact, a study elegantly demonstrated that
roughly 80% of N-cadherin junctions between adjacent cardiomyocytes are located
in the peripheral (lateral) membrane (Pedrotty et al., 2008). Furthermore, these lat-
eral junctions allow cardiomyocytes to adhere to other cell types as intercalated
discs are only formed between cardiomyocytes.
Aside from propagating mechanical force, adherens junctions have also been
postulated to interact with PI3K to mediate AKT signaling (Tran et al., 2002).
Increased and decreased adherens junctions were shown to respectively improve
and weaken basal AKT phosphorylation (Tran et al., 2002). However, it is possible
that this loss of AKT stimulation could be indirectly due to weakened mechanosen-
sitivity as there is still little evidence of direct interactions between cadherins and
the PI3K pathway.

6.4.2 Autocrine and Paracrine Signaling

While the conversion of mechanical signals into chemical signals occurs in the
intracellular compartment, the spreading of this chemical signal from cell to cell
occurs through autocrine and paracrine signaling; hence, these processes play key
roles in stretch induced cardiac hypertrophy (Cingolani et al., 2001; van Wamel
et al., 2001). For instance, Ang II, ET-1, and TGF- have all been implicated in
mediating cardiac hypertrophy in hemodynamic overload models (Arai et al., 1995;
Ishiye et al., 1995; Ito et al., 1994; Ruzicka et al., 1995; Takahashi et al., 1994). In
response pressure overload, the heart increases expression of both endogenous ET-1
and ET-1 receptors appropriating ventricular hypertrophy (Arai et al., 1995), which
can be attenuated by applying ET-1 receptor blockers (Ito et al., 1994). Similarly, in
response to volume overload, the heart develops ventricular hypertrophy as a result
of increased Ang II levels (Ruzicka et al., 1995), but can be reversed via pharma-
cological inhibition of Ang II receptors (Ishiye et al., 1995). TGF- is primarily
expressed by non-cardiomyocytes during basal condition, but in response to pres-
sure overload, cardiomyocytes surprisingly become the primarily secretor of TGF-
(Takahashi et al., 1994). TGF- paracrine signaling also stimulates excess colla-
gen deposition by fibroblasts leading to fibrosis and stiffness of the myocardium
(Eghbali et al., 1991).
Cardiac hypertrophy following mechanically induced paracrine signaling has
also been demonstrated in vitro. Cultured cardiomyocytes exposed to stretch release
ET-1 and TGF-, which in turn causes an increased expression of the hypertrophy
marker ANP (van Wamel et al., 2001). Whether cardiomyocytes express ET-1 in
the absence of stretch appears to be under debate (Suzuki et al., 1993; van Wamel
et al., 2001), but in either case, the level of ET-1 excretion in the absence of stretch
is likely insufficient for stimulating the expression of hypertrophic markers (van
Wamel et al., 2001). Similarly, studies investigating whether Ang II is expressed
by acutely stretched cardiomyocytes have yielded mixed results (Sadoshima et al.,
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 155

1993; van Wamel et al., 2001; Yamazaki et al., 1999). Nevertheless, multiple studies
have confirmed that endogenous secretion of Ang II mediates, but is not neces-
sary for inducing, hypertrophy in stretched cardiomyocytes (Sadoshima and Izumo,
1993; Sadoshima et al., 1993; van Wamel et al., 2001; Yamazaki et al., 1999).
Furthermore, it should be noted that Ang II mediated hypertrophy occurs partially
via increased production and excretion of ET-1 (Cingolani et al., 2001).
The release of Ang II by stretched cardiac fibroblasts can also induce hypertrophy
in unstretched cardiomyocytes (van Wamel et al., 2001, 2000). Similarly, stretched
cardiac fibroblasts also produce TGF-, altering gene expression in neighbouring
cardiomyocytes (van Wamel et al., 2001). Recently, a co-culture study demonstrated
that paracrine signaling from cardiac fibroblasts can induce cardiomyocyte prolif-
eration (Ieda et al., 2009). Together, these in vitro and in vivo studies describe the
prominent potential of intercellular dependence and regulation between cardiomy-
ocytes and cardiac fibroblasts in mechanosensitivity. An array of other factors, such
as ANP, is also excreted to mediate cellular changes in response to mechanical stress
(described in Sections 6.3.1 and 6.3.2) (Ruwhof et al., 2000; van Wamel et al., 2001,
2000). Hence, given the current knowledge of autocrine and paracrine effectors in
mechanically stimulated pathways in the heart, it is clear that diffusible chemical
factors are crucial in mediating mechanotransduction.

6.5 Myocardial Remodelling

6.5.1 Extracellular Remodelling

The loss of cell-ECM linkage proteins in the heart almost always leads to cardiac
dysfunction due to the inability to tolerate stressful conditions. Similar results are
observed from the loss of signaling proteins such as FAK and ILK. As previously
discussed, the inability to detect and to respond to mechanical stress can incapacitate
the hearts ability to initiate proper compensatory mechanisms ultimately leading
to heart failure. Increased mechanical work load and hemodynamic stress is often
closely correlated with concomitant intracellular and extracellular remodelling. In
the heart, like in many other organs and tissues, extracellular remodelling is primar-
ily regulated by MMPs (Kassiri and Khokha, 2005; Spinale, 2007). MMPs are a
family of potent proteolytic enzymes that digest, among others, collagen, laminin
and fibronectin and are inhibited by interactions with tissue inhibitors of metallo-
proteinases (TIMPs) (Kassiri and Khokha, 2005). Indeed, loss of TIMP3 leads to
an exacerbation of pressure-overload induced adverse myocardial remodelling and
heart failure (Kassiri et al., 2005).
Several mechanically induced signaling pathways, specifically PI3K pathways,
stimulate MMP expression and activation (Grote et al., 2003; Hess et al., 2003;
Ispanovic and Haas, 2006; Zahradka et al., 2004). For instance, stretch induces
NAD(P)H mediated formation of reactive oxygen species (ROS), increasing expres-
sion and activation of pro-MMP2 and the release of active MMP2 (Grote et al.,
156 D. Guo et al.

2003). As this suggests, chronic exposure to stressful hemodynamic conditions


may lead to over-expression and over-activation of MMPs resulting in excess ECM
degradation. In turn, this could reduce the efficiency and effectiveness of cell-ECM
adhesions making the heart unable to respond to subsequent increases in biome-
chanical stress and increasing the susceptibility to the development of a dilated
cardiomyopathy and heart failure.
Aside from their influence on the ECM, several MMP isoforms also cleave the
extracellular portion of cadherins (Ito et al., 1999; Uglow et al., 2003), proteins that
make up cell-cell adhesion complexes such as adherens junctions and desmosomes
(Stokes, 2007; Zuppinger et al., 2000). In addition to the reduction of cadherin-based
intercellular junctions, this process also releases -catenin, an intracellular compo-
nent of cadherin complexes which translocates to the nucleus to stimulate, among
others, cell proliferation and survival (Table 6.1) (Dwivedi et al., 2009; Uglow et al.,
2003). A recent study illustrated that -catenin shedding is necessary for post-
infarction adverse left ventricular remodelling (Zelarayan et al., 2008) implying
that excess or perhaps even normal level of mechanical stress post-infarction may
exacerbate ventricular damage through differential gene expression and imbalanced
matrix remodelling.

6.5.2 Intracellular Remodelling

6.5.2.1 Actin
Intracellular remodelling of the actin cytoskeleton plays an important role in
mechanotransduction because many cell-cell and cell-ECM adhesion complexes
rely on coupling to cytoskeletal actin filaments to gain anchorage and to main-
tain location. Cytoskeletal actin regulation is mediated via a combination of
polymerization, depolymerisation, and modifications of actin crosslinking proteins
(Pollard, 2007; Vicente-Manzanares et al., 2005), maintaining actin structures in
a dynamic equilibrium. Cytoskeletal actin can also relay extracellular force to the
nucleus, altering its morphology (Maniotis et al., 1997). This immediate transmis-
sion of mechanical stimulus to the nucleus has been demonstrated to be capable
of directly inflicting changes in gene expression (Guilak et al., 2000), though this
hypothesis still remains to be elucidated. More details of the mechanical properties
of ECM-nuclear connectivity are described elsewhere (Dahl et al., 2008).
Disruption of actin polymerization prevents cell adhesion (Sanger and Holtzer,
1972), whereas cellular adhesion to matrix stimulates actin reorganization (DeMali
et al., 2003); albeit, the resulting actin infrastructure depends on the cells external
environment (DeMali et al., 2003; Hannigan et al., 2005). For instance, fibrob-
lasts in 2D cultures spread out and cover a large surface area while fibroblasts in
3D cultures have enhanced migration and adopt a star like shape (DeMali et al.,
2003) suggesting that environmental differences detected via cell-ECM adhesion
help direct migration and modulate morphology. Interestingly, the formation of focal
adhesions and the association of FAK at focal adhesions are much more prominent
in 3D cultures than 2D cultures (Berrier and Yamada, 2007; DeMali et al., 2003)
6 Role of Signaling Pathways in the Myocardial Response to Biomechanical Stress 157

suggesting restricted FAK signaling in 2D cultures. In addition to adhesion and


nuclear changes, stretch also stimulates changes in actin structure (Simpson et al.,
1999). For instance, pressure overload has been observed to induce changes in actin
isoform expression prior to ventricular decompensation (Berni et al., 2009). Also,
stretched cardiomyocytes induces reinforcement and thickening of actin filaments
and increase actin turnover in vitro via proteins such as zyxin and gelsolin (Liepina
et al., 2003; Simpson et al., 1999; Sun et al., 1999b; Yoshigi et al., 2005; Yu et al.,
1992) as discussed below.

6.5.2.2 Zyxin
Zyxins are subcellular proteins found at the cellular substratum attached to cell
adhesion complexes, such as focal adhesions, as well as to cytoskeletal actin
(Beckerle, 1998; Bershadsky et al., 2003; Hoffman et al., 2003). Zyxins molec-
ular structure reveals its role as both a scaffold for focal adhesion complexes as
well as a docking site for the ENA/VASP family of proteins (Hoffman et al., 2003;
Krause et al., 2003), which are abundantly found in intercalated discs of cardiomy-
ocytes (Brancaccio et al., 2006). Under resting conditions, zyxins are found bound to
vinculin and -actinin at focal adhesions close to Z-discs (Crawford and Beckerle,
1991; Yoshigi et al., 2005). Interestingly, unlike other integrin complex proteins
such as FAK, ILK, vinculin, or paxillin, (Li et al., 1999; Sawada and Sheetz, 2002;
Sharp et al., 1997; Torsoni et al., 2003), zyxins move away from integrin-linked
complexes toward actin filaments in response to stretch, causing realignment of
actin and thickening of actin filaments according to the direction of stretch (Yoshigi
et al., 2005). Interestingly, zyxin can localize between the nucleus and the cytoplas-
mic membrane and can thus potentially mediate gene regulation (Nix and Beckerle,
1997; Nix et al., 2001). For instance, exposure of cardiomyocytes to atrial natriuretic
peptide accumulates zyxin at the nucleus inducing anti-apoptotic effects (Kato et al.,
2005).
Zyxin is not necessary for survival as zyxin knockouts are both viable and fertile
(Hoffman et al., 2003), yet the lack of zyxin compromises the tolerance to mechani-
cal stimuli (Yoshigi et al., 2005). When stretched, zyxin mutant cells have impaired
thickening, but not reorganization of actin filaments, which can be rescued via the
addition of exogenous zyxin (Yoshigi et al., 2005). Reorganization of actin filaments
was likely not impaired due to compensation from other actin remodelling proteins
such as gelsolin.

6.5.2.3 Gelsolin
Gelsolin is a calcium dependent monomeric protein that regulates actin reorganiza-
tion by severing and capping actin filaments (Fig. 6.4b) (Liepina et al., 2003; Sun
et al., 1999b; Yu et al., 1992). Increases in intracellular calcium concentrations stim-
ulates gelsolin to sever actin, following by its binding to the truncated actin filament
acting as a cap to prevent subsequent regeneration and repolymerization (Sun et al.,
1999b). The removal of the gelsolin cap requires the binding of PIP2 (Liepina et al.,
2003; Yu et al., 1992). Cell adhesion causes gelsolin to co-localize with members
158 D. Guo et al.

of the integrin complex such as FAK, PI3K, c-Src, paxillin, vinculin, and talin sug-
gesting that mechanical stimulation recruits gelsolin to integrin-linked complexes
(Chellaiah et al., 2001).
Gelsolin is up-regulated in DCM, ventricular hypertrophy and heart failure (Yang
et al., 2000). Moreover, gelsolin null mice have better contractile function post
infarction compared to wildtypes (Li et al., 2009) suggesting that gelsolin activity
may be necessary in mediating ventricular damage and various forms of cardiomy-
opathy (Nishio and Matsumori, 2009). However, gelsolin activity has been argued
to be beneficial to the survival of cardiac cells as it can stimulate anti-apoptotic sig-
naling (Azuma et al., 2000; Koya et al., 2000). Considering gelsolins potency in
cardiac cells as well as other cell types and the central role of actin in both cell-
cell and cell-ECM adhesion complexes, Gelsolins role in cardiac remodelling and
mechanotransduction poses an important target for future investigation.

6.6 Conclusion and Perspectives


Mechanosensitive signaling pathways in cardiac cells orchestrate the dynamic
process of intracellular and extracellular cardiac remodelling, stimulating an abun-
dance of physiological processes such as hypertrophy. However, when exposed to
pathological stress, such as pressure- and/or volume-overload, the same signaling
pathways act adversely causing cardiomyopathy and heart disease. In conclusion,
mechanical signaling in the heart is a fundamental adaptation to biomechanical
stress and suggests that a shift of focus toward repairing damage in cell adhesion
complexes and restoring normal mechanotransduction may have therapeutic bene-
fits. Ultimately, the manipulation of these various signaling cascades may indeed
provide novel therapeutic avenues for the prevention and/or treatment of heart
failure.
Acknowledgements GYO is a Clinician-Investigator Scholar of the Alberta Heritage Foundation
for Medical Research and ZK is a New Investigator of the Heart and Stroke Foundation of Canada.
We acknowledge the financial support from the Canadian Institute for Health Research (GYO
grant 86602, ZK grant 84279), the heart and Stroke Foundation of Canada (GYO) and the Alberta
Heritage Foundation for Medical Research (GYO).

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Chapter 7
Atomistic Molecular Simulation of Gating
Modifier Venom Peptides Two Binding
Modes and Effects of Lipid Structure

Kazuhisa Nishizawa

Abstract GsMTx4, a gating-modifier peptide obtained from tarantula venom has


been a valuable tool for investigating the gating mechanisms of mechanosensitive
channels. GsMTx4 is thought to act at the channel/lipid interface by modifying
the structure of the surrounding lipid molecules. However, the atomistic details of
these actions are poorly understood. Here, the studies of GsMTx4 and related pep-
tide toxins that inhibit the voltage activation of various ion channels are reviewed,
with emphasis on the results of molecular dynamic (MD) simulation analyses. Free
energy profile analyses suggest that these toxins exhibit two modes of binding to
lipid membrane, namely, the shallow mode and the deep mode. These toxins favor
the deep mode, especially in membranes rich in saturated lipid acyl chains, which
make the headgroup layer tight. It is hypothesized that in the case of HaTx the
deep mode is the action mode, while for GsMTx4 the two modes can explain
the concentration-dependent (biphasic) effect of GsMTx4 that has recently been
reported. The possibility that such toxins seek out specific types of lipid molecules
is discussed. Simulation results support the view that the channel/GsMTx4 (or
HaTx)/lipids make a tertiary complex crucial to the effectiveness of the toxin and
therefore binding of the toxin to channels occurs only in the presence of lipid
molecules with appropriate structures.

Keywords GsMTx-4 MscS MscK Stretch-activated channels


Mechanosensitive channels Inhibitory cysteine knot peptides Spider
venom Molecular dynamics simulation HaTx

K. Nishizawa (B)
Department of Laboratory Medicine, Teikyo University School of Medical Technology, Kaga,
Itabashi, Tokyo 173-8605, Japan
e-mail: kazunet@med.teikyo-u.ac.jp

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 167


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_7,

C Springer Science+Business Media B.V. 2011
168 K. Nishizawa

7.1 Introduction
Since the identification and characterization of the tarantula toxin GsMTx4 by Sachs
and coworkers (Chen et al., 1996; Suchyna et al., 2000), this cationic hydropho-
bic polypeptide has been utilized as a specific blocker of stretch-activated cation
channels (SACs). As such, GsMTx4 has proven to be a valuable tool in studies
analyzing the involvement of SACs in various phenomena. The seminal papers by
Chen et al. (1996) and Suchyna et al. (2000) have reported a role for SACs in hypo-
tonic cell swelling-induced Ca2+ increase with the use of the venom from which
GsMTx4 was later isolated. More recently, several additional targets of GsMTx4,
including TRPC6 (Spassova et al., 2006) and MscK and MscS of E. coli have
been reported (Hurst et al., 2009). The number of studies showing pharmacolog-
ical potentials of GsMTx4 is also increasing (for review, Bowman et al., 2007). In
addition to such medical interests, GsMTx4 has drawn much interest from biophysi-
cists after the elucidation of a wide variety of target channels of different phyla of
organisms. GsMTx4 inhibits cationic SACs in a variety of vertebrate cell types such
as chick heart, rat astrocytes and skeletal muscle and human smooth muscle. How
does it modify the gating of such different channels with different kinetic proper-
ties and presumably different structures? It is likely that, after GsMTx4 adsorbs to
lipid bilayer membranes, it modifies their mechanical properties, thereby modifying
the energy from bilayer tension required for gating SACs. It is also likely that the
effects of GsMTx4 on membrane proteins are dependent on its partition into the
interface between channels and lipid molecules. However, structural and dynamical
features enabling these effects on various channels and motor molecules are not well
understood.
This review primarily focuses on recent studies using MD simulation analyses
and experimental results of GsMTx4 and related gating modifier peptides includ-
ing HaTx, a gating modifier of Kv channels. Based on these findings, we propose a
model of membrane behaviors of these toxins and the mechanisms by which such
toxins affect the gating of channels. A limited amount of information on the poten-
tial medical applications of GsMTx4 can be covered here, thus interested readers are
referred to more comprehensive reviews on GsMTx4 and ICK peptides. Bowman
et al. (2007) reviewed topics of history, properties, mechanisms and pharmacology
of GsMTx4. HaTx and other gating modifier toxins are detailed in Swartz (2007).
Recent reviews on ICK peptide gating modifiers include Sollod et al. (2005) and
Escoubas and Rash (2004). Computational methods for ion channels, including
homology modeling and MD simulations, have been reviewed by Tai et al. (2008).
Computer modeling and simulation studies of bacterial mechanosensitive channels
have been reviewed by Corry and Martinac (2008a, b). Recent progress on MD
simulations of membrane proteins is covered by Lindahl and Sansom (2008).

7.2 Discovery and Functions of GsMTx4

GsMTx4 is a 34 amino acid residue peptide isolated from the venom of a spider,
the Grammostola spatulata tarantula. The inhibitory activity of the crude venom on
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 169

SACs was originally discovered in a study to evaluate the importance of L-type Ca2+
channels and SACs in the hypotonic cell swelling-induced Ca2+ increase (HICI)
in GH3 cells (Chen et al., 1996). In their study, the finding that the extracellular
Ca2+ is the source of the Ca2+ influx of HICI led the authors to determine the route
for the Ca2+ influx; both L-type Ca2+ channels and SACs were likely involved in
the HICI. Importantly, the venom was found to block the HICI without blocking
L-type Ca2+ channels, suggesting the sensory role for SACs in the events of HICI.
Sachs and coworkers continued their study and isolated the molecule responsible
for the inhibition and named it GsMTx4 (Grammostola mechanotoxin number 4)
(Suchyna et al., 2000). They further showed that GsMTx4 inhibits SACs in astro-
cytes and heart cells (Suchyna et al., 2000). The group also determined the structure
of GsMTx4 in solution using NMR (Oswald et al., 2002).
Important insights into the mechanism for gating modification by GsMTx4
were provided by an experiment using the enantiomer. GsMTx4 and its enan-
tiomer, enGsMTx4, exhibit similar efficacy in inhibiting stretch-activated channels
of rat astrocyte, suggesting that membrane distortion, rather than specific molecular
recognition, plays an important role (Suchyna et al., 2004). Despite that GsMTx4 is
unlikely related to gramicidin A, GsMTx4 causes a 1025 fold increase in the chan-
nel appearance rate of gramicidin A, leading the authors to conclude that GsMTx4
exerts its effect on the gramicidin A channel-formers by altering the lipid packing at
the bilayer/solution/channel interface. Clearly, the action of GsMTx4 is not depen-
dent on the traditional lock-and-key model of ligand protein interactions postulated
for a number of inhibitors of enzymes (Suchyna et al., 2004). This finding also drew
the interest of researchers with respect to how GsMTx4 can modify the mechanical
properties of bilayer membranes.
While the potential pharmacological value of GsMTx4 cannot be fully covered
here, notable findings include GsMTx4-dependent inhibition of atrial fibrillation
(Bode et al., 2001) and Yeung et al. (2005) have shown that GsMTx4 inhibits
Ca2+ elevation in muscle fibers from the mdx mouse, a mouse model of Duchenne
Muscular Dystrophy. This suggests that GsMTx4 may be effective in protecting
dystrophic muscle cells in which SACs activation, leading to Ca2+ influx, is easily
triggered due to muscle cells defective in the dystroglycan complex (Yeung et al.,
2005).
As reviewed in the next section, GsMTx4 is a member of a peptide family named
ICK (inhibitory cysteine knot) peptides (Narasimhan et al., 1994). Different ICK
peptides modify the gating property of a variety of channels (Swartz, 2007). Among
ICK peptides, GsMTx4 is unique in its high specificity to SACs. Recently, several
genes belonging to the TRP (transient response potential) channel family have been
cloned and a consensus is emerging that TRP channels form an important group of
SACs (Yin and Kuebler, 2009). A recent example of the specificity of GsMTx4 for
SACs was also obtained from the TRC family channels. GsMTx4 inhibits TRPC1
(Maroto et al., 2005, Bowman et al., 2007), although the presence of endogenous
TRPC1-like SACs confounded the effect of GsMTX4 on TRPC1 (Bowman et al.,
2007). On the other hand, inhibitory effects of GsMTx4 on TRPC6 channels were
reported in a more reliable system (Spassova et al., 2006). In their study, HEK293
cells were transfected with the TRPC6 cDNA under the control of an inducible
170 K. Nishizawa

promoter. TRPC6 was shown to be indeed a direct sensor of the mechanically


and osmotically-induced membrane stretch. These stretch responses are blocked
by GsMTx4. TRPC6 channel is also activated by diacylglycerol (DAG), which in
turn is induced by the receptor-induced phospholipase C activation. Moreover, they
showed that GsMTx4 blocks DAG-induced TRPC6 activation, suggesting that both
chemical and mechanical lipid sensing by the channel have a common molecular
basis (Spassova et al., 2006).
Intriguingly GsMTx4 also has an effect on prokaryotic mechanosensitive chan-
nels. Using E.coli spheroplasts, Martinac and coworkers reported that GsMTx4
exhibits a biphasic effect on the gating of MscS and MscK (Hurst et al., 2009). That
is, at low concentrations of 24 M the pressure sensitivity of the gating of MscS
and MscK was decreased and therefore gating was hampered, whereas at concentra-
tions of 1220 M the pressure sensitivity was increased and gating was facilitated
(Hurst et al., 2009). This finding suggests that there are at least two different kinds
of interaction between GsMTx4 and lipid membrane or interaction among GsMTx4,
lipid membrane and channels as considered below.
While it is reasonable to argue that GsMTx4 is specific to the stretch-activated
channels, an even wider spectrum of membrane proteins may turn out to be targets
of GsMTx4 in the future. GsMTx4 has recently been shown to have effect on the
voltage sensing of a Kv channel; Elinder and coworkers found that 5 M GsMTx4
causes a positive shift (+5.7 mV) in the G(V) curve of the Shaker K channel)
(Brjesson and Elinder, 2008), meaning that depolarization to the higher voltage
(by +5.7 mV) is necessary to induce the similar level of conductance . Strikingly,
Fang and Iwasa (2006) found that GsMTx4 has an inhibitory effect on the outer hair
cell (OHC) motor; the presence of 5 M GsMTx4 causes a positive shift (39.8
to 18.4 mV) in the bell-shaped curve of the membrane capacitance. Let us briefly
review the OHC motor. It is well known that the primary function of the organ of
Corti of cochlea is to change sound-induced vibrations into electrical signals. OHCs,
however, not only detect and convert sound in this way but also they convert the elec-
trical signals into changes in cell length. This response is considered to be essential
to mechanical tuning unique to mammalian ears. The mechanical response is accom-
panied by a current similar to the gating current observed for voltage-activated ion
channels, although in the OHC case the current is manifested as nonlinear mem-
brane capacitance. This transient capacitive current reflecting changes in membrane
voltages is dependent on motor proteins such as the membrane protein prestin
(Zheng et al., 2000).
Therefore, while GsMTx4 is relatively specific toward SACs, the range of target
proteins may be wider. Overall findings thus far suggest that the structural require-
ment of the target molecule is not stringent. Rather, GsMTx4 appears to be located at
the protein/lipid boundary for many membrane proteins and a large part of its action
is likely to be related to the change in structural and/or mechanistic properties of
lipid molecules in the vicinity of the channel.
Lipid partition has been investigated from the point of view that it enhances the
binding or the local concentration of toxins. Posokhov et al. (2007) have shown that
GsMTx4 bonds strongly to both anionic and zwitterionic membranes. By contrast,
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 171

SGTx1, another member of the ICK peptide family (see below) acts only with
anionic lipids. However, this study used POPC (palmitooleoyl-phosphatidylcholine)
and POPC-POPG (palmitooleoyl-phosphatidylglycerol) membranes that did not
contain DPPC (dipalmitoyl-phosphatidylcholine), sphingomyelin or other lipid
mixtures that have potential to create domains rich in saturated lipid tails. As consid-
ered below, our MD simulation study showed that the use of DPPC has a dramatic
increase of the binding energy ( G = 100 kJ/mol) compared with POPC,
indicating the necessity to take the lipid tail structure into account.

7.3 Gating Modifier Toxins Related to GsMTx4


Besides GsMTx4, a variety of peptide toxins isolated from the venom of spi-
ders, scorpions, sea anemone, cone snails and snakes have drawn the attention of
researchers because of their ability to inhibit voltage-gated ion channels (Catterall
et al., 2007; Swartz, 2007). In a recent thorough review, Swartz (2007) classified
such toxins into two groups. The first group consists of toxins, such as charybdo-
toxin and agitoxin, which inhibit Kv channels by binding to the pore forming regions
(MacKinnon and Miller, 1989). The second group of toxins, often called gaiting-
modifier toxins, influences the gating mechanism by altering the relative stability
of the closed, open or inactivated states. For example, -scorpion toxin is a well-
studied example of the second group and specifically enhances activation of sodium
channels by holding the S4 segment in domain II in its outward position. (Cestle
et al., 1998; Catterall et al., 2007). For -scorpion toxins, homology modeling and
extensive docking analyses have been done by Cestle et al. (2006). Such toxins
have been utilized to study the structure and function of voltage sensing machinery
of ion channels. ICK peptides constitute a well-known subclass of gating-modifier
toxins. The name ICK comes from the fact that these peptide toxins contain a com-
mon structural motif known as inhibitory cysteine knot (ICK) (Narasimhan et al.,
1994; Takahashi et al., 2000; Sollod et al., 2005; Bosmans et al., 2006). Table 7.1
shows examples of ICK peptides and their targets. (Table 7.1 is not an exhaustive list
of ICK peptides but rather focuses on the cases where the time for the development
of the effects or the affinity to the channel in the membrane can be inferred at least
approximately.) GsMTx4 is unique in terms of its specificity to SACs rather than
voltage-gated ion channels (Table 7.1). Figure 7.1d shows an alignment of amino
acid sequences of several ICK peptides. ICK peptides have of six cysteine residues
forming three disulphide bonds (Fig. 7.1d), creating the cysteine knot motif. All
these peptides share a common structural feature, a hydrophobic patch (or protru-
sion) surrounded by a unique charge belt (Fig. 7.1a, b) (Oswald et al., 2002;
Takahashi et al., 2000). The hydrophobic patch contains either a F or W residue
(shown in green in Fig. 7.1d). The overall feature of the structure is likely simi-
lar to that of GsMTx4, yet the sequence similarities are generally low. Amino acid
identity of GsMTx4 is 47% with GxTX-1E, 35% with VSTx1 and 21% with HaTx
(Fig. 7.1d;, Herrington et al., 2006; Swartz, 2007)
172

Table 7.1 Target and time for onset of ICK peptide toxins

Toxin Targeta Kd Time for onsetb Reference

HaTx Kv2.1, Xenopus Kd = 42 nM (1:1) = 114 s at 200 nM Swartz and MacKinnon


(1995)
HaTx Kv2.1, Xenopus Kd = 42 nM (1:1) = 500 s at 50 nM Swartz and MacKinnon
(1997)
HaTx Kv2.1, Xenopus = 4 s at 5 M Swartz and MacKinnon
(1997)
HaTx 1A-Cav , Xenopus Kd = 40 M = 20 s at 10 M Li-Smerin and Swartz
(4 sites) (1998)
HaTx Kv2.1/F274A, Kd = 2.8 M = 5.1 s at 2 M Phillips et al. (2005b)
Xenopus
GrTx-SIA Kv2.1 in Xenopus Kd = 19 M = 5 s at 25 M Li-Smerin and Swartz
(1998)
AgaIVA 1A-Cav , Xenopus Kd = 160 nM (1:1) = 200 s at 125 nM Winterfield and Swartz
(2000)
AgaIVA 1A-Cav , Xenopus = 50 s at 1 M Winterfield and Swartz
(2000)
SGTx Kv2.1, Xenopus Kd = 2.7 M = 10 s at 2.5 M Wang et al. (2004)
(4 sites)
SGTx Kv2.1, Xenopus Kd 2 M (4 = 20 s at 500 nM Lee et al. (2004)
sites)
VSTx1 KvAP, IC50 = 30 nM = 3000 s at 20 nM Jung et al. (2005) and Lee
POPE/POPG and MacKinnon (2004)
ScTx1 Kv4.2, COS cell IC50 = 1.2 nM steadyc at 93 s, 30 nM Escoubas et al. (2002)
(0 mV)
ScTx1 Kv2.1, COS cell IC50 = 12.7 nM steadyc at 101 s,100 nM Escoubas et al. (2002)
(0 mV)
ScTx1 Kv2.2, COS cell IC50 = 21.4 nM steadyc at 113 s,100 nM Escoubas et al. (2002)
(0 mV)
K. Nishizawa
7

Table 7.1 (continued)

Toxin Targeta Kd Time for onsetb Reference

GxTX-1E Kv2.1, CHO cell IC50 = 1.5 nM = 50 s at 43 nM Herrington et al. (2006)


(80 mV)
HpTx2 Ito1, rat heart IC50 = 15.8 nM Sanguinetti et al. (1997)
myocyte (10 mV)
HpTx3 Kv4.2, Xenopus IC50 = 67 nM Sanguinetti et al. (1997)
(5 mV)
PaTx1 Kv4.3, Xenopus IC50 = 28 nM steadyc at 90 s, at 50 nM Diochot et al. (1999)
(0 mV)
CcoTx2 Nav1.2/1, IC50 = 8 nM = 2050 s at 50 nM Bosmans et al. (2006)
Xenopus
GsMTx4 SACs, astrocytes Kd = 630 nM =594 ms at 5 M Suchyna et al. (2000)
GsMTx4 capacitance, outer Kd =3.1 M = 17.6 s at 5 M Fang and Iwasa (2006)
hair cell

Note that this list is not exhaustive, but focuses on the toxins/targets for which the time course of effect can be inferred from the
literature, at least approximately. For quick reference, readers are referred to the SwissProt file (http://au.expasy.org/sprot/) for each
Atomistic Molecular Simulation of Gating Modifier Venom Peptides

toxin using the accession number shown in the Fig. 7.1 legend
a Targetchannel or current shown along with the expression system used for the experiment. Artificial bilayer system. b Time
constant, , based on exponential curve fitting is shown along with the toxin concentration in the experiment. Some data are rough
estimations from the reference figure. c The time at which the steady state was reached
173
174 K. Nishizawa

Fig. 7.1 (ac) Structure of GsMTx4 and HaTx. The bottom and side view of GsMTx4 (a) and
HaTx1 (b) and the initial orientation used for simulations (c). The hydrophobic patch is oriented
around the center of the normal view and at the bottom of the side view. (ac) are similar to
Figure 1 of Nishizawa and Nishizawa (2007), in which the following coloring scheme is used;
Hydrophobic residues (Ala, Cys, Ile, Leu, Met, Phe, Pro, Trp, Tyr and Val) are green; basic (Arg
and Lys) are blue and acidic (Asp and Glu) residues are red. (d) Amino acid sequence alignment
of ICK peptides. SwissProt accession numbers are as follows: GsMTx4 (Grammostola mechan-
otoxin number 4, Q7YT39), GxTx-1E (Guangxitoxin-1E, P84835), -Aga4A (P30288), CcoTx2
(P84508), HaTx1 (P56852), SGTx1 (P56855), VSTX1 (voltage sensor toxin 1, P60980), ProTx2
(Beta-theraphotoxin-Tp2a, P83476), HmTx1 (heteroscordatoxins, P60992), ScTx1 (stromatoxin
1, P60991), HpTx3 (heteropodatoxin3, P58427), PaTx1 (phrixotoxin1, P61230), -GrTx-SIA
(Omega-grammotoxin-SIA, P60590)

Hanatoxin (HaTx) is a 35 amino acid residue gating-modifier of Kv channels


(Swartz and MacKinnon, 1995, 1997). HaTx was isolated from the venom of the
Chilean tarantula (Grammostola spatulata), the same venom from which GsMTx4
was found. HaTx (a mixture of HaTx1 and HaTx2, which are identical except for
residue 13 which is Ser in HaTx1 and Ala in HaTx2) can bind to VSDs of closed
Kv2.1 channels and dramatically shift the channels voltage dependence towards
more positive voltages (Lee et al. 2003). It exhibits very slow action ( = 114 s)
despite its high affinity to the voltage sensor (Kd = 42 nM) (Swartz and MacKinnon,
1995).
Besides GsMTx4 and HaTx, the ICK peptide gating modifiers include: SGTx1
from Scodra griseipes, which resembles HaTx and inhibits Kv2.1 (Marvin et al.,
1999; Wang et al., 2004), VSTx1 isolated from Grammostola spatulata by screen-
ing inhibitory activity against KvAP (Ruta et al., 2003; Lee and MacKinnon, 2004),
ProTx2 (Beta-theraphotoxin-Tp2a) from Thrixopelma pruriens which inhibits var-
ious Nav s (voltage-gated Na channels) and Cav s (Middleton et al., 2002), HmTx1
(heteroscordatoxins) from Heteroscodra maculate and ScTx1 (stromatoxin 1) from
Stromatopelma calceata which are Kv2 and Kv4 inhibitors (Escoubas et al., 2002),
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 175

and GxTx1E (guangxitoxin) from Plesiophrictus guangxiensis, which is a high


affinity inhibitor of Kv2.1 (Herrington et al., 2006). Other ICK peptides include;
GrTx-SIA, which is another gating modifier toxin from Grammostola spatulata
and inhibits voltage gating of Cav2.1 (Li-Smerin and Swartz, 1998); Aga-IVA
from Agelenopsis aperta, which is an inhibitor of P-type Ca2+ channels (Winterfield
et al., 2000; Mintz et al., 1992); CcoTx2 (-theraphotoxin-Cm1b) from Ceratogyrus
marshalli (Straighthorned baboon tarantula), which is an inhibitor of several sub-
types of Nav s (Bosmans et al., 2006); HpTx2 and HpTx3 (heteropodatoxin-2 and -3)
from Heteropoda venatoria (Brown huntsman spider), which are inhibitors of dif-
ferent sets of Kv4 channels (Sanguinetti et al., 1997); PaTx1 (phrixotoxin-1) from
Paraphysa scrofa (Chilean copper tarantula), an inhibitor of Kv4.2 and Kv4.3
(Diochot et al., 1999).
For some ICK peptide gating modifiers, the time required for the development
of the effect is known at least approximately (Table 7.1). In general, gating modi-
fiers have a high affinity for voltage-activated ion channels, the dissociation constant
(Kd ) or the half maximal inhibitory concentration (IC50 ) being in the 10100 nM
range. Such is the case with HaTx (Kd = 42 nM), VsTX1 (IC50 = 30 nM), GxTX-
1E (IC50 = 1.5 nM), HpTx2 (IC50 =15.8 nM) and PaTx1 (phrixotoxin1) (IC50 =28
nM). GsMTx4 is unique in its relatively low degree of affinity with Kd >500 nM; in
the case of the OHC motor (Fang and Iwasa, 2006), Kd is 3 M. It is somewhat
puzzling that, even in the case of the OHC motor, the time for action, =17.6 s
at 5 M, is approximately in the same range as for HaTx.
The affinity of VsTx1 to KvAP in the absence of membranes is quite low
(100500 M) (Lee and MacKinnon, 2004). Nonetheless, VSTx1 inhibits KvAP in
membranes at a low concentration in solution (10 nM). This discrepancy led the
authors to suggest that VSTx1 first binds to the membrane and then diffuses laterally
until it finds its binding site on the channel (Lee and MacKinnon, 2004). For HaTx,
the binding site in the Kv2.1 channel has been determined to be several residues in
the S3b and S4 helices of the voltage-sensing domain, and that at least a large part
of the interaction is mediated by a hydrophobic interaction (Li-Smerin and Swartz,
2000). (Of note, S4 is the helix which mainly carries the gating charges and consti-
tutes a main part of votlage-sensing domain. (Brjesson and Elinder, 2008; Swartz,
2008) Swartz and coworkers have also shown using SGTx1 that the binding site is
made up of a small number of residues and the most critical two residues, based on
their alanine-scanning analysis, are F6 and W30 of SGTx1, implying the importance
of hydrophobic interactions. (SGTx1, being similar to HaTx1 and easily expressed,
was used for amino acid alteration analysis.) More recently, HaTx is thought to
influence the interaction between the voltage sensing domain and lipids, suggesting
the formation of a tertiary complex made up of the toxin, voltage sensor and lipids
(Milescu et al., 2007). These findings point to a view that, while the toxin/channel
interaction is mediated by only a small number of mainly hydrophobic residues (as
in the case of SGTx1), the membrane/toxin interaction in the vicinity of the channel
may cause deformation facilitating the toxin/channel interaction. Thus, the strength
of the interaction between the ICK peptide toxin and channel maybe greatly altered
by and even dependent on the interaction between the toxin and membrane.
176 K. Nishizawa

7.4 Unanswered Questions About HaTx, VsTx and GsMTx4


Because of the variety of functions of ICK peptides, researchers studying differ-
ent ICK peptides tend toward quite different views about this family of toxins. For
example, while GsMTx4 has low affinity and the target specificity also appears
weak (Table 7.1), several toxins including HaTx and VSTx1 have higher affinity
and specificity toward the target. Notwithstanding such differences, the following
five qualities appear to be relevant to many of these toxins:

(1) Slow action. They have small apparent association and dissociation rate con-
stants, 103 104 times slower than the pore blocking toxins (Swartz and
MacKinnon, 1995; Ruta et al., 2003). In the case of HaTx, despite of its
high affinity Kd = 42 nM, action is on the order of minutes. The action
tends to become slow especially when the concentration is low (Table 7.1).
One example is the slow equilibration rate of VSTx1 in inhibition of KvAP
(60 min for 20 nM VSTx1) reconstituted in a planer membrane (POPE
(palmitooleoyl-phosphatidylethanolamine): POPG=3:1) (Lee and MacKinnon,
2004). Intriguingly, in a recent experiment with the Xenopus oocyte expression
system, after the removal of HaTx from the aqueous solution, the Kv2.1 chan-
nel activity recovered very slowly, requiring 1,000 s (Milescu et al., 2007),
suggesting the presence of an energy barrier between the aqueous phase and the
action site.
(2) Small binding site and high affinity. It is puzzling why the apparent bind-
ing affinity of HaTx (and grammotoxin and VSTx1) to the voltage sensor is
so high, considering the binding site on the channel consists of only a few
amino acids on S3b and S4. However, from the VSTx1 study by MacKinnon
and coworkers, a consensus is emerging that, not only the toxin partition into
the lipid membrane enriches the toxin in the membrane, the tertiary com-
plex toxin/channel/lipid is created and this strengthens the binding of toxin to
channel proteins (Schmidt and MacKinnon, 2008).
(3) Not activation, but inhibition. Unlike -scorpion toxin that stabilizes activated
states of Nav channels (Cestle et al., 1998), HaTx and VSTx1 (and most other
ICK peptide toxins) inhibit channel activation. It is unknown how they do so.
(4) Effects of lipid mechanical states. Lipid mechanical state or structure influ-
ences the inhibition efficacy of these peptides. A recent study by Schmidt and
MacKinnon (2008) employing a Kv1.2 channel with a voltage sensor paddle
from Kv2.1 showed that VsTx1 activity depends on the mechanical state of
the membrane. While VSTx1 has no effect on channel analyzed in whole cell
recording of oocytes, VSTx1 causes a slowing of voltage activation similar to
the effect observed on the channel embedded in planar bilayers. This suggests
that voltage sensor toxins exert their effect by perturbing the interaction between
the channel and the membrane lipid. The importance of lipid head groups is also
shown by Milescu et al. (2007) in the finding that sphingomyelinase D, which
removes choline from sphingomyelin, dramatically reduces the inhibitory effect
of HaTx1.
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 177

(5) Concentration-dependent effect. Martinac and coworkers showed that GsMTx4


action is biphasic; at low concentration they enhance but at high concentrations
they inhibit the SACs (Hurst et al., 2009).

With respect to (1) The membrane diffusion coefficient for a molecule like ICK
peptides is probably 108 cm2 s1 (Lee and Mackinnon, 2004). The authors also
speculate that the collision frequency between VSTX1 and the KvAP channel is
likely to be orders of magnitude faster than the observed kinetics of inhibition in
their experimental system. Also with respect to (1), note that the time for action
depends on the concentration of toxin; the lower concentration leads to slower action
(e.g., HaTx, AgaIVA and SGTx in Table 7.1). Although it is speculative, one
possibility is that the toxin concentration influences the location of toxin, which in
turn influences the rate of its access to the site of action. From (5) (concentration-
dependent effects of GsMTx4) and the consideration given below, it is possible that
HaTx at high concentration may be located at a distinctly different position from
the position preferred at low concentration. On the other hand, even with the same
concentration, the time for action differs significantly among the systems examined.
For example, while GsMTx4 at 5 M inhibits SACs on outside-out patch of astro-
cyte within 0.6 s, the effect of the same concentration of GsMTx4 on the OHC
motor takes 17.6 s and the Kd is also high. It is currently difficult to address this
issue because both lipid composition and peptide-protein interaction are influence
the dynamics.
With respect to (5), a recent study by Martinac and colleague (Hurst et al., 2009)
using E. coli spheroplast is of particular interest. They measured two channel activi-
ties MscS (small conductance) and MscK (small conductance, K+ dependent), both
activities being mediated by mechanosensitive channels but with distinct gating
characteristics where the membrane tension required for activation is different. For
both MscS and MscK, when GsMTx4 is applied to the outer leaflet of the bilayer
at concentrations of 24 M, channel activity is inhibited, while at higher con-
centrations of 1220 M, channel activity is potentiated. Moreover they reported
that the initial inhibiting effect of 24 M GsMTx4 becomes less pronounced with
increasing time of exposure. They repeated the pressure application three times,
each lasting 30 min including lag-times of 20 min. Their free-energy parameter
analyses showed that the initially inhibiting 4 M GsMTx4 had only a minimal
degree of effect during the third pressure cycle (Hurst et al., 2009). As the authors
discuss, these puzzling findings can be accounted for by considering the two binding
modes, which we proposed based on our MD simulation analyses (see below).

7.5 The Deep Mode Hypothesis an Insight from MD


Simulations
When we performed numerous simulations of a HaTx/DPPC membrane system with
various positions and orientations of HaTx in the membrane, HaTx exhibited a ten-
dency to move inward such that some of the charged residues interact with the inner
178 K. Nishizawa

leaflet of the membrane (Nishizawa and Nishizawa, 2006). This result prompted us
to apply the free energy analysis to HaTx and GsMTx4 in lipid membranes. In this
and the next sections, technical issues for such free energy analyses are considered.

7.5.1 Free Energy Analysis


The free energy profile of a molecule along the membrane normal represents the
density corresponding to the probability of localization of the molecule at different
depths in the lipid bilayer membrane. For most atomistic peptide/membrane simu-
lations, the timescale covered is within the range of 100 ns which is too short to
sample the entire conformation/orientation of the peptide and lipids to the degree
necessary for free energy analysis. Fortunately, a free energy profile can be obtained
with use of several efficient methods. Current MD simulation packages such as
NAMD (Phillips et al., 2005a) and Gromacs (Lindahl et al., 2001) provide a module
for such free energy profile analyses.
For small molecules or ions, the umbrella sampling may be the most popular
approach (Roux, 1995). With this method, the position of the center of the harmonic
potential that restrains the molecule (or atom) is successively varied among the sim-
ulations such that the successive distribution of the molecule overlaps. The free
energy profile can be recovered from the resulting overlapping distributions using
the weighted-histogram analysis method (Kumar et al., 1992). Recently, adaptive
biasing force simulations have also been utilized, in which, instead of the harmonic
potential, a potential that counteracts the free energy profile is used (Hnin and
Chipot, 2004). However, while these methods are useful for very small molecules,
the amount of computation necessary for peptide/membrane systems is not clear.
Another approach is the one based on the steered molecular dynamics (Izrailev
et al., 1998). This procedure allows one to move a set of particles in a specified
direction. From the work done to move the particle, the free energy can be ana-
lyzed. The steered dynamics is effective in systems with strong binding such as
avidin-biotin binding (Izrailev et al., 1998). However, the binding energy of most
peptide/membrane interactions is so weak that the force measured is no stronger
than the range of the frictional and elastic force. The lipids are so viscous that very

slow pulling (e.g., 1 /ns) is necessary to make the frictional and elastic forces
negligible (Nishizawa, unpublished result). This is inconvenient because it is often
difficult to establish whether the pulling rate is sufficiently slow.
Yet another approach is the mean force measurement. We chose this method for
the toxin/membrane free energy analyses (Nishizawa and Nishizawa, 2006, 2007).
As described in Marrink and Berendsen (1994), the average force exerted on a
particle constrained at a point can be measured directly. The constraining proce-
dure is repeated at different positions and is briefly represented in Fig. 7.2ad. The
z-position (i.e., the vertical position with respect to the membrane plain) of the
center of mass (COM) of the two lysine residues (K8/K28) in GsMTx4 is con-
strained and the average force acting on them measured (the circle of Fig. 7.2c). The
advantage of this approach is that there is no complication from the frictional force.
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 179

However, unlike the mean force on a water molecule in a membrane (Marrink and
Berendsen, 1994), the mean force acting on a peptide in a membrane depends on the
strong electrostatic interactions between the peptide and the lipid head groups and
on the energy associated with lipid packing. Therefore, how to avoid the membrane
deformation represents a major problem, as discussed in the next section. Another
weakness of this method is that the requirement of a long equilibration time for each
position necessitates the coarse discretization, which may lead to inaccuracy in the
free energy analyses.

7.5.2 Technical Consideration of Free Energy Analysis


of GsMTx4/Membrane System
For both the steered dynamics analyses and the mean force measurement analyses,
it is crucial to avoid the concerted movement of the proximal part of the mem-
brane, which makes the meaning of the position within the membrane obscure
(Nishizawa and Nishizawa, 2006, 2007). Related to this, elasticity of the lipid
molecules and the membrane may cause a bias in the mean force measurement. For
example, if the mean force is measured immediately after the peptide is pulled to
the position of interest, the force in the opposite direction becomes dominant due to
the elasticity of the membrane lipid. Pre-equilibration required for this may become
too long. To reduce such complications, introduction of some restraints on the lipid
head group could be a reasonable compromise.
In our analyses, we chose to restrain the z-coordinate of C13 of DPPC (C13
forms an O-ester with the sn-2 acyl chain) in order to avoid the gross deforma-
tion of the membrane when the constraining position was varied (Nishizawa and
Nishizawa, 2006, 2007). In practice, the template frame was chosen from a tra-
jectory exhibiting a mild but not intense membrane thinning (Fig. 7.2b). The mean
force measurement in the presence of the restraint on C13 atoms was carried out
with the use of the template frame and GsMTx4. In practice we chose the mem-
brane for which the thickness of the proximal part of membrane was 2.4 nm based
on the average distance of the C13 atoms between the leaflets (i.e., l2 =1.2 nm of
Fig. 7.2b). Our preliminary mean force measurement showed that, with the presence
of GsMTx4 in the shallow mode, the membrane thinning (from 2.8 to 2.4 nm) per
se requires energetic cost about 15 kJ/mol (Nishizawa and Nishizawa, 2007). This
value is much smaller than the energy difference between the shallow and deep
binding modes.
Even with the use of such a method, further care may become necessary to avoid
a systematic bias arising from the preparation of the initial structures for the mean
force measurement. One possibility, which we have not explored as of yet, is that
charged residues may be neutralized by protonating Glu and Asp (or deprotonating
Lys and Arg) sidechains so as to smoothen the peptide movement during the setting-
up procedure. Another possibility is to avoid the pulling procedure. The peptide may
be placed and restrained, then the lipid molecules moved horizontally so that they fit
to the peptide. These techniques may improve the free energy analyses in the future.
180 K. Nishizawa

In addition, the difference in the density of lipid molecules between the two
leaflets may cause inaccurate measurement of the mean force. In our first paper
in ICK peptides (Nishizawa and Nishizawa, 2006), a set of DPPC bilayers, namely
58/64, (number of DPPC of the upper/lower leaflets) was used. We also tested 61/64
but this alteration did not have a significant effect on the result. Although we have
not tested this extensively, embedding the GsMTx4 in one of the leaflets a 64/64
DPPC membrane may lead to too high a density of lipid on the side containing the
GsMTx4, which may in turn artificially promote the deep positioning of the toxin.
To minimize such a density effect, we also tried a 6 nm 12 nm wide membrane
and embedded two GsMTx4 molecules on the two sides of the membrane to approx-
imate the C2 symmetry. In this way the lipid density of the two leaflets become the
same. This consideration also has a bearing on the issue of the local lateral pres-
sure of the membrane and the effect of peptide toxins on the local pressure profile.
Lindahl and Edholm (2000) developed an important method to calculate the pres-
sure profile of membranes, which shows the lateral pressure at different depths of
the lipid membrane. Gullingsrud and Schulten (2004) applied this method to sev-
eral membranes with and without the applied tension. In the future, the effect of
GsMTx4 on the pressure profile may well be investigated. In such studies, the num-
ber of lipid molecules in two leaflets may have to be carefully varied. The flip-flop
of lipid molecules (i.e., the translocation of lipid molecules from one leaflet to the
other leaflet of the bilayer) is a slow process (minutes) even with the help of flli-
pase (Zachowski and Devaux, 1990) and therefore, the uneven distribution of the
lateral pressure among the two leaflets may be of relevance also in the real experi-
mental system. After the binding of GsMTx4 to the membrane, the lateral pressure
profile of the membrane may slowly change during the experiment as such flip-flop
events occur.


Fig. 7.2 (continued) Our measurement showed that, with the presence of GsMTx4 in the shallow
mode, the membrane thinning (from l1 (1.4 nm) to l2 (1.2 nm)) requires 15 kJ/mol of energy for
DPPC and 14 kJ/mol for POPC (Supplementary File 6 of Nishizawa and Nishizawa, 2007). This
value is much smaller than the energy difference between the shallow and deep binding modes
(Fig. 7.2). (c) The mean force measurement. The z-position of the center of mass (COM) of the
sidechain atoms of K8 and K28 was restrained and the mean force experienced by the COM was
measured. (d) Deep mode binding. (e) Free energy profile of GsMTx4 and HaTx in a DPPC mem-
brane. For GsMTx4, K8/K28 COM was used as the reaction coordinate (Nishizawa and Nishizawa,
2006). For HaTx, R24/K26 COM was used as the reaction coordinate. (f) Free energy profile same
as (e) but with the POPC membrane (Nishizawa and Nishizawa, 2007). Cartoons below (e) and (f)
represent our hypothesis on the factors influencing the stability of the deep mode relative to the
shallow mode and our hypothesis on the transition from the shallow to the deep mode. Movements
of GsMTx4 toward saturated acyl chain-rich domains and/or movements of such lipids toward
GsMTx4 may promote the transition toward the deep binding mode. The transition may also be
facilitated by binding of GsMTx4 to the membrane at a high density. (g) Two mode-based model
for the GsMTx4 biphasic effect on E.coli MscK and MscS (Hurst et al., 2009). Note that in this
illustration the Msc channel is represented by a simple tetramer composed of four transmembrane
cylinders. (h) A deep mode-based model for the inhibition of the voltage sensor movement by
HaTx
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 181

Fig. 7.2 Free energy analyses and two-binding-mode model of GsMTx4 effects. (ad) Steps
assumed for energy analyses of GsMTx4 binding and penetration into the membrane. A simple
illustration of the GsMTx4 structure is used; the shaded area represents the charge band, whereas
the Lys and Arg residues are shown by long sidechains (zigzag lines). (a) GsMTx4 binds to the
membrane from bulk water, forming the shallow mode. l1 is the distance from the bilayer center
based on the average positions of phosphorus of DPPC for representative simulations. In our study,
l1 was 1.4 nm. (b) A mildly deformed membrane was subjected to the membrane fixation, which
was useful for the mean force measurement (see text). To make the interpretation easy, a mem-
brane with a mild degree of thinning (l2 = 1.2 nm) was used. Of note, the approximate free energy
associated with the membrane thinning can also be measured by the mean force measurement.
182 K. Nishizawa

7.6 Two Binding Modes of GsMTx4/Lipid Bilayer Membrane


Results show that there are at least two binding modes (the shallow and deep mode)
in the HaTx (and GsMTx4)-lipid bilayer membrane interaction (Nishizawa and
Nishizawa, 2006, 2007) (Fig. 7.2e, f). This result is different from the MD simu-
lation analyses of VSTx1 by other researchers (Bemporad et al., 2006; Wee et al.,
2008), in which only the shallow mode was reported. However, their simulations
appear to have been started with a limited range of position of the toxin relative to
the membrane and used limited types of lipids, giving rise to a condition favorable
to the shallow mode. In the following, we discuss from the viewpoint that the many
interesting behaviors the toxins exhibit can be better explained by considering the
two bonding modes rather than only the shallow mode binding.
Phillips et al. (2005b) has shown that the fluorescence of W30 of HaTx was
quenched by C9, C10, which are located near the middle of the hydrocarbon tail of
the outer leaflet lipid of bilayer made of POPG:POPE=1:1. The quenching effect
of C6, C7 was weaker than C9, C10 and even weaker than C11, C12, i.e., carbon
atoms deeper into the hydrocarbon core. This finding does not support the shallow
mode as the only binding mode; our simulations that started from different positions
showed that the data was more consistent with the deep mode than with the shallow
mode (Nishizawa and Nishizawa, 2006).
In our view, the simple interfacial positioning (i.e., shallow mode) does not
explain the very slow action of the toxin at least for HaTx. At least in our simu-
lations, there is no substantial energy barrier between the extracellular space and
the shallow mode (Nishizawa and Nishizawa, 2007). Although orientation can be
varied exhaustively, our analyses on HaTx and Kv2.1 VSD interaction in the DPPC
membrane suggest that there is no substantial barrier between the extracellular space
and the shallow mode. Moreover, the shallow binding mode alone does not provide
a good explanation for the stabilization by HaTx of the Kv2.1 channel in the resting
state. Current understanding of the VSD structure in the resting state places S4 at a
position lower than the HaTx binding site can access. Therefore, the shallow bind-
ing does not appear to significantly hinder the S4 upward movement. Also puzzling
is the concentration-dependent effect of GsMTx4 (see above).
To our knowledge, MD simulation analyses of ICK peptide carried out thus
far have not directly addressed the structural features of the tertiary complex
of toxin/channel/lipids. Nonetheless, based on the currently available findings, we
hypothesize that the following model may be relevant to many of the ICK peptide
toxins. (1) First, the toxins bind to the membrane at the interface (the shallow mode)
(Fig. 7.2a, e). Due to the high energy barrier between the two modes, a spontaneous
transition to the deep mode is a rare event, initially. (2) Several factors act to pro-
mote the transition to the deep mode (Fig. 7.2e). As the surface density of the toxin
increases, which may happen with a high concentration of toxins in the solutions,
pressure imbalance between membrane leaflets may become so high that it facili-
tates the transition of some toxins to the deep mode. Lipid structure and mechanical
state may also be a factor. In our analyses, DPPC, which contains saturated acyl
chains only, favored the deep mode over the shallow mode (Fig. 7.2e), whereas
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 183

POPC membrane favored the two modes equally (Fig. 7.2f). (3) It is possible that,
after several minutes, the toxin moves into the domains rich in the saturated acyl
chains and/or the toxin attract such lipids in its vicinity (Fig. 7.2e). In this way,
deep mode binding may acquire stability. (4) Then, the toxin may interact with the
binding site on the channel, if such a site is accessible (Fig. 7.2 g, h). In the case
of HaTx, primarily the effect of (3) drives the toxin downward and this force may
inhibit the S4 upward movement (Fig. 7.2 h).
The concentration-dependent (biphasic) effect of GsMTx4 can be explained by
our two mode-based model (Hurst et al., 2009). As we hypothesize in Fig. 7.2 g,
GsMTx4 in the shallow mode may inhibit the opening of SACs. In the deep
mode, further thinning of the membrane induced by the GsMTx4 molecules may
facilitate opening the SACs. As our free energy analyses have shown, an energy
barrier between the two binding modes explains the slow equilibration and recovery
(after removal) for GsMTx4, HaTx and VSTx1 (Nishizawa and Nishizawa, 2007).
Moreover, given the recent finding showing the low position of S4 in the resting
state (Brjesson and Elinder, 2008) and the finding that VSTx1 interact at S4 of the
KvAP, the deep binding mode appears to be more suitable to stabilize the resting
state than the shallow mode. Finally, extensive chimera analyses showed that, for
the Kv2.1 channel containing KvAP S4, in the presence of VSTx1, strong depo-
larization (100 mV) allows channel opening to some extent and this opening is
accompanied by the dissociation of VSTx1 from the S4 of the chimera channel
(Alabi et al., 2007). This finding is also consistent with the view that the deep
mode is the action mode of VSTx1; considering that the partial channel opening
is accompanied by upward movement of S4, it is difficult to explain, based only on
the shallow mode, why VSTx1 exhibits the higher affinity in the resting (down) state
than in the activated state. Shiau et al. (2002) carried out modeling by the docking
method and suggested a binding model for HaTx1 and the Kv2.1 channel, although
their analyses were based on a lipid-free system (for review, Huang et al., 2007).
Intriguingly, our preliminary analyses show that the model by Shiau et al. (2002) is
more consistent with the deep binding mode (our manuscript in preparation). Further
simulation analyses are underway. In general, for ICK peptides other than GsMTx4,
more experimental and modeling studies are necessary before a model based on the
two modes of interaction can be proposed.

7.7 Conclusion and Perspectives


The above considerations show that there is an array of gating-modifier peptides
for voltage-dependent channels, but gating-modifiers specific to SACs are very few.
Therefore, GsMTx4 is a valuable tool to study the structural and dynamical details
of SACs. There is some evidence supporting the view that ICK peptides change
the mechanical properties of lipid membranes in the proximity of channels (e.g.,
Schmidt and MacKinnon, 2008; Suchyna et al., 2004). To characterize the structure
of the GsMTx4/lipid membrane/channel complex, MD simulation will continue to
be useful. Although the number of studies of ICK peptides utilizing MD simulation
184 K. Nishizawa

is currently limited, importance of MD simulation as a powerful tool to integrate


physiological and structural data is increasing. On the other hand, it is always true
that the value of MD simulation depends on the structural and molecular physiolog-
ical information, given the weaknesses and limitations of current MD simulations.
Here, general limitations of MD analyses are considered first.
Despite a wide array of technical advances, there are a number of limitations in
simulation that impede faithful representation of the experiments. A major drawback
is the limited simulation time that can be covered by MD simulations. The limi-
tation in timescale is severe for analyses of protein-lipid interactions. Large-scale
motions of lipids, internal dynamics of peptides in a viscous lipid environment, or
peptide-peptide association in a lipid environment are beyond the timescale of tens
of nanoseconds that can be achieved in many of simulation studies (Mtyus et al.,
2007). For the purpose of the free energy analyses, combinations of the aforemen-
tioned techniques may help perform more meaningful simulations, but we cannot be
free of the essential problem. For example, combination of the membrane restraints
and the temporary neutralization of charged residues may be worth consideration.
Recent developments on coarse-grained (CG) models of lipids and peptides are also
promising for long timescale simulations involving channel peptides embedded in a
membrane (e.g., Marrink et al., 2004; Bond and Sansom, 2007). Since CG models
have a limited degree of accuracy in the representation of interactions, for exam-
ple, between charged residues of the peptide and the lipid headgroups, it is always
desirable that the results from CG analyses are evaluated with atomistic simulation
systems.
Another technical limitation is the accuracy of parameters. Given that ion channel
activation/deactivation depends on many interactions between amino acid residues
and interactions between amino acid and lipids, the accuracy of the parameters used
to describe lipids, peptides and their interaction are critical. It should be kept in
mind that these parameters are not perfect, but intensive research for improvements
is on going (Kandt et al., 2007; Mtyus et al., 2007). Therefore, to draw useful con-
clusions from simulation results, one usually has to take into account the potential
effects of the force field chosen and other simulation parameters used. The good
news is that, while the validity of an individual simulation result is often difficult
to show, a set of simulations for comparative analyses (variation in mutations, ini-
tial configurations, lipid compositions, etc.) often become more effective than one
isolated simulation.
Our analyses suggested that GsMTx4 and HaTx can interact with lipid mem-
branes in at least two different modes. The two-mode hypothesis explains the
biphasic effect by Hurst et al. (2009) well. In the future, many more analyses includ-
ing mechanosensitive channels themselves are necessary. To this end, structural
information of SACs in membranes is crucial. The crystal structure of MscL from
Mycobacterium tuberculosis (Tb-MscL) in the closed state (Chang et al., 1998) has
become a useful starting point for computational modeling and simulation studies.
The crystal structure of E.coli MscS was solved by the Rees group at a resolution
of 3.9 A (Bass et al., 2002). This structural information facilitated many modeling
and simulation studies (for review, Corry and Martinac (2008a, b)). More recent
computational efforts on MscL include Yefimov et al. (2008) and Jeon and Voth
7 Atomistic Molecular Simulation of Gating Modifier Venom Peptides 185

(2008), and those on MscS include Anishkin et al. (2008a) and Anishkin et al.
(2008b).
Recently, Yefimov et al. (2008) used the CG model of protein and lipid (DOPE).
In the study, starting from the closed crystal structure, considerable opening of the
channel was observed close to the rupture tension of the bilayer. This demonstrates
the potential usefulness of the CG approach, but it should be noted that the CG rep-
resentation introduces artificial smoothness into the channel gating. In fact, in the
atomistic simulations by Jeon and Voth (2008), the membrane tension and/or the
structural asymmetry (i.e., due to the difference in the number of lipid molecules
between two leaflets) of the membrane was rather ineffective in inducing MscL
structural changes within 20 ns. Recent determination of the structure of a truncated
MscL from Staphylococcus aureus is illuminating because it likely corresponds
to the pre-expanded state based on the tilted transmembrane helices and overall
flattening of the channel (Liu et al., 2009). This flattening is accompanied by an
increase in the cross sectional area of the MscL channel. Flattening accompany-
ing channel opening has also been pointed out in several simulation studies (e.g.,
Anishkin et al. 2008a, b). These studies should deepen our understanding of the gat-
ing mechanism by membrane stress, and also provide opportunities for simulation
studies of the tertiary complex ICK peptide/channel/lipid membrane.
In future studies, lipid composition should also be varied, since different compo-
sitions will likely alter the relative stability of the two binding modes. It is possible
that the presence of ICK peptides influences the formation of microdomains con-
taining acyl chains with a distinct degree of saturation. Besides acyl chains, lipid
headgroup structure/composition may also be influenced by ICK peptides. Such
considerations are interesting because the phospho-headgroups of membrane lipids,
together with certain acidic channel residues, are believed to provide the necessary
counter-charges for the positively charged residues of S4 during individual steps of
the voltage-sensor movement (Xu et al., 2008; Ramu et al., 2006). Therefore, some
ICK peptides may have direct effects on lipid headgroups, which then modify the
gating property of channels.
Besides such potential effects of ICK peptides on channel-lipid interaction, the
effect of the peptides on the lateral pressure profile of the membrane could exerts
indirect effects on the gating of mechanosensitive channels. The binding of GsMTx4
to the membrane may change the lateral pressure profile. For the pure bilayers of
POPE or POPC, the layer of the lipid headgroups has a tension trough that has a
weak lateral pressure (Lindahl and Edholm, 2000; Gullingsrud and Schulten, 2004).
The presence of GsMTx4 in the shallow mode may nullify the tension trough. The
GsMTx4 transition to the deep mode may further alter the pressure profile, thereby
changing the free energy required to open the gate. Clearly, many more analyses are
necessary to discuss this issue quantitatively.
To improve future simulation studies of ICK peptides, the importance of compar-
ing simulations starting from various positions/orientations is again emphasized. In
particular, when studying the deep binding mode, it is important to take into account
that various combinations of two or three charged residues may interact with the
inner leaflet of the membrane to form the deep mode. Therefore, reorientation may
be necessary until the peptide finds the orientation/position with the lowest free
186 K. Nishizawa

energy. Although, for both experiments and simulations, the reorientation may be
a time-consuming step because interaction between the charged residues and lipid
headgroup has to be remodeled.
Notwithstanding such difficulties, homology modeling and MD simulations pro-
vide useful measures to study many themes of mechanosensitive channels. In many
channel studies, it is difficult to say that MD simulations can stand alone as a
method, yet they further the understanding of how ion channels work in atom-
istic details. At the same time, ICK peptide analyses suggest that understanding
the mechanics of mechanosensitive channels at the atomic level will help us design
molecules that can modify gating dynamics in a channel-specific manner. On the
other hand, it can be anticipated that many future MD simulation studies suffer
from discrepancies between the simulation results and experimental findings. As is
the case with large membrane proteins in general, careful consideration is necessary
when applying computational methods to ion channels; for example, how close is
the simulation system to reality, or how robust is the result? As with the experi-
mental approaches, deliberate designing of a series of simulations should always be
helpful.

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Part III
Mechanosensitivity
and Mechanotransduction
in Vascular Cells
Chapter 8
Cellular and Molecular Effects of Mechanical
Stretch on Vascular Cells

Kou-Gi Shyu

Abstract The vascular endothelium is a dynamic cellular interface between the


vessel wall and the blood stream. It plays an important role by sensing the alterations
in biological, chemical, and physical properties of blood flow to maintain home-
ostasis. Cells in the cardiovascular system are permanently subjected to mechanical
forces due to pulsatile nature of blood flow and shear stress, created by the beat-
ing hearts. These haemodynamic forces play an important role in the regulation
of vascular development, remodeling, wound healing, atherosclerotic lesion forma-
tion, and endothelial progenitor cell function. Mechanical stretch can modulate cell
alignment and differentiation, migration, survival or apoptosis, vascular remodeling,
and autocrine and paracrine functions in smooth muscle cells. Laminar shear stress
exerts anti-apoptotic, anti-atherosclerotic, and anti-thrombotic effects on endothe-
lial cells. However, low shear stress or high laminar shear stress exerts atherogenic
effect on endothelial cells. Knowledge of the impact of mechanical stretch on the
cardiovascular system is vital to the understanding of pathogenesis of cardiovascu-
lar diseases and is also crucial to provide new insights in the prevention and therapy
of cardiovascular diseases.

Keywords Mechanical stretch Shear stress Smooth muscle cell Endothelial


cell Cardiac myocyte

8.1 Introduction

Cells in the cardiovascular system are permanently subjected to mechanical forces


due to pulsatile nature of blood flow and shear stress, created by the beating heart.
These haemodynamic forces play an important role in the regulation of vascular
development, remodeling, wound healing, and atherosclerotic lesion formation, and

K.-G. Shyu (B)


Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Graduate
Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
e-mail: shyukg@ms12.hinet.net

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 193


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_8,

C Springer Science+Business Media B.V. 2011
194 K.-G. Shyu

endothelial progenitor cell function. For the cardiovascular systems, endothelial


cells (ECs) and smooth muscle cells (SMCs) are the major cells that face mechanical
forces. Blood pressure is the major determinant of vessel stretch. Vascular smooth
muscle cells (VSMCs) are a main cellular component of the blood vessel wall.
They are subjected to a dynamic mechanical environment modulated by pulsatile
pressure and oscillatory shear forces. VSMCs are primarily subjected to the cyclic
stretch resulting from pulsatile changes in blood pressure. ECs are mainly sub-
jected to shear stress by the flowing blood. Knowledge of the impact of mechanical
stretch on the cardiovascular system is vital for the understanding of pathogene-
sis of cardiovascular diseases and can provide new insights in the development of
therapeutic strategies of cardiovascular diseases. In the past years, several review
articles have been published discussing the molecular mechanisms of mechanical
stretch on ECs and VSMCs (Kakisis et al., 2004; Gunningham and Gotlieb, 2005;
Haga et al., 2007; Lehoux, 2006; Lehoux et al., 2006; Li and Xu, 2007; Orr and
Helmke, 2006; Riha et al., 2005; Shyu, 2009). In this book chapter, I summarize
the recent findings about the cellular and molecular effect of mechanical stretch
on vascular cells, including ECs and VSMCs. Because several types of devices
have been applied to induce mechanical stretch in vitro (Brown, 2000; Reinhart-
King et al., 2008), the cellular and molecular responses in each type of vascular
cells may be different. Most of the effects of shear stress on the ECs are beneficial
(including anti-apoptotic, anti-atherosclerotic, and anti-thrombotic effects) because
atherosclerosis preferentially occurs in areas of disturbed flow or low shear stress,
whereas regions with steady laminar flow and physiological shear stress are pro-
tective. However, the effect of mechanical stretch on SMCs may be beneficial or
detrimental. Although various models of mechanical stimuli (static or dynamic)
have been used in the past, the majority of research used the Flexercell Stress Unit
(Flexercell Corp., USA). This model, a 2-dimensional cell culture, is controlled by a
computer program and provides a physiological representation of the in situ environ-
ment of repetitive mechanical stimuli. However, this model is a poor representation
of natural tissue environment of vascular cells, which is 3-dimensional, mechani-
cally dynamic, and involves the interaction of multiple cell types (Nikolovski et al.,
2003). Organ culture model for study the effect of mechanical stretch of vascular
cells is the best in vitro representation of the vessel in its in vivo environment, where
multiple cell types and the extracellular matrix participate in response to mechanical
stimuli (Lehoux et al., 2000). Regardless of the 2- or 3-dimensional model used, in
vitro studies do not allow easy distinction between stretch effects due to transmem-
brane force transfer and stretch effects due to a global change in cell morphology
which causes generalized deformation of the plasma membrane and the cytoskeleton
(Wang et al., 1993).

8.2 Effect of Mechanical Stress on Endothelial Cells


The vascular endothelium is a dynamic cellular interface between the vessel wall
and the blood stream. It plays an important role by sensing the alterations in
biological, chemical, and physical properties of blood flow to maintain homeostasis.
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 195

In addition to interacting with blood constituents and circulating cells, the vascular
endothelium is exposed to a distinct mechanical environment consisting of haemo-
dynamic shear stress and mechanical stretch from blood pressure. Disturbance
of normal haemodynamic load can contribute to cardiovascular diseases includ-
ing hypertension, intimal hyperplasia, vascular restenosis, and atherosclerosis
(Cummins et al., 2007). Laminar shear stress, the frictional force created by the
flowing blood, exerts a variety of cellular and molecular effects on endothelial
structure and functions. The molecular mechanism of shear stress on ECs has
been extensively reviewed (Cummins et al., 2007; Kakisis et al., 2004; Lehoux,
2006; Lehoux et al., 2006; Li et al., 2005) In addition to anti-apoptotic and anti-
atherosclerotic effects of laminar shear stress on ECs, laminar shear stress has a
profound impact on endothelial metabolism and can alter gene expression lead-
ing to changes in the endothelial phenotype and vessel wall homeostasis. The
genes regulated by shear stress can modulate several endothelial functions including
vessel diameter, cell proliferation, migration and angiogenesis, cell-cell commu-
nication, coagulation and fibrinolysis, anti-inflammation, and immune modulation
(Cummins et al., 2007; Kakisis et al., 2004; Lehoux, 2006; Li et al., 2005). In this
book chapter, additional novel findings about the impact of shear stress on ECs are
discussed.

8.2.1 Effect of Shear Stress on ECs Protein Alteration


DNA mircoarrays have been used to analyze a large number of genes in ECs exposed
to shear stress (Wasserman et al., 2002; Andersson et al., 2005). Proteomic analysis
shows that a broad spectrum of proteins is altered by shear stress. Wang et al. found
142, 213, and 186 candidate proteins up- or down-regulated at least two-fold after
10 minutes, 3 hour, and 6 hour of shear stress, respectively (Wang et al.,
2007). These proteins include transcriptional regulators, enzymes, protein kinases,
G-protein-coupled receptors, cytokines, protein degradation related proteins,
cytoskeletal and matrix proteins. These findings suggest that shear stress has pro-
found effects on the molecular response and the physiological function of the
vascular endothelium.

8.2.2 Vasculoprotective Effect of Shear Stress on ECs

The vasculoprotective effects of shear stress on ECs have been documented more
than one decade ago (Traub and Berk, 1998). Novel findings extending this aspect
have been reported recently. Bone morphogenetic protein (BMP) is a TGF- family
member cytokine that exerts proinflammatory effect on the endothelium and plays
a role in atherogenesis. BMP is upregulated at athero-prone regions in blood ves-
sels and may contribute to vascular calcification and development of atherosclerotic
plaques (Csiszar et al., 2006). Csiszar et al. have reported that laminar shear stress
activates cAMP and cAMP-dependent protein kinase and downregulates the BMP-4
expression in coronary artery ECs (Csiszar et al., 2007). This finding supports the
196 K.-G. Shyu

antiatherogenic and vasculoprotective effect of shear stress because BMP-4 elicits


endothelial activation, dysfunction, hypertension, and vascular calcification.
The transcription factor Kruppel-like factor-2 (KLF2) is an important mediator
of the anti-inflammatory and anti-thrombotic properties of the endothelium (Dekker
et al., 2005; Lin et al., 2005). Prolonged shear stress stabilizes the KLF2 mRNA and
induces KLF2 protein expression, especially in the presence of pro-inflammatory
cytokine TNF- stimulation (van Thienen et al., 2006). The atheroprotective effect
of prolonged shear stress is superior to statin, a lipid lowering agents, in the presence
of TNF- in endothelial cell culture model. This finding also supports the atheropro-
tective effect of prolonged shear stress on ECs. Vascular injury and atherogenesis
can be induced by complement activation. The complement inhibitory protein CD59
can be upregulated by shear stress through KLF2 activation (Kinderlerer et al.,
2008) in venous and aortic ECs, indicating a vascular protection by shear stress
in complement-mediated injury. Laminar shear stress also activates nuclear factor-
erythroid 2-related factor 2 (Nrf2) which regulates redox levels by activation of
numerous anti-oxidant genes including heme oxygenase-1 and ferritin (Ridger et al.,
2008). Both KLF2 and nrf2 are candidate regulators of the anti-inflammatory effects
of laminar shear stress. Elevated shear stress has been shown to enhance classi-
cal pathway complement activation on vascular ECs in vitro (Yin et al., 2007).
Shear stress actually can enhance C4 deposition on ECs surface (Yin et al., 2008).
Regulation of complement activation at the cell surface by complement regula-
tory proteins may modulate the inflammatory response and attendant pathological
sequelae.
Stearoly-CoA desaturase-l (SCD1) is a rate-limiting enzyme in the biosynthesis
of monounsaturated fatty acids. SCD1 converts palmitate and stearate into palmi-
toleate and oleate by catalyzing the 9-cis desaturation of saturated fatty acids
(Ntambi and Miyazaki, 2004). Palmitoleate and oleate are the predominant unsat-
urated fatty acids in membrane phospholipids. Qin et al. recently have reported
that shear stress increases SCD1 expression in human vascular EC through a per-
oxisome proliferator-activated receptor- (PPAR) mechanism (Qin et al., 2007).
The metabolic effect of shear stress provides another evidence for its atheropro-
tective effect. The liver X receptors participates in cholesterol transport and lipid
metabolism causing atheroprotective effect. Laminar shear stress increases liver X
receptor function via a PAPAR-sterol 27-hydroxylase dependent mechanism (Zhu
et al., 2008), further supporting the atheroprotective role of laminar shear stress
in ECs.
Li et al. provide additional evidence for the atheroprotective effect of shear stress
in ECs by demonstrating that shear stress decreases TNF--mediated vascular adhe-
sion molecule-1 by inhibiting JNK through MEK5-BMK1 signaling pathways (Li
et al., 2008). They found that flow inhibits TNF--mediated signaling events in ECs
by a mechanism dependent on activation of MEK5-BMK1, but not MEK1-ERK1/2.
Heme oxygenase-1 catalyzes the degradation of heme to liberate free iron, carbon
monoxide and biliverdin in mammalian cells. Heme oxygenase-1 induction repre-
sents a cytoprotective defense mechanism against oxidative insults. Recently, Di
Francesco et al. reported that laminar shear stress up-regulates heme oxygenase 1
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 197

Fig. 8.1 Schematic summary of vasculoprotective effects of laminar shear stress on endothelial
cells. A diagram summarizing the laminar shear stress-induced mechanosensing and intracellu-
lar signaling that lead to the modulation of gene expression and cellular function, resulting in
vasculoprotection, as discussed in the text

by cyclooxygenase2-dependent prostacyclin induction in human umbilical vein ECs


(Di Francesco et al., 2009). The up-regulating heme oxygenase-1 attenuates TNF-
generation. The cyclooxgenase2 and heme oxygenase-1 are the vasoprotective genes
up-regulated by steady laminar shear stress, which characterized atherosclerotic
lesion-protected areas. The mechanism and signal pathways for vasculoprotective
effects of laminar shear stress on endothelial cells are summarized in Fig. 8.1.

8.2.3 Effect of Shear Stress on ECs Polarity and Morphology

Mechanical forces regulate ECs polarity and directional migration. Mechanical


stress also affects ECs morphology and function (Chien, 2007). This is important
for vascular function, remodeling and wound repair (Chien et al., 2005). Shear stress
can modulate microtubule-organizing centers polarity and microtubule stability in
vitro and in vivo by activation of glycogen synthase 3 signaling pathway (McCue
et al., 2006). The application of shear stress causes of EC elongation in the direction
of flow. Shear stress induces reorientation of the microtubule organizing center to
the leading edge of migrating cells in a cdc42-dependent manner (Simmers et al.,
2007). Vascular ECs respond to laminar shear stress by aligning in the direction of
flow. Goldfinger et al. have reported that localized 4 integrin phosphorylation leads
localized Rac1 activation and subsequent stress fiber alignment and ECs elongation
parallel to the flow direction in response to shear stress (Goldfinger et al., 2008).
The shear-induced 4 integrin phosphorylation is dependent on cAMP-dependent
protein kinase A. The 4 integrin and cAMP-dependent protein kinase A regulate
198 K.-G. Shyu

vascular ECs adhesion, migration and survival, and angiogenesis. The shear-induced
cAMP-dependent protein kinase A-dependent 4 integrin phosphorylation may be
an important regulatory step in endothelial function during vascular development
and remodeling (Goldfinger et al., 2008). The reorientation of ECs after cyclic strain
has been related to activation of transient receptor potential vanilloid 4 (TRPV4)
ion channels (Thodeti et al., 2009). TRPV4 is one of the stretch-activated channels.
Cyclic stretch of capillary ECs activates mechanosensitive TRPV4 ion channels that
stimulate PI3K-dependent activation and binding of additional 1 integrin receptors,
which promotes cytoskeletal remodeling and cell reorientation. Simmers et al. have
also demonstrated that shear stress-induced directed migratory polarity is modulated
by exogenous growth factors and dependent on Par6 activity, a major downstream
effector of Cdc-42-induced polarity, and shear stress direction (Simmers et al.,
2007). Shear stress regulates ECs bulk migratory characteristics as well as mor-
phology. Lee et al. reported adaptation of ECs shape in arteries under axial stretch
using an organ culture model. They demonstrated that ECs were initially elon-
gated by the axial stretch but eventually adapted to the axial stretch, regaining their
normal shape (Lee et al., 2008). The capillary ECs reorientation after mechani-
cal stress is important for angiogenesis. These data indicate that mechanical stress
regulates ECs bulk migratory characteristics as well as morphology. These mechan-
ical sensitive ion channels may represent new targets for therapeutic intervention in
angiogenesis-dependent disease.

8.2.4 Anti-Inflammatory and Anti-Oxidant Effect of Shear Stress


on ECs

Endothelial inflammation is a major initiator of atherosclerosis. ECs exposed to


disturbed flow experience oxidative stress, increased expression of markers of
inflammation, and monocyte recruitment as early signs of atherosclerosis (Harrison
et al., 2006). Anti-inflammatory and anti-oxidant defenses are critical for the pro-
tection of cellular macromolecules and progression of atherosclerosis. Laminar
shear stress upregulates peroxiredoxins as important antioxidants in ECs (Mowbray
et al., 2008). Laminar shear stress increases ERK5 and PPAR transcriptional activ-
ity and decreases adhesion molecule expression in ECs. The laminar shear stress
increases eNOS expression through ERK5 to inhibit the formation of ROS (Woo
et al., 2008). Shear stress also activates AMP-activated protein kinase in ECs,
which contributes to elevated eNOS activity and subsequent NO production (Zhang
et al., 2006). AMP-activated protein kinase is the primary kinase phosphorylat-
ing eNOS Ser633/635, which is functionally linked to NO bioavailability (Chen
et al., 2009). Shear stress also activates phosphatidylinositol 3 (PI-3) kinase to
increase eNOS expression. Sud et al. recently reported a key role for decrease in
protein kinase C (PKC) signaling in the up-regulation of eNOS in response to
shear stress was also due to inhibition of phosphorylation of STAT3 (Sud et al.,
2009). The decrease in PKC caused by shear stress was mediated by a decrease in
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 199

STAT3 binding to the eNOS promoter. Laminar shear stress up-regulates antioxidant
genes and activates the transcriptional factor, NF-E2-related factor-2, which is a
major transcriptional factor for EC redox homeostasis (Dai et al., 2007). Laminar
shear stress alters the functions of NF-B by inhibiting its capacity to induce pro-
inflammatory molecules such as VCAM-1, e-selectin, interleukin-8, and ICAM-1
and simultaneously enhancing the induction of NF-B-dependent cytoprotective
transcripts such as manganese superoxide dismutase, Bcl-2 and A1 (Patridge et al.,
2007). High glucose and arachidonic acid synergistically decrease cell viability
and increase glutathione oxidation and lipid peroxidation in human umbilical ECs,
while laminar shear stress attenuates the oxidative stress induced by high glucose
and arachidonic acid (Mun et al., 2008). The antioxidant effect of laminar shear
stress could be attributed to increased biosynthesis of tetrahydrobiopterin and glu-
tathione. These findings strongly support the crucial role of laminar shear stress as
anti-inflammatory and anti-oxidative force. The mechanism and signal pathways for
anti-inflammatory and anti-oxidative effects of laminar shear stress on endothelial
cells are summarized in Fig. 8.2.
High wall shear stress haemodynamics mediate adaptive outward remodeling and
cerebral aneurysm development. High wall shear stress stimulates ECs proliferation
and suppresses apoptosis (Metaxa et al., 2008). The stimulation of EC proliferation
by high wall shear stress is dependent on NO signaling pathways. The mechanism
by which ECs sense high wall shear stress is through stretch-activated calcium chan-
nels on the luminal surface of the endothelium (Brakemeier et al., 2002). High wall
shear stress by stimulation of ECs proliferation plays an important role in maintain-
ing continuity of the endothelium in regions where the vessel wall is subjected to
high levels of haemodynamic stress.

Fig. 8.2 Schematic summary of anti-inflammatory and anti-oxidative effects of laminar shear
stress on endothelial cells. A diagram summarizing the laminar shear stress-induced mechanosen-
sing and intracellular signaling that lead to the modulation of gene expression and cellular function,
resulting in anti-inflammatory and anti-oxidative stress, as discussed in the text
200 K.-G. Shyu

8.2.5 Effect of Disturbed Flow on ECs


While laminar shear stress plays an atheroprotective role on ECs, cyclic stretch
or oscillatory shear stress induces different cellular responses (Fig. 8.3). Pulsatile
flow is steady and laminar in the straight part of vessels, whereas disturbed flow
is not steady with large oscillation near bifurcations and curvatures. Disturbed
flow stimulates the pro-inflammatory transcription factor NF-B through integrin-
and Rac-dependent production of ROS. Cyclic stretch is the repetitive mechanical
deformation of the vascular cells as it rhythmically distends and relaxes with the
cardiac cycle. These biomechanical forces promote atherosclerosis by increasing
formation of reactive oxygen species in ECs and by upregulating pro-atherogenic
cytokine expression. We have reported that cyclic stretch augments TNF- produc-
tion and matrix metalloproteinase expression in human umbilical vein ECs (Wang
et al., 2003). CD40 is a co-stimulatory molecule playing an important role in con-
trolling inflammatory responses, including atherosclerosis. Recently, Korff et al.
have demonstrated that cyclic stretch increases the abundance of CD40 in ECs
co-cultured with VSMCs through TGF-1/activin-receptor-loke kinase-1 (ALK1)
signaling, whereas EC CD40 abundance is down-regulated by exposure to cyclic
stretch in ECs alone (Korff et al., 2007). Cyclic stretch also activates Akt, glycogen
synthase kinase (GSK)-3 to enhance survival of ECs (Nishimura et al., 2006). Akt
is important in preventing apoptosis but is not involved in EC proliferation. These
findings indicate that haemodynamic forces present in atherosclerosis-resistant and
-susceptible region of the vasculature induce different responses in the vessel wall.

Fig. 8.3 Schematic summary


of atherogenic effects of
disturbed flow on endothelial
cells. A diagram summarizing
the disturbed flow-induced
mechanosensing and
intracellular signaling that
lead to atherogenesis, as
discussed in the text
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 201

Atherosclerotic lesions frequently develop in areas of the vasculature exposed to


disturbed flow, whereas areas that experience pulsatile laminar flow are relatively
protected from plaque formation. Actually, prolonged high laminar shear stress
suppress endothelial tissue-type plasminogen activator expression through down-
regulation of JNK pathway (Ulfhammer et al., 2009) and may contribute to the
enhanced risk of arterial thrombosis in hypertensive disease. Therefore, high lami-
nar shear stress is atherogenic on endothelial cells. The role of JNK by mechanical
stress in ECs is controversial. Li et al. reported that laminar flow inhibits JNK
activation in ECs by inflammatory cytokines such as TNF- (Li et al., 2008).
Inhibition of JNK is mediated through MEK5 and ERK5/BMK1. Recently, Hahn
et al. reported that JNK activation by laminar flow and long-term oscillatory flow
is matrix-specific, with enhanced activation on fibronectin compared to basement
protein or collagen (Hahn et al., 2009). The JNK activation is mediated by MKK4
and p21-activated kinase. The JNK activation is also seen in vivo at atheroprone
regions of arteries coincident with fibronectin in the subendothelial matrix. P21-
activated kinase acts as a critical upstream mediator of matrix-specific NF-B
activation by disturbed flow (Orr et al., 2008). Low shear stress has been shown
to induce interleukin-8 gene expression through ERK1/2, JNK1/2 and p38 MAP
kinase, which triggers a cascade of events that lead to inflammatory events, which
in turn contribute to the initialization of atherosclerosis (Cheng et al., 2008). More
recently, Thomas et al. reported that human EC exposed to an atherosclerosis-prone
flow pattern, as in vascular regions susceptible to the development of atherosclero-
sis, exhibited a significant increase in PDGF-DD expression (Thomas et al., 2009).
PDGF-DD inhibits expression of multiple SMCs genes, including SM -actin and
SM myosin heavy chain, and up-regulated expression of the potent SMC differ-
entiation repressor gene Kruppel-like factor-4 at the mRNA and protein levels.
These findings establish a link between low-oscillatory shear stress blood flow,
EC-mediated PDGF-DD expression, and SMCs phenotype modulation.
Circulating leukocytes are recruited into the tissue mainly in small vessels such
as capillaries and venules. Glutamate-leucine-arginine (ELR) tripeptide motif plays
an important role in leukocyte trafficking into the tissues. ELR expression is higher
in microvascular endothelium than in aortic ECs (Shaik et al., 2009). Low intensity
shear stress at 4 dynes/cm2 activated endothelial ELR chemokine production via cell
surface heparin sulfates, 3 -integrins, focal adhesion kinase, MAPK p38, mitogen-
and stress-associated protein kinase-1 and NF-B. The preferential activation of
endothelial chemokine expression by low shear is consistent with the concentra-
tion of endothelial-leukocyte interaction in capillaries and post-capillary venules.
These chemokines may provide a mechanism for the high neutrophil concentrations
seen in the capillaries in the normal state. Low shear stress increases the expres-
sion of adhesion molecules including VACM-1 and ICAM-1 which engage integrins
expressed by activated leukocytes thus reducing the rolling speed of inflammatory
cells over the endothelium (Ridger et al., 2008). The mechanism and signal path-
ways for atherogenic effects of low shear stress on endothelial cells are summarized
in Fig. 8.4.
202 K.-G. Shyu

Fig. 8.4 Schematic summary of atherogenic effects of low shear stress on endothelial cells. A
diagram summarizing the low shear stress-induced mechanosensing and intracellular signaling
that lead to the modulation of gene expression and cellular function, resulting in atherogenesis, as
discussed in the text

8.2.6 Effect of Shear Stress on Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs), mobilized from bone marrow, contribute post-
natal neovascularization. EPCs are exposed to shear stress generated by flowing
blood and tissue fluid flow during the process of EPC incorporation into tissues and
neovascularization (Obi et al., 2009). EPCs appear to be responsible to shear stress
and the vasculogenic activities of EPCs may be modulated by shear stress. The effect
of shear stress on progenitor cell fate has been reviewed by Stolberg and McCloskey
(Stolberg and McCloskey, 2009). Shear stress can accelerate the proliferation, dif-
ferentiation, and capillary-like tube formation of EPCs (Yamamoto et al., 2003).
The endothelial differentiation of EPCs by shear stress has been demonstrated by
activation of Akt (Ye et al., 2008) and by increasing ephrinB2 expression and Sp1
activation (Obi et al., 2009). Shear stress increases the gene expression of arterial
endothelial markers in EPCs, such as ephrin B2, Notch1/3, Hey1/2 and ALK1 but
decreases the gene expression of venous endothelial markers such as EphB4 and
NRP2 (Obi et al., 2009). Shear stress within physiological range can enhance tis-
sue type plasminogen activator and prostaglandin I2 secretion (Yang et al., 2007)
and decrease plasminogen activator inhibitor-1 (Yang et al., 2006) in human EPCs,
which improves the antithrombogenic potential of human EPCs. Shear stress has
also been demonstrated to increase Cu/Zn superoxide dismutase activity and NO
production, as well as superoxide dismutase and eNOS mRNA expression (Tao
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 203

et al., 2007). Shear stress also induces endothelial differentiation in a mouse mes-
enchymal progenitor cell line (Wang et al., 2005). Wang et al. reported that TGF-1
was down-regulated in mesenchymal progenitor cell line by shear stress (Wang
et al., 2008). The negative regulation of the TGF-1 system may be involved in
shear-induced endothelial cell differentiation in the mouse mesenchymal progenitor
cell line.

8.3 Effect of Mechanical Stretch on VSMC Function

Mechanical stretch can modulate several different cellular functions in VSMCs.


These functions include, but are not limited to cell alignment and differentiation,
migration, survival or apoptosis, vascular remodeling, and autocrine and paracrine
functions. However, different kinds of VSMCs (venous or arterial type) and sev-
eral species of animals (mouse, rat, rabbit, swine, and others) were used in different
studies, resulting in sometimes controversial findings. Most of the studies used in
vitro models. The cellular functions induced by in vitro mechanical stretch may
not really represent the in vivo cellular function. Further and detailed studies are
needed to elucidate the real effect and mechanisms of mechanical stress on VSMC
functions.

8.3.1 Effect of Mechanical Stretch on VSMC Alignment and


Differentiation

Arterial SMCs are aligned primarily in the circumferential direction in the media
of artery. The mechanical stretch from pulsatile blood flow is one of the key fac-
tors in regulating vascular remodeling (Taber, 1998). Mechanical environment in
vivo modulates the distinct patterns of VSMC orientation in the arterial wall. The
predominant mechanical force influencing VSMCs structural organization and sig-
naling is cyclic stretch (Halka et al., 2008). There are at least three elements
included in the cyclic stretch: magnitude, frequency, and duration. Cultured VSMCs
in vitro can be induced to reorient to a uniform alignment almost perpendicular
to stretch vector alignment by mechanical stretch (Lehoux et al., 2000; Standly
et al., 2002). The response of cell reorientation depends on the stretching mag-
nitude and frequency (Dartsch et al., 1986; Liu et al., 2008; Wang et al., 1995).
The signaling pathways involved in the stretch-induced VSMCs alignment include
p38 mitogen-activated protein (MAP) kinase (Liu et al., 2008), nitric oxide and
reactive oxygen species (Chen et al., 2003; Standly et al., 2002). The mechanosen-
sor and outside-in signal of integrin-1 is also involved in the stretch-induced
VSMCs alignment (Chen et al., 2003). An intact cyctoskeleton is important for
the stretch-induced VSMCs alignment. Destroying the actin filament system by
cytochalasin D inhibits the effect of stretch-induced alignment (Liu et al., 2008).
H1-calponin, a family of actin-associated protein, is a specifically differentiated
204 K.-G. Shyu

marker in SMCs. Cyclic strain can upregulate the expression of Rac and down-
regulate its negative regulator Rho-GD dissociation inhibitor alpha in a nonlinear
frequency-dependent pattern, then cause the activation of p38 pathway followed
by increasing expression of h1-calponon, which marked VSMC differentiation (Qu
et al., 2008). Rho-GD dissociation inhibitor alpha, a member of Rho-GD disso-
ciation inhibitor, can negatively regulate the activities of small G proteins of the
Rho family by shutting off their GDP/GTP cycling and cytosol/membrane translo-
cation. Not only VSMCs alignment is affected by stretch frequency, but also the
phenotype of VSMCs. Cyclic strain increases smooth muscle and decreases non-
muscle myosin expression in VSMCs (Reusch et al., 1996). Mechanical stretch
increases both smooth muscle -actin protein expression and promoter activity
(Tock et al., 2003). The induction of smooth muscle -actin is mediated by acti-
vation of JNK and p38 MAP kinase pathways. Mechanical stretch could promote
a frequency-dependent redifferentiation of synthetic VSMCs in vitro, mediated at
least in part by the activation of p38 MAP kinase (Qu et al., 2007). Mechanical
stretch modulates cell shape, cytoplasmic organization and intracellular processes
leading to migration, proliferation, or contraction. Rho and intact actin filaments
play an important role in mechanical stretch-induced extracellular signal-regulated
kinase (ERK) activation and cell growth (Numaguchi et al., 1999). RhoA signal-
ing plays a major role in the serum responsive factor (SRF)-dependent regulation
of SMC differentiation marker gene expression (Mack et al., 2001). The primary
genes encoding SMCs contractile proteins are regulated by the stretch-induced
RhoA pathway and associated transcription factors, most importantly the SRF
(Hellstrand and Albinsson, 2005). In vitro, RhoA enhances actin polymerization and
stimulates the SRF homodimer binding to their CArG boxes (Mack et al., 2001).
SRF binds to the serum response element region containing the 10-bp CArG-box
sequence facilitating activation of this motif alone or as a macromolecule bound to
myocardin, its specific co-activator (Halka et al., 2008). Myocardin increases the
promoter activity of the CArG-dependent VSMCs contractile markers. Stretch of
the vascular wall can stimulate increased actin polymerization, activating synthesis
of smooth muscle-specific proteins via Rho-associated kinase and cofilin down-
stream of Rho (Albinsson et al., 2004). Rho/Rho kinase, p44/p42 MAP kinase and
phosphatidylinositol-3 (PI-3) kinase pathways are all involved in the stretch-induced
human saphenous vein SMC proliferation and inhibition of either of them prevents
stretch-induced SMCs proliferation (Kozai et al., 2005). The effect of mechani-
cal stretch on SMCs phenotype has been reviewed by Halka et al. (Halka et al.,
2008).

8.3.2 Effect of Mechanical Stretch on VSMCs Migration

VSMCs migration is important in the development of vascular diseases including


atherosclerosis and post angioplasty restenosis. VSMCs migration is found more
frequently in curved and bifurcating blood vessels, which are exposed to non-
laminar blood flow, than in straight arterial segments exposed to laminar blood flow
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 205

(Liu, 1999). In a vein graft model of SMC study, vortex blood flow induces VSMCs
migration and neointimal hyperplasia in control vein graft, whereas reduction of
vortex blood flow in the vein graft strongly suppresses the migration and hyperpla-
sia (Goldman et al., 2007). In this model, VSMCs migration is regulated through the
mediation of ERK1/2 and myosin light chain kinase. In vitro, mechanical stretch of
arterial VSMCs translocates PKC from membrane to cytoskeleton and increases
the migration of VSMCs (Li et al., 2003). In our laboratory, we could also demon-
strate that mechanical stretch increased migration of VSMCs (Shyu et al., 2005).
The increased migration of VSMCs by mechanical stretch involves p38 MAP kinase
and transforming growth factor-1 (TGF-1) (Li et al., 2003). Inhibition of p38
MAP kianse and TGF-1 activity decreased the migration activity. Qi et al. used
proteomic analysis to demonstrate that low shear stress-induced VSMC migra-
tion and apoptosis are mediated by a down-regulation of Rho-GDP dissociation
inhibitor alpha (Qi et al., 2008). The effect of Rho-GDP dissociation inhibitor
alpha on VSMCs migration is dependent on the PI3K/Akt signal transduction
pathway.

8.3.3 Effect of Mechanical Stretch on Proliferation, Survival


and Apoptosis of VSMCs

The effect of mechanical stretch on survival and apoptosis of VSMCs has been
extensively reviewed by Kakisis et al. (2004) and Haga et al. (2007). In this chapter, I
would like to extend these studies by data published recently. In a mouse SMCs cul-
tured model, Cheng et al. have reported that mechanical stretch prevents apoptosis
of VSMCs in response to oxidized low-density lipoprotein (Cheng et al., 2007). The
mechanism of increased survival of VSMCs induced by mechanical stretch includes
V3 integrin expression, stabilization of PINCH-1, a survival protein that is linked
with integrin and the cytoskeleton (Xu et al., 2005), and remodeling of cytoskeleton.
Small interfering RNA (siRNA) against integrin 3 as well as VSMC isolated from
integrin 3 knockout mice abolishes the antiapoptotic effect of mechanical stretch.
Down-regulation of PINCH-1 by siRNA enhanced the ability of oxidized low den-
sity lioporotein to cause apoptosis of VSMCs, while up-regulation of integrin 3
stabilizes PINCH-1 and protects VSMCs from apoptosis. Disruption of cytoskeleton
also abolishes the antiapoptotic effect of stretch. In cultured rabbit VSMCs, mechan-
ical stretch also stimulates SMCs growth and hypertrophy (Richard et al., 2007).
The increased SMCs survival by mechanical stretch is induced by nuclear protein
import and nuclear pore protein expression that is mediated via MAP kinase. In
cultured bovine pulmonary artery VSMCs, mechanical stretch stimulates prolifera-
tion of VSMCs and RhoA is essential for stretch-induced VSMCs proliferation (Liu
et al., 2007). Blocking Rho completely inhibits the proliferation of VSMCs induced
by stretch. In cultured vein VSMC, mechanical stretch stimulates proliferation of
venous SMCs (Cheng and Du, 2007). The proliferation of venous SMCs induced
by mechanical stretch is mediated by activation of insulin-like growth factor-1
(IGF-1) and IGF-1 receptor. When IGF-1 receptor is knocked out, the mechanical
206 K.-G. Shyu

stretch-induced increase in VSMCs proliferation is blocked. IGF-1 receptor level


is increased in neointima in vein grafts and IGF-1 receptor deletion reduces neoin-
tima formation in vein grafts. Cysteine- and glycine-rich proteins (CRP) regulate
SMCs proliferation and differentiation and are important for the maintenance of
the contractile apparatus of SMC. Campos et al. reported that CRP3/muscle LIM-
domain protein is expressed mainly in arteries and can be induced in veins during
the arterialization process in vitro and in vivo (Campos et al., 2009). The induc-
tion of CRP3/muscle LIM-domain protein is dependent on increased stretch in
SMCs, rather than increased shear stress in ECs. Pulsatile mechanical pressure
has been shown to provoke proliferation of human aortic SMCs and mechanical
pressure initiates an up-regulation of angiotensin-converting enzyme protein, activ-
ity and mRNA expression (Iizuka et al., 2008). The mechanism of up-regulating
angiotensin-converting enzyme by pulsatile pressure is regulated by ERK-related
signal transduction cascades.
In a porcine VSMCs cultured model, Su et al. have demonstrated that mechan-
ical stretch-induced VSMCs apoptosis is phenotype dependent (Su et al., 2006).
Mechanical stretch induces apoptosis in differentiated VSMCs, but not in prolif-
erating VSMCs. The stretch-induced apoptosis in VSMCs is associated with Bcl-
2-associated death factor expression. Vascular endothelial growth factor (VEGF)
and overexpression of the antiapoptotic protein Bcl-2 decrease Bcl-2-associated
death factor expression and apoptosis induced in response to stretch. Recently, we
also have demonstrated that mechanical stretch induces apoptosis in VSMCs from
rat thoracic aorta (Cheng et al., 2008). The mechanical stretch induced VSMCs
apoptosis is load-dependent. In contrast to 10% stretch, stretch of 20% induces
apoptosis. Cyclic stretch enhances GADD153 protein and mRNA expression in
VSMCs. Stretch-induced GADD153 protein expression in VSMCs is mediated
by JNK Cyclic stretch increases AP-1 binding activity. Cyclic stretch increases
GADD153 promoter activity through AP-1 (Fig. 8.5).
The mechanism of apoptosis induced by mechanical stretch in our study is
mediated by GADD153, a component of endoplasmic reticulum stress-mediated
apoptosis factor (Oyadomari and Mori, 2004). Caspase 3 is involved in the
GADD153-induced apoptosis of VSMCs after mechanical stretch. An in vivo
model of aorta-caval shunt also increases aortic GADD153 protein expression.
These results indicate that GADD153 plays an important role in stretch-induced
VSMCs apoptosis. Fitzgerald et al. also confirm that laminar shear stress stimulates
SMCs apoptosis (Fitzgerald et al., 2008). SMCs respond to laminar shear stress
with diminished Akt activity that in turn promotes the intrinsic apoptotic pathway.
Pulsatile equibiaxial stretch, acting through NO and cGMP, can prevent the ability
of thrombin to stimulate Rho signaling pathways that contribute to pathophysio-
logical proliferative and inflammatory responses (Haga et al., 2008). The inhibition
of thrombin-induced RhoA activation by stretch suggests that mechanical forces
provide an additional level of regulation of SMCs proliferation and inflammatory
signaling. Using cells from different species and modifications in intensity and dura-
tion of stretch may cause the differences observed in previous studies. Therefore,
the controversial effect of mechanical stretch on survival and apoptosis of VSMCs
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 207

Fig. 8.5 Schematic summary of regulation of mechanical stretch on GADD153 expression in


VSMCs. The increased GADD153 expression by mechanical stretch induces apoptosis of VSMCs

Fig. 8.6 Schematic summary of various effects of mechanical stretch on VSMCs. A diagram
summarizing mechanical stretch-induced mechanosensing and intracellular signaling that lead to
the modulation of gene expression and cellular function, resulting in migration, differentiation,
proliferation, and survival of VSMCs, as discussed in the text

under mechanical stretch needs further investigation. The mechanism and signal
pathways for effects of mechanical stretch on migration, proliferation, and survival
in VSMCs are summarized in Fig. 8.6.
208 K.-G. Shyu

8.3.4 Effect of Mechanical Stretch on Vascular Remodeling


Vascular inward remodeling results in decreased lumen size and increased vessel
resistance. The signaling cascades that modulate vascular remodeling process in
response to mechanical stretch include reactive oxygen species (ROS), nitric oxide
(NO), nuclear factor B (NF-B), epidermal growth factor receptor (EGFR), MAP
kinase and protein kinase C (PKC) (Kouri and Eickelberg, 2006). Mechanical force
can be transduced via ROS-dependent autocrine and paracrine EGFR activation and
may regulate VSMCs proliferation and synthetic activity through NF-B pathway
(Lemarie et al., 2006). TGF- is a potential specific target for vascular remodeling
induced by mechanical stretch. In vivo, increased hemodynamic forces in a model
of hypertension by angiotensin II (AngII) infusion, activation of NF-B and asso-
ciated cell proliferation and wall thickening are reduced in TGF-- mutant mice,
compared with wild-type animals. Syndecan-1 and -4 belong to a family of tranas-
membrane proteoglycans, acting as co-receptors for growth factor binding as well
as cell-matrix and cell-cell interactions, and are induced in neointimal SMCs after
balloon injury (Julien et al., 2007a, b). Both syndecan-1 and -4 expression and shed-
ding are upregulated by mechanical stretch (Julien et al., 2007a, b), which may
contribute to the vascular pathology induced by mechanical microenvironment in
vivo. Recently, Albinsson and Hellstrand have reported that remodeling of SMCs
to stretch requires a dynamic cytoskeleton (Albinsson and Hellstrand, 2007). The
stabilization of actin filaments is essential for the growth and synthesis of contrac-
tile proteins in response to physiological levels of mechanical stretch. Mechanical
stretch enhances VEGF and hypoxia-inducible factor-1 (HIF-1) gene expression
through transcriptional regulation in VSMCs (Chang et al., 2003; Shyu et al., 2001).
The transient increase in VEGF and HIF-1 gene expression induced by mechanical
stretch may be relevant to pathological complications in the cardiovascular system,
including atherosclerosis, plaque stability and hypertension. The induction of VEGF
and HIF-1 gene by mechanical stretch may play a role in vascular remodeling.
Prolonged cyclic strain produced up-regulation of small proline-rich repeat pro-
tein (SPRRP3), a protein highly expressed in advanced atheromas of human arteries,
RNA and protein in VSMCs (Pyle et al., 2008). SPRR3 regulation by cyclic strain
required type I collagen, whereas VSMCs grown on poly-L-lysine or pronectin F
failed to regulate SPRR3 with cyclic strain. The 11 collagen-binding integrin is
required for mechanoregulation of SPRR3. The data presented by Pyle et al. indi-
cated that SPRR3 may play a role in altered biomechanical compliance of SMCs
within an atheromatous lesion because SPRR3 is exclusively enriched in VSMC
within atheromas in response to mechanical stress.
EC can regulate VSMCs proliferation. Heparin and EC soluble heparin sulfate
proteoglycans (HSPG) are potent inhibitors of VSMCs proliferation and fibroblast
growth factor-2 induced mitogenesis. Baker et al. reported that mechanical strain
stimulated the production of perlecan and HSPGs by EC (Baker et al., 2008).
HSPGs are a key component in an integrated feedback control loop regulating
vascular remodeling through the modulation of paracrine endothelial inhibition of
VSMCs growth. ERK and TGF- signaling were required for pressure-induced
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 209

up-regulation of endothelial HSPG using in vitro and ex vivo studies. Net arterial
remodeling to haemodynamic forces is controlled by a dynamic interplay between
growth stimulatory signals from VSMCs and growth inhibitory signals from ECs.

8.3.5 Autocrine and Paracrine Effect of Mechanical Stretch


on VSMCs

Mechanical stretch may induce secretion or synthesis of bioactive molecules from


VSMCs. These secreted bioactive molecules can act on neighboring cells or the
cells secreting them. The autocrine and paracrine effect of mechanical stretch has
been demonstrated recently and these effects regulate individual intracellular sig-
naling pathways, VSMCs growth and initiate the cellular and molecular effect of
mechanical stretch on VSMCs. Platelet derived growth factor was initially found to
play autocrine function in VSMCs after mechanical stretch (Wilson et al., 1993).
Mechanical stretch using the portal vein SMCs induces endothelin-1 release and
promotes synthesis of smooth muscle specific proteins by a mechanism requir-
ing an intact cytoskeleton (Zeidan et al., 2003). Mechanical stretch also stimulates
autocrine IGF-1 production from arterial and venous VSMCs (Cheng and Du, 2007;
Standley et al., 1999). TGF- has been shown to modulate the NF-B activation
and vascular remodeling under stress (Lemarie et al., 2006) and TNF- has been
reported to modulate the GADD153 expression and VSMCs apoptosis in VSMCs

Fig. 8.7 Schematic summary of different effects of mechanical stretch on VSMCs. A diagram
summarizing mechanical stretch-induced mechanosensing and intracellular signaling that lead
to the modulation of gene expression and cellular function, resulting in vascular remodeling,
autocrine, and paracrine effects in VSMCs, as discussed in the text
210 K.-G. Shyu

under mechanical stretch (Cheng et al., 2008). The intracellular signaling path-
ways of autocrine and paracrine effect of TGF- under mechanical stress involve
ROS and NFB. The intracellular signaling pathways of stretch-induced GADD153
expression involve JNK and AP-1 pathway. AngII and TGF-1 play autocrine and
paracrine action on discoidin domain receptor 2 (DDR2) expressions in VSMC
under mechanical stretch (Shyu et al., 2005). The stretch-induced DDR2 is mediated
by p38 MAP kinase and Myc-Max pathway. DDR2 can regulate cell proliferation
and extracellular matrix remodeling mediated by MMP activities. The intracellu-
lar signaling pathways of stretch-induced DDR2 involve p38 MAP kinase and Myc
pathway. The mechanism and signaling pathways for effects of mechanical stretch
on vascular remodeling, autocrine and paracrine effects in VSMCs are summarized
in Fig. 8.7.
In an ECs-SMCs co-culture system, SMCs secrete interleukin-1 and
interleukin-6 after application of shear stress resulting in inhibition of E-
selectin expression (Chiu et al., 2007). In this model, SMC induces endothelial
E-selectin expression, while shear stress inhibits the SMC-induced E-selectin
expression via the inhibition in SMCs activation of interlukin-1 receptor associ-
ated kinase/glycoprotein-130, JNK/p38 MAP kinase, and NF-K (Haga et al., 2007;
Lehoux, 2006).

8.4 Conclusions and Perspectives


Mechanical stretch activates in ECs, VSMCs, and cardiac myocytes multiple intra-
cellular signaling networks and regulates gene expressions and functional responses.
Specific cell types may respond differently to mechanical forces. Different mecha-
nisms of response may be observed by using different duration, load, and frequency
of mechanical forces. Although the in vitro mechanical stretch model is assumed
to mimic the in vivo haemodynaimc overload, the findings obtained from the in
vitro mechanical models must be considered with caution, because the in vivo
haemodynamic overload is more complex than the in vitro mechanical stretch
model. The cellular and molecular effects of mechanical stretch on vascular cells
may provide new insights in the pathogenesis of vascular diseases and therapeutic
options. Understanding the molecular mechanisms regulating electrical remodeling
under mechanical stretch supports the clinical application of angiotensin converting
enzyme inhibitor and ARB in the cardiac protection and in the prevention of atrial
fibrillation (Healey et al., 2005). Recently, we have used siRNA technology in the
carotid artery balloon injury model to demonstrate the potential therapeutic utility
of DDR2 siRNA for prevention of neointimal formation induced by balloon injury
(Shyu et al., 2008). This finding supports previous studies indicating that DDR2
increases SMCs migration and proliferation in response to mechanical stretch (Shyu
et al., 2005). Therefore, knowledge of the impact of mechanical stretch on the ECs
and VSMCs is vital for the understanding of pathogenesis of cardiovascular diseases
and crucial to provide new insights in the prevention and therapy of cardiovascular
diseases.
8 Cellular and Molecular Effects of Mechanical Stretch on Vascular Cells 211

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Chapter 9
Role of Proteoglycans in Vascular
Mechanotransduction

Aaron B. Baker

Abstract Vascular mechanotransduction is the process through which the arterial


system adapts to changing hemodynamic and pathophysiological stimuli to main-
tain homeostasis. This adaptation is made possible by the major cellular components
of the artery including vascular smooth muscle cells of the arterial wall and
endothelial cells that line the luminal surface. This review focuses on the role
of proteoglycans in vascular mechanobiological responses of the arterial system.
Proteoglycans are proteins that are post-translationally modified with polysac-
charide glycosaminoglycan chains. These molecules are intimately involved in
controlling cellular organization, proliferation and migration. In this chapter, we
discuss how these complex molecules allow cells to sense mechanical forces and
alter arterial structure.

Keywords Vascular smooth muscle cells Endothelial cells


mechanotransduction Proteoglycans Heparan sulfate Syndecans Vascular
remodeling Shear stress Mechanical stretch Integrins Cytoskeleton

9.1 Introduction

The vascular system is under continual exposure to a complex mechanical


environment due to variations in blood flow and pressure during the cardiac cycle.
Biomechanical signals are potent regulators of the growth, structure and function
of the cardiovascular tissues (Dzau and Gibbons, 1993). Blood flow through the
arterial conduits leads to cyclical distension of the arterial wall and concomitant
shear stress (Humphrey, 2008). Shear stresses are regulators of endothelial nitrous
oxide production and, consequently, vasomotor tone (Smiesko et al., 1985). When
the mechanics of the vascular system are perturbed by disease processes or changes

A.B. Baker (B)


Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology,
77 Massachusetts Avenue, E25-442, Cambridge, MA 02139, USA
e-mail: abbaker@mit.edu

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 219


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_9,

C Springer Science+Business Media B.V. 2011
220 A.B. Baker

in tissue metabolism the system must adapt to seek a new level of homeostasis. For
example, hypertension is the most common clinical finding in the United States and
is associated with increased risk of stroke, atherosclerosis and myocardial infarction
(Whisnant, 1996; Rosendorff, 2007). While the fundamental mechanisms causing
essential hypertension remain elusive, the consequences to the vascular system are
profound and include arterial stiffening and vascular smooth muscle cell hypertro-
phy (van den Akker et al., 2009). Fundamental to the processes vascular adaptation
are the mechanosensing mechanisms of the vascular cells themselves. Numerous
in-vitro studies have been done examining vascular cell mechanotransduction in
response to alterations in shear stress or mechanical stretch (Davies, 2009). Vascular
cells respond to mechanical forces and are able to translate them into biochem-
ical events including gene expression and protein secretion. Various mechanisms
have been proposed to explain vascular mechanotransduction at the cellular and
molecular levels. Vascular cells have a variety of cell surface receptors including the
integrins that allow the artery detect and respond to mechanical forces (Hahn and
Schwartz, 2009). In addition, studies have implicated signaling mechanisms such as
receptor tyrosine kinases, mechanosensitive ion channels, and G proteins in cellu-
lar mechanotransduction (Li et al., 2005; Orr et al., 2006). Also implicated are the
cytoskeleton and other structural components that can transmit and modulate cellu-
lar tension through integrins/focal adhesion complexes that link to the extracellular
environment (Li et al., 2005; Orr et al., 2006).
This chapter focuses on the role of proteoglycans in vascular mechanotransduc-
tion. Proteoglycans are complex biomolecules that consist of a core protein that
is post-translationally modified to have one or more glycosaminoglycan chains.
Glycosaminoglycans are linear polymers of repeating disaccharides that is mod-
ified by multiple enzymes to have a complex sulfation and acetylation pattern
(Sasisekharan and Venkataraman, 2000). The addition of the glycosaminoglycan
chains occurs in the transgolgi and proceeds in a processive manner to create a het-
erogeneous structure and with intricate microdomains. These complex molecules
are known to play an important role in controlling vascular homeostasis and have
an emerging role in mechanobiology. We first describe the various types of pro-
teoglycans in the artery and then discuss how these molecules are involved in the
sensing and adaptation of the vascular system to mechanical stimuli. Finally, future
directions in examining the role of proteoglycans in vascular mechanobiology are
discussed.

9.2 Proteoglycans of the Cardiovascular System

9.2.1 Glycosaminoglycans
The most common types of glycosaminoglycans in the artery include heparan
sulfate, chondroitin sulfate and hyaluronic acid (Fig. 9.1). Of these four, only
hyaluronic acid is synthesized without being attached to a protein core. The other
9 Role of Proteoglycans in Vascular Mechanotransduction 221

Fig. 9.1 The chemical structure of glycosaminoglycans found in the vascular system. Shown in
include one potential structure for: (a) heparan sulfate, (b) chondroitin sulfate and (c) hyaluronan

glycosaminoglycans are found as post-translational modifications to core proteins.


In the case of heparan sulfate, proteins are targeted for glycosylation in the trans-
golgi by having a particular amino acid sequence Ser-Gly (Ala)-X-Gly (Ala). This
site accepts an initial tetrasaccaride synthesized by four enzymes (Esko and Zhang,
1996). Heparan sulfate synthesis is initiated by the heparan sulfate copolymerase
that adds glucuronic acid and N-acetylglucosamine to produce the initial heparan
sulfate structure (Rosenberg et al., 1997a). This initial chain is then heterogeneously
modified by deacetlyation, epimerization and sulfation to create a intricate fine
structure (Rosenberg et al., 1997b).

9.2.2 Heparan Sulfate Proteoglycans

9.2.2.1 Perlecan
In the vascular system heparan sulfate is found on several core proteins including
perlecan, glypicans, and syndecans. Perlecan is a large heparan sulfate proteogly-
can found in the basement membrane (Fig. 9.2a). In humans it is the product of the
HSPG2 gene with a molecular weight of 470 kDa and approximately 800 kDa after
222 A.B. Baker

Fig. 9.2 The predominant proteoglycans of the vascular system: (a) perlecan, (b) glypican-1 (c)
the syndecans (d) versican

post-translational glycosylation (Iozzo, 2005). It has a modular structure possessing


a myriad of interactions with growth factors, extracellular matrix molecules and
adhesion molecules. Its name is derived from its pearls on string appearance under
9 Role of Proteoglycans in Vascular Mechanotransduction 223

rotary shadowing electron microscopy (Noonan et al., 1991). Perlecan has been
shown to have a role in angiogenesis, atherosclerosis and vascular injury (Nugent
et al., 2000; Iozzo and San Antonio, 2001). Heparin and heparan sulfate have also
been shown to inhibit neointimal proliferation in animal models of vascular injury
and disease (Clowes and Karnovsky, 1978; Guyton et al., 1980; Hoover et al., 1980;
Lindner et al., 1992; Edelman et al., 1993; Volker et al., 1995). This inhibition is
dependant on heparan sulfate proteoglycans but also requires a protein component
(Ettenson et al., 2000).

9.2.2.2 Glypicans
The glypicans are a family of cell surface heparan sulfate proteoglycans having a
globular structure with membrane association due to a glycosylphosphatidylinositol
(GPI) anchor (Fig. 9.2b) (Fransson, 2003). Predominantly heparan sulfate groups
are attached to serine residues in consensus sequences located between the central
domain and the C-terminal GPI-anchor. The GPI anchor can be cleaved phospholi-
pase C or D and leads to shedding of glypican from the cell surface. The GPI anchor
also localizes the protein to cholesterol and sphingolipid-rich lipid rafts within the
cell membrane (Fransson, 2003).

9.2.2.3 Syndecans
The syndecans are a family of transmembrane heparan sulfate proteoglycans found
on the cell surface and shed in a soluble form (Bernfield et al., 1999). Each synde-
can consists of an extracellular domain that contains glycosaminoglycan attachment
sites, a single pass transmembrane domain, and a short cytoplasmic domain with
multiple phosphorylation sites (Fig. 9.2c). The heparan sulfate and chondroitin sul-
fate glycosaminoglycan chains allow syndecans to interact with a large number of
ligands including FGF-2, VEGF, PDGF and TGF- (Tkachenko et al., 2005). The
interaction of syndecans with FGF-2 is probably the most characterized of these
interactions. Syndecans and the attached heparan sulfate proteoglycan are essen-
tial for effective binding and signaling of the FGF receptor (Nugent and Iozzo,
2000). On the cell surface syndecans stabilize the FGF-2/FGFR complex and are
essential for downstream signaling (Nugent and Iozzo, 2000). When shed from the
surface, syndecan-1 can inhibit FGF-2 induced cell proliferation (Mali et al., 1993).
However, physiologic degradation of syndecan by heparanase may lead to heparan
sulfate fragments that enhance FGF-2 signaling (Kato et al., 1998). Syndecan-4 has
also been shown to interact with FGF-2 and promote FGF-2 signaling (Volk et al.,
1999). Recent work has also found that syndecans can act independently of the FGF
receptor to act as a transmembrane receptor of FGF (Chua et al., 2004).
The transmembrane domain and two regions of the short cytoplasmic domain
of the syndecans are highly conserved. Conserved region 1 and 2 (C1 and C2)
are separated by a variable domain (V) that is specific to each syndecan type
(Fig. 9.2c). The C1 region has been found to bind src (Kinnunen et al., 1998),
ezrin (Granes et al., 2000), and tubulin (Brockstedt et al., 2002). The C2 region
224 A.B. Baker

contains a binding site for PDZ-domain proteins linking the syndecans to CASK,
syntenin and other molecules (Grootjans et al., 1997; Cohen et al., 1998; Hsueh
et al., 1998; Ethell et al., 2000; Gao et al., 2000). This region has been shown to
control trafficking of syndecan-1 in epithelial cells (Maday et al., 2008). The vari-
able region has differential functions in each syndecan type. The variable region
of syndecan-4, in particular, is involved in focal adhesion formation, protein kinase
C activation and binding of -actinin (Couchman, 2003). The cytoplasmic variable
region of syndecan-1 is known to affect cellular adhesion, migration and spreading
(Chakravarti et al., 2005).
Transforming growth factor- (TGF-) has been shown to interact with the hep-
aran sulfate chains on syndecans. Syndecan-2, in particular, has been shown to
interact with TGF- via a protein-protein interaction (Chen et al., 2004). The exact
nature and role of this interaction is complex and still remains to be elucidated. The
presence of syndecan-2 may serve to compete with betaglycan (TGF receptor type
III) for the binding of synectin. Synectin stabilizes betaglycan on the cell surface
and, consequently, syndecan-2 may serve to reduce signaling in the TGF pathway
(Chen et al., 2004; Tkachenko et al., 2005).
The syndecans have an intricate role in orchestrating development and are known
to be involved in cell-cell and cell-matrix adhesion. Syndecan-1 has been shown
to be important for cell adherence to type-I collagen (Sanderson et al., 1989). In
addition, syndecan-1 stabilizes the interactions of vitronectin with v3 integrin
(Beauvais et al., 2004). During migration syndecan-1, syndecan-4 and calveolin are
directed to the region of cell contraction (Baciu and Goetinck, 1995; Borset et al.,
2000; Beardsley et al., 2005). Syndecan-4 has also been shown to be an essen-
tial component for the activation of focal adhesion kinase and is known to bind
fibronectin with its heparan sulfate chains (Mukai et al., 2002; Wilcox-Adelman
et al., 2002; Hsia et al., 2003). In vascular smooth muscle cells exposed to shear
stress syndecan-4 has been shown to dissociate from focal adhesions (Li and
Chaikof, 2002).
Several studies have revealed differential regulation of syndecans by various
growth factors and cytokines. Fibroblast growth factor-2 (FGF-2) has been shown
to increase syndecan-4 expression in vascular smooth muscle cells (Cizmeci-Smith
et al., 1997). Arterial injury and myocardial infarction have also been shown to
increase syndecan-4 expression (Geary et al., 1995; Li et al., 1997; Li and Chaikof,
2002). Stimulation with tumor necrosis factor- (TNF-) increases syndecan-2
expression and decreases syndecan-1 in endothelial cells (Halden et al., 2004).
Transforming growth factor 2 increases syndecan-4 and decreases syndecan-1 in
epithelial cells (Dobra et al., 2003).

9.2.3 Chondroitin Sulfate/Dermatin Sulfate Proteoglycans

9.2.3.1 Versican
Versican is the major extracellular chondroitin sulfate bearing proteoglycan in arter-
ies (Wight and Merrilees, 2004). This molecule has multiple sites for chondroitin
9 Role of Proteoglycans in Vascular Mechanotransduction 225

sulfate attachment and reaches a molecular weight exceeding 1000 kDa (Fig. 9.2d).
Versican has been linked to controlling the viscoelastic behavior in many tissues
(Ludwig, 2007). It also has a pro-migratory affect in vascular smooth muscle cell-
sand various tumor cell types. Versican has multiple molecular interactions with
other extracellular matrix components including hyaluronan, CD44, tenascin R and
the fibulins (Wu et al., 2005).

9.2.3.2 Biglycan
Biglycan is the predominant chondroitin sulfate-bearing proteoglycan found on the
endothelial cell surface. It has been found to be associated with lipid deposition and
atherosclerosis. Biglycan binds to TGF-, collagen I/IV, fibronectin and other extra-
cellular matrix molecules. It is thought to have pro-inflammatory activity and has
a role in activating TNF- and MIP-2 in macrophages. Biglycan can be positively
regulated by TGF- in response to abnormal mechanical stresses. In addition, nitric
oxide (NO) can down regulate biglycan and can thereby link bilgycan expression to
shear stress-induced NO levels (OBrien et al., 1998; Williams, 2001).

9.2.3.3 Decorin
Decorin is another arterial proteoglycan that is similar to biglycan in that it is a mem-
ber of the small leucine-rich proteoglycan (SLRP) gene family. Decorin, howver,
has different biological activity in comparison to biglycan. It can bind to IGF-R and
IGF-I and regulate this pathway in endothelial cells. In addition, decorin induces
expression of p21WAF1, a cyclin-dependent kinase inhibitor, that can arrest the cell
cycle (Williams, 2001).

9.2.4 Hyaluronan

Hyaluronan is a glycosaminoglycan with several distinct properties including


its lack of attachment to a protein core, broad range of molecular weights
(520000 kDa) and lack of sulfated groups (Pure and Assoian, 2009). It is
a non-branching polymer of repeating disaccharides of D-glucuronic acid and
N-acetylglucosamine. Hyaluronan is strongly expressed in the extracellular matrices
of cells that are proliferating or migrating. In particular, it is highly expressed during
healing, inflammation and development (Fraser et al., 1997). One of the major recep-
tors for hyaluronan is CD44, a transmembrane glycoprotein. Through its binding to
CD44 hyaluronan can be linked to the actin cytoskeleton, rho family GTPases and
the ERM (ezrin/radixin/moesin) proteins (Wight and Merrilees, 2004; Cain et al.,
2005).

9.2.5 Sialic Acid


In addition to the glycosaminoglycans/proteoglycans mentioned above there are a
number of sialic acid modified surface proteins found on the vascular cell surface
226 A.B. Baker

(von Gunten and Bochner, 2008). While sialic acid is a term that encompasses either
the N- or O-substituted derivatives of neuraminic acid, N-acetylneuraminic acid is
the predominant form found in mammalian cells. Sialic acid modification is found
in gangliosides and glycoproteins located at the cell membrane. Gangliosides are
glyocolipids anchored in the cell membrane that have been found to mediate cell-
cell recognition, inflammation and growth factor signaling. Sialic acid groups serve
as ligands for sialic acid-binding, immunoglobulin-like lectins (Siglecs). These
lectins bind to specific forms of sialic acids mediating a number of immunological
processes (von Gunten and Bochner, 2008).

9.2.6 Arterial Distribution of Glycoproteins


9.2.6.1 Glycocalyx
The glycocalyx is a structure found on the luminal surface of the endothelium
consisting of the glycoproteins and glycosaminoglycans (Fig. 9.3). In-vivo, the gly-
ocalyx varies from 100 to 500 nm in thickness depending on location within the
vascular tree and the local flow environment (Weinbaum et al., 2007). Notably,
this structure is almost completely lost in in-vitro endothelial cell lines (Potter and

Fig. 9.3 Arterial distribution of proteoglycan in the artery. Boxes show magnified regions of the
artery including the composition for the basement membrane underlying the endothelial layer and
the glycocalyx that extends into the lumen of the artery
9 Role of Proteoglycans in Vascular Mechanotransduction 227

Damiano, 2008; Chappell et al., 2009). The glycocalyx interacts directly with blood
flow and is thus a prime candidate for the mechanotransduction of shear forces in
the endothelial cells. In-vivo, this relatively thick structure is sufficient to reduce the
fluid shear forces to nearly zero at the endothelial surface (Weinbaum et al., 2003).

9.2.6.2 Basement Membrane


The basement membrane of the endothelial layer consists of multiple components
(Fig. 9.3). Most notably there is a high concentration of perlecan that serves to
inhibit vascular smooth muscle proliferation and migration as well as inhibit the
proliferation of the endothelial cells themselves (Ettenson et al., 2000; Nugent et al.,
2000; Baker et al., 2008; von Gunten and Bochner, 2008). In addition there are
many protein constituents that include laminins, nidogens, agrins and nonfibrillar
collagens (Iozzo et al., 2009).

9.2.6.3 Arterial Wall


The arterial wall contains versican, biglycan, decorin and heparan sulfate proteo-
glycans (Fig. 9.3). Chondroitin sulfate proteoglycans are distributed throughout the
wall in areas not occupied by fibrous components. Versican and biglycan both bind
to lipoproteins and are markedly enhanced in atherosclerosis and neointimal hyper-
plasia following vascular injury (OBrien et al., 1998; Williams, 2001; Wight and
Merrilees, 2004).

9.3 Proteoglycans in Sensing Mechanical Forces

9.3.1 Proteoglycans in the Control of Flow-Induced Vasodilation


Increases in blood flow cause arteries to dilate in an endothelium dependent manner
(Kamiya et al., 1984; Pohl et al., 1991). This process is predominantly mediated by
the release of nitric oxide (NO). There is a rapid response to the rate of change of
the shear stress that is both G-protein and Ca2+ -dependent. The later phase of the
response to shear depends on absolute shear level and is independent of G-protein
and Ca2+ signaling pathways (Kuchan and Frangos, 1994; Kuchan et al., 1994).
Several studies have supported the role of elements of the glycocalyx in control of
the shear induced vasodilator response of arteries and are discussed below.
Hecker et al. examined the vasoconstriction of rabbit femoral arteries in ex-vivo
organ culture and found that incubation with neuraminidase, an enzyme that digests
sialic acid moieties, reduced shear stress-induced NO release (Hecker et al., 1993).
In contrast, neuraminidase digestion did not have an effect on acetylcholine-induced
NO release. They also examined the release of the eicosanoid prostacyclin 2 (PGI2)
in response to shear stress. This molecule is an important inhibitor of the platelet
aggregation in blood clot formation and also has vasodilatory properties. Luminal
228 A.B. Baker

neuraminidase digestion did not attenuate the shear stress dependent release of PGI2
(Hecker et al., 1993).
In a parallel study, Pohl et al. examined diameter changes in response to flow
and pressure changes in the mesenteric arteries of the rabbit (Pohl et al., 1991).
They found that in-situ perfused mesenteric arteries had a three stage response to
changes in perfusion flow and pressure. The first phase was a passive distention
due to increased transmural pressure. The second phase is the myogenic response
to the arterial stretch (Johnson, 1986). The myogenic response is the propensity of
blood vessels to respond to transmural pressure elevation with constriction and to
pressure reduction with dilation. This behavior is inherent to smooth muscle and
is most pronounced in arterioles but can be demonstrated occasionally in arteries,
veins, and lymphatics (Johnson, 1986). The late phase of the response to changes
in perfusion is a flow-dependent dilation that overcomes the myogenic constriction
leading to increased vessel diameter. Neuraminidase digestion completely inhibited
the late phase of flow-induced vasodilation and enhanced the myogenic constriction
of the artery (Pohl et al., 1991).
More recently, Florian et al. studied the production of NO in cultured endothe-
lial cells under shear stress (Florian et al., 2003). The cells were treated with an
enzyme to digest heparan sulfate groups (heparinase) and then exposed to various
shear stress regimes. This study demonstrated that heparan sulfate digestion leads
to about a three-fold decrease in the NO production after exposure to steady shear
stress as well as a four-fold decrease in the NO production following treatment with
oscillatory shear stress. Similar studies found that pre-digestion with heparinase
did not inhibit bradykinin-induced increases in NO production. Additional stud-
ies on endothelial cells in culture from this group also demonstrated that digestion
with neuraminidase and hyaluronidase both attenuated shear induced NO release but
digestion with chondroitinase did not. In addition, shear induced increases in PGI2
were not affected by removal of heparan sulfate, sialic acid, chondroitin sulfate or
hyaluronan (Pahakis et al., 2007).
In a recent study, VanTeefelen et al. hypothesized that the binding of heparin to
the glycocalyx would alter the mechanotransduction of shear stress in the endothe-
lium (Van Teeffelen et al., 2007). They found that treatment with clinical levels of
heparin led to a reduction in the duration of vasodilation in response to occlusion-
induced reactive hyperemia in mice. This again adds to the evidence that heparan
sulfate is involved in NO-mediated vasodilation in response to changes in fluid flow.
While there are multiple studies supporting a role for glycocalyx proteins in the
arterial mechanotransduction of shear forces, the mechanisms remain to be uncov-
ered. The majority of studies have used digestive enzymes whose activity is not
necessarily restricted to the luminal surface or to endothelial cells versus vascular
smooth muscle cells. Thus, it remains unclear if the model of fluid shear acting
on the glycocalyx is correct in the case of heparan sulfate proteoglycans or if these
effects are due to alterations in heparan sulfate mediated extracellular matrix interac-
tion. Another notable complexity is that heparan sulfate can mediate the vasodilatory
effects of released factors such as FGF and can consequently have indirect effects
on these processes (Cuevas et al., 1991). One recent study examined the role of
9 Role of Proteoglycans in Vascular Mechanotransduction 229

the glycocalyx and the reactive oxygen species in the shear-induced NO release
(Kumagai et al., 2009). In this work, it was found that the superoxide dismutase
mimic superoxide scavenger (Tempol) restored the vasodilatory response and NO
increase in heparinase or neuraminidase digested arteries. However, Tempol did not
inhibit the vasodilation/NO response in hyaluronidase digested arteries. Endothelial
superoxide dismutase (SOD) catalyses the dismutation of superoxide into oxygen
and hydrogen peroxide and thus has anti-oxidative properties. Heparan sulfate binds
to endothelial SOD and can localize in the extracellular matrix. It has an important
role in maintaining endothelial NO as it diffuses to the vascular smooth muscle
cells in the arterial wall. Thus, the SOD binding properties of heparan sulfate may
represent a potential mechanism of involvement of heparan sulfate in flow-induced
vasodilatory responses.

9.3.2 Glycocalyx in Shear Stress Induced Vascular


Smooth Muscle Cell Contraction
While shear-stress induced responses are most often thought of as being associ-
ated with endothelial cells, vascular smooth muscle cells can also be exposed to
fluid flow shear stress after endothelial denudation and through transmural intersti-
tial flow (Cohen et al., 1995). Although vascular stretch has been proposed as the
major mechanism in determining myogenic response, other studies suggest that this
response can occur in the presence of increased pressure without vascular stretch.
Ainslie et al. examined the contraction of vascular smooth muscle cells in culture
after expose to a step increase in shear stress (Ainslie et al., 2005). Digestion of the
chondroitin sulfate or heparan sulfate chains inhibited contraction after exposure to
shear stress, suggesting a potential role of chondroitin sulfate/heparan sulfate in this
behavior.

9.3.3 Role of HSPGs in Controlling Mechanically-Induced


Changes in Cell Migration, Proliferation and Adhesion

Moon et al. found that disruption of HSPGs by heparinase decreased endothelial


adhesion and strength of adhesion (Moon et al., 2005). Further, heparinase digestion
decreased actin stress fiber formation, focal adhesions and enhanced cellular motil-
ity. Under flow conditions cells with digested HSPGs lost the directional migration
induced by fluid flow. In addition, loss of heparan sulfate lead to a reduction in focal
adhesion formation and recruitment of focal adhesion kinase (FAK) aligned with
fluid flow. Fluid flow is also known to reduce the proliferation of endothelial cells
and induce alignment in the direction of flow. Yao et al. showed that both of these
phenomenons did not occur after heparinase III digestion of the cultured endothe-
lial cells (Yao et al., 2007). In addition, they found that fluid flow induced a cellular
redistribution of HSPGs to the cell-cell interface of the endothelial layer.
230 A.B. Baker

9.4 Mathematical Models of Glycocalyx-Based


Mechanotransduction

Weinberg and colleagues have proposed a general model of fluid glycocalyx interac-
tion and force transmission (Weinbaum et al., 2003). Their model assumed that the
glycocalyx has a quasiperiodic structure based on experimental observations (Squire
et al., 2001). These studies revealed the ultrastructural details of the glycocalyx as
a network with a characteristic spacing of 20 nm with periodic centers spaced at
1012 nm. In their model the glycocalyx elements are linked to the cortical layer of
actin that links the dense peripheral actin band. Their model predicts that the gly-
cocalyx is fairly rigid and reduced the fluid shear stress at the surface of the cell to
almost zero. As fluid flows over the surface of the glycocalyx the resultant torque
is applied through first ~25 nm of the glycocalyx. This torque is transmitted to the
cortical actin and applied to the dense peripheral actin band causing disruption of
this actin network. Their theory was consistent with the observations of fluid stress
causing disruption of this actin network (Weinbaum et al., 2003).

9.5 Long Term Adaptations of the Vascular System


to Alterations in Mechanical Stresses
While the majority of studies have examined the role of proteoglycans in acute cel-
lular and arterial vasodilatory responses, the role of these molecules in the long
term changes that occur in vascular remodeling has been less well studied. Tissue
level vascular remodeling is highly dependent on the mechanical environment. An
increase in shear stress in arteries and arterioles causes the artery to remodel to
expand the luminal diameter. Conversely, reduction in flow leads to decreased arte-
rial diameters in the long term. These observations lead Murray to suggest that the
artery adapts to normalize the wall shear stress to a preferred, homeostatic level
(Murray, 1926). Murrays Law has been generally validated for most arteries
(Kamiya et al., 1984). Cyclic circumferential arterial stress arises due to blood pres-
sure during the cardiac cycle. Similar to the control of luminal diameter to normalize
arterial shear stress, the artery adjusts the number of elastic laminae and arterial
thickness to obtain a preferred wall stress due to arterial pressure.
The role of proteoglycans in these long term adaptations to mechanical stimuli
are relatively unknown. The normal arterial structure of the vessel results from the
dynamic communication between the endothelium, vascular smooth muscle cells,
tissue resident/recruited inflammatory cells and adventitial cells surrounding the
artery. In the context of controlling arterial structure, endothelial cells produce sev-
eral inhibitory factors that limit the growth of vascular smooth muscle cells within
the artery (Ettenson et al., 2000; Nugent et al., 2000). Among these, the heparan
sulfate proteoglycans are potent inhibitors of vascular smooth muscle proliferation
and migration. Both heparin and endothelial cell heparan sulfate proteoglycans are
potent inhibitors of vascular smooth muscle cell proliferation and FGF-2 induced
9 Role of Proteoglycans in Vascular Mechanotransduction 231

mitogenesis (Clowes and Karnowsky, 1977; Ettenson et al., 2000; Nugent et al.,
2000). Heparan sulfate-induced regulation is growth state dependent, with sub-
confluent cultures of endothelial cells stimulating vascular smooth muscle cell
growth and postconfluent cultures inhibiting vascular smooth cell growth (Ettenson
et al., 2000). Consistent with these findings, both, perlecan and endothelial-derived
heparan sulfate proteoglycans have been shown to be essential in inhibiting the
neointimal response to vascular injury (Nugent et al., 2000). A recent study from our
group aimed to examine how mechanical forces alter the dynamic interplay of reg-
ulatory signals between endothelial and vascular smooth muscle cells (Baker et al.,
2008). In this work, we applied mechanical stretch to cultured endothlelial cells
and examined their production of growth-inhibitor heparan sulfate proteoglycans.
Mechanical stretch stimulated increase endothelial inhibition of vascular smooth
cell proliferation. Heparan sulfate proteoglycans and the core protein perlecan were
both in creased by exposure to mechanical stretch. Knock down studies demon-
strate that perlecan was required to increase vascular smooth muscle cell inhibition.
Experiments using small molecule inhibitors and neutralizing antibodies demon-
strated that a complex autocrine signaling cascade controlled the mechanical-load
induced production of the growth inhibitory heparan sulfate proteoglycan perlecan
(see Fig. 9.4). This pathway involved autocrine TGF-, ERK and p38 MAPK signal-
ing to induce perlecan expression in endothelial cells and lead to increased vascular
smooth muscle growth inhibition. Using ex-vivo arteries exposed to high pressure
under physiological flow conditions, these pathways were found to regulate the
production of heparan sulfate proteoglycans in intact arterial segments.

Fig. 9.4 Mechanical feedback control mechanisms of arterial structure in response to mechanical
stretch. The molecular signaling pathway shown refers to the pathways involved in controlling the
mechanical load-induced endothelial production of growth inhibitory heparan sulfate proteogly-
cans. These factors are increased with mechanical load and partially inhibit the pro-growth signals
in vascular smooth muscle cells
232 A.B. Baker

The finding that endothelial cells produce more inhibitory factors to vascular
smooth muscle cells with mechanical load is in contrast with the behavior of vas-
cular smooth muscle cells under similar mechanical strain conditions. Mechanical
stretch induces vascular proliferation (Hishikawa et al., 1997; Morita et al., 2004),
release of FGF (Cheng et al., 1997) production of PDGF (Ma et al., 1999), and pro-
duction of cell surface associated heparan sulfate proteoglycans (Li and Chaikof,
2002) (Fig. 9.4). Thus, under mechanical stimulation the endothelium provides
negative feedback control inhibiting the progrowth signals produced by vascular
smooth muscle cells under mechanical strain. Consequently, in the absence of an
intact endothelium due to disease of injury mechanical stretch would induce vascu-
lar smooth muscle cells to produce growth stimulatory factors that are unchecked by
load-enhanced endothelial cell paracrine growth inhibition. With endothelial repair,
the inhibitory responsiveness to mechanical strain is restored as well as additional
means of responding to hemodynamic forces can be applied including regulation of
vascular tone and inhibition of vascular smooth muscle cell proliferation.

9.6 Conclusion and Perspectives


There remain many unanswered questions in terms of the role of proteoglycans in
mechanotransduction. While there is ample evidence that these molecules partici-
pate in sensing and responding to mechanical forces, there is little data that directly
addresses the role of individual core proteins in mechanotransduction. Several
studies have demonstrated that the expression of syndecan-1 and syndecan-4 are
increased in mechanically stimulated cells. The syndecans are good candidates for
mechanotransduction pathways as they have multiple interactions with integrins,
focal adhesion, cytoskeletal elements and growth factor signaling (Morgan et al.,
2007). While the last decade has yielded many new insights into the basic biology
of proteoglycans, future studies will hopefully be able to shed additional light on
the roles these molecules play in mechanotransduction and vascular remodeling.

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Part IV
Mechanotransduction in the Lung
Chapter 10
Control of TRPV4 and Its Effect on the Lung

James C. Parker and Mary I. Townsley

Abstract The transient receptor potential vanilloid 4 (TRPV4) non-selective cation


channel has emerged as a critical channel for initiating the increased vascular
permeability induced by high airway or vascular pressures in the lung. TRPV4 gat-
ing is regulated by multiple factors: mechanical stress, heat, epoxyeicosatrienoic
acids (EETs) the arachidonic acid metabolites of P450 epoxygenases, and phor-
bol esters. Increased pulmonary venous pressure and ventilation with high peak
inflation pressures increase endothelial calcium influx, nitric oxide production, and
vascular permeability in a TRPV4 dependent fashion in intact lungs. The perme-
ability response to excess mechanical stress is attenuated by inhibition of cytosolic
phospholipase A2 or P450 epoxygenases, and permeability increases in response
to infusion of EETs. Various molecular mechanisms have been implicated for
regulating TRPV4 gating, including channel translocation, direct ligand binding
and phosphorylation. However, the mechanisms for EET dependent regulation of
TRPV4 or amplification of TRPV4 by phosphorylation in intact lungs subjected to
mechanical stress have not been clarified.

Keywords Transient receptor potential vanilloid Ventilator induced lung injury


Calcium Pulmonary hypertension Pulmonary edema Epoxyeicosatrienoic acids

10.1 Mechanical Stress in Lung and TRPV4

The lung is continuously subjected to mechanical movement and cellular strain


during breathing. As the lung inflates, extra-alveolar vessels, airways and alveolar
sacs elongate and/or distend. Similarly, with an increase in alveolar volume, lateral
forces in the alveolar septal wall lead to expansion of its surface area and elongation

J.C. Parker (B)


Department of Physiology and Center for Lung Biology, MSB 3074, University of South Alabama,
Mobile, AL 36688, USA
e-mail: jparker@usouthal.edu

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 239


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_10,

C Springer Science+Business Media B.V. 2011
240 J.C. Parker and M.I. Townsley

and flattening of capillaries. The pulmonary circulation is also in constant motion


due to circumferential and shear stresses which accompany pulsatile blood flow.
While lung function is maintained over a physiological range of mechanical stress,
excessive stress is associated with acute lung injury.
The transient receptor potential vanilloid 4 (TRPV4) channel is an important sen-
sor for detecting mechanical stress induced by hypotonicity (cell swelling), shear,
and stretch (Nilius et al., 2004; Cohen, 2005; Nilius and Voets, 2005; Liedtke, 2006;
Townsley et al., 2006). TRPV4, a member of the TRPV family of cation channels,
is expressed in lung epithelium, endothelium, fibroblasts and macrophages (Alvarez
et al., 2006). Among other members of this TRPV family, TRPV4 has the great-
est responsiveness to mechanical perturbation and is likely involved in cell volume
regulation and mechanical sensing in all lung cells. Single channel conductance for
TRPV4 is approximately 100 pS for outward currents and 60 pS for inward cur-
rents; inward currents can be blocked by ruthenium red. The channel permeability
for Ca2+ is 6- to 10-fold higher than that for sodium (Everaerts et al., 2010; Nilius
et al., 2004). Yin et al. (2008) reported inward calcium currents in whole cell patch
clamp recordings in pulmonary microvascular endothelial cells after treatment with
the TRPV4 agonist, 4-phorbol-12,13-didecanoate (4PDD) which was inhibited
with 8Br-cGMP.
TRPV4 channels are gated by multiple stimuli and likely integrate a num-
ber of external stimuli for cell responses. Activation occurs during hypotonic
stress and at temperatures above 27 C. Vreins et al. (2006) demonstrated that
cell swelling-induced activation of TRPV4 required phospholipase A2 -mediated
release of arachidonic acid and subsequent metabolism of arachidonic acid by P450
epoxygenases to epoxyeicosatrienoic acids (EETs). Further, the P450 epoxygenase
products 5,6- and 8,9-EET appear to activate TRPV4. Watanabe et al. (2003a)
reported Ca2+ transients via heterologously expressed TRPV4 in HEK293 cells in
response to arachidonic acid, which were completely abolished by blockade of P450
epoxygenase, indicating that EETs were involved in TRPV4 activation. Both 5,6-
and 8,9-EET evoked Ca2+ responses via the heterologously expressed channel and
the endogenous TRPV4 in endothelium, with a smaller response to other EETs.
Blockade of the cyclooxygenase and soluble epoxide hydrolases which degrade
EETs also enhanced the swelling- and arachidonic acid-induced Ca2+ transients in
mouse aortic endothelial cells (Vriens et al., 2005). Similar to hypotonicity-induced
activation of TRPV4, shear stress activates heterologously expressed TRPV4 as
well as endogneous TRPV4 in systemic endothelium in a phospholipase A2 -and
EET-dependent manner (Hartmannsgruber et al., 2007; Loot et al., 2008; Wegierski
et al., 2009). The activation of TRPV4 elicited by by 4-phorbol-12,13-didecanoic
acid (4PDD) and heat does not appear to require EETs (Vriens et al., 2004).

10.1.1 Stretch Activated Cation Channels and Lung Injury


Calcium entry through stretch activated cation channels and arachidonic acid
metabolites have been previously implicated in acute lung injury. Parker et al.
10 Control of TRPV4 and Its Effect on the Lung 241

(1998a) reported a 3.7-fold increase in the filtration coefficient (Kf ), a sensitive mea-
sure of lung endothelial permeability, in isolated perfused rat lungs ventilated with
30 cmH2 O peak inflation pressure (PIP). This mechanical stretch-induced injury
was completely prevented after treatment with gadolinium, a blocker of stretch
activated cation channels. Subsequent studies confirmed that the increases in Kf
and lung edema induced by mechanical ventilation were prevented by gadolinium
(Parker and Yoshikawa, 2002). Although these studies implicated a mechanically
gated Ca2+ permeable channel, the molecular nature of this stretch activated channel
was unknown at the time.
Previous studies had also implicated several signaling pathways now acknowl-
edged as important for regulation of TRPV4 channel gating. Townsley et al. (1990)
observed a synergistic impact of arachidonic acid and high vascular pressure on
increases in Kf in isolated, perfused dog lungs. In both intact mice and isolated
mouse lungs, the permeability lesion and edema resulting from high PIP ventila-
tion were attenuated after blockade of cytosolic phospholipase A2 (Miyahara et al.,
2008; Yoshikawa et al., 2005). Alvarez et al. (2004) reported an increase in Kf in
isolated rat lungs of approximately 2.5-fold after infusion of 5,6- and 14,15-EET,
whereas 8,9- and 11,12-EET had no significant impact. The permeability responses
to 5,6- and 14,15-EET were attenuated by low extracellular Ca2+ but not by phos-
pholipase A2 or P450 epoxygenase inhibitors. Figure 10.1 summarizes the impact of
extracellular Ca2+ on Kf measurements in isolated rat lungs treated with the TRPV4

Fig. 10.1 Filtration coefficients (Kf ) in isolated rat lungs showing the effect of treatment with 4-
phorbol-12,13-didecanoate (4PDD), epoxyeicosatrienoic acids (EETs), and thapsigargin (TG).
During baseline (BL) Kf and the initial Kf measurement after addition of channel agonists, a low
Ca2+ perfusate was used. Subsequently, Ca2+ addback in the same lungs brought extracellular Ca2+
to a physiologic concentration (Alvarez et al., 2006)
242 J.C. Parker and M.I. Townsley

agonists 4PDD, 5,6-EET or 14,15-EET, compared to that in lungs treated with


thapsigargin, which activates store-operated Ca2+ channels (Alvarez et al., 2006).
Kuebler et al. (2002) demonstrated that increases in venous pressure as small
as 5 cmH2 O evoked Ca2+ entry in venular endothelium in isolated perfused
lungs. These Ca2+ increases were abolished by gadolinium and Ca2+ -free per-
fusate. In other studies this group found that elevated left atrial pressure in isolated
lungs induced increases in endothelial Ca2+ accompanied by P-selectin expression
(Kuebler et al., 1999). However, Wu and colleagues recently documented a depen-
dence of P-selectin expression on the voltage-gated T-type Ca2+ channel, rather than
TRPV4 (Wu et al., 2009). Lung stretch induced by either high vascular pressure or
by ventilation with high airway pressure induced production of endothelial nitric
oxide (Kuebler et al., 2003), which could be blocked by inhibitors of nitric oxide
synthase (NOS) or phosphatidylinositol-3-0H kinase. Kuebler et al. (2003) demon-
strated a cumulative, linear, time dependent NO production measured by DAF-FM
fluorescence in venular capillary endothelium in situ in isolated rat lungs induced by
an increase in venous pressure of only 10 cmH2 O. Finally, Ito et al. (2010) reported
that stretch of cultured pulmonary microvascular endothelial cells induced increases
in intracellular Ca2+ that were sensitive to the TRPV antagonist, ruthenium red.

10.1.2 TRPV4 is Critical for Pressure Induced Lung Injury

The TRPV4 channel has now been identified as a stretch-activated cation channel
responsible for lung injury produced by both high vascular and airway pressures.
Hamanaka et al. (2007) observed that high airway pressure increased Kf in isolated
lungs of TRPV4+/+ mice (solid bars) but not in lungs of TRPV4-/- mice (shaded
bars). Heat (40 C) amplified the mechanical injury only in TRPV4+/+ mice. The
endothelial Ca2+ transients observed in TRPV4+/+ lungs during distention by high
airway pressure were absent in TRPV4/ mice and in TRPV4+/+ mice treated with
ruthenium red, a TRPV channel antagonist. Lung injury was also blocked by inhi-
bition of anandamide hydrolysis or cytochrome P450 epoxygenases. Figure 10.2
indicates the effects of high PIP ventilation, increased temperature, and inhibition
of these pathways on Kf in isolated mouse lungs from both phenotypes.
Jian et al. (2008) also measured a 4.5-fold increase in Kf in isolated lungs
from TRPV4+/+ mice after venous pressure elevations of 30 cmH2 O, which was
attenuated by approximately 80% in lungs from TRPV4/ mice and ruthe-
nium red-treated lungs from TRPV4+/+ mice. Significant attenuation of Kf was
also achieved by phospholipase A2 and cytochrome P450 epoxygenase inhibitors.
Endothelial Ca2+ transients and alveolar edema volumes were attenuated after pres-
sure stress in TRPV4/ lungs and by inhibition of Ca2+ entry, TRPV channels,
and P450 epoxygenases. Figure 10.3 shows the increase in intracellular Ca2+ after
an increase in pulmonary venous pressure in isolated mouse lungs from wild type
mice untreated, treated with ruthenium red or treated with the P450 epoxygenase
inhibitor (PPOH), and lungs from TRPV4 knockout mice either untreated or treated
with gadolinium.
10 Control of TRPV4 and Its Effect on the Lung 243

Fig. 10.2 Impact of high


airway pressure ventilation on
filtration coefficients in lungs
isolated from TRPV4+/+ and
TRPV4/ mice; (a) at 35 C,
(b) at 40 C, and (c) after
treatment of TRPV4+/+ lungs
with inhibitors of TRPV,
P450 epoxygenase and
arachidonic acid (Hamanaka
et al., 2007). PIP, peak
inflation pressure

In situ fluorescence studies of lung endothelium by Yin et al. (2008) also indi-
cated an increase in intracellular Ca2+ after a venous pressure increase of only 15
cmH2 O. Calcium entry was enhanced by blockade of soluble guanylate cyclase
(sGC) or nitric oxide synthase (NOS), but decreased by the nitric oxide generator,
S-nitrosoglutathione (GSNO), a sGC activator drug, or the cGMP analog, 8Br-
cGMP. Kf measurements in the isolated rat lungs mirrored the changes in endothelial
Ca2+ . Using a nitric oxide (NO) sensitive dye, endothelial production of NO also
244 J.C. Parker and M.I. Townsley

Fig. 10.3 Relative Ca2+


fluorescence in lungs isolated
from TRPV4+/+ (WT) and
TRPV4/ (KO) mice
exposed to increased venous
pressure (Pv) with and
without treatment with P450
epoxygenase and TRPV
inhibitors (Jian et al., 2008)

increased as venous pressure was increased, but was attenuated by the sGC activator
or cGMP analogue. The vascular pressure-induced increase in Kf was attenuated by
gadolinium or the NO donor GSNO, but was further increased by blockade of nitric
oxide synthase (NOS). The increase in endothelial Ca2+ elicited by 4PDD was
also blocked by both ruthenium red and the cGMP analogue. Sildenafil, an inhibitor
of PDE5, also attenuated the pressure-induced increases in lung Kf , protein leak
and edema formation. These data indicate a TRPV4-mediated Ca2+ increase in lung
endothelium after venous pressure elevation which increased vascular permeabil-
ity, but which simultaneously activates a negative feedback control of the Ca2+ and
permeability increases mediated by cGMP.

10.1.3 Phosphorylation and Mechanical Injury

Activation of Src family kinases or myosin light chain kinase (MLCK) during
mechanical distention of the lung appear to mediate the increased vascular per-
meability. Yin et al. (2008) found that inhibition of myosin light chain kinase
(MLCK) protected against the permeability response to increased vascular pressure
in isolated rat lung. Inhibition of MLCK or tyrosine kinases also attenuated air-
way pressure induced injury, whereas inhibition of tyrosine phosphatase augmented
injury (Miyahara et al., 2007; Parker et al., 1998b). The mechanism by which
MLCK amplifies TRPV4-mediated Ca2+ entry and lung injury is unknown, but may
relate to the dependence of TRPV4 channel activity on actin cytoskeleton attach-
ments and tension within the cytoskeleton (Becker et al., 2009; Ramadass et al.,
2007). Alternatively, direct phosphorylation of TRPV4 may play a role. For exam-
ple, the Src family kinases have been shown to amplify TRPV4 activity through
phosphorylation of tyrosine 253 (Xu et al., 2003). Involvement of other kinases
such as Akt/PKB may play a protective role. In isolated mouse lung, inhibition of
Akt amplified the high airway pressure vascular permeability lesion (Miyahara et al.,
2007), suggesting a protective pathway that likely acts through NO feedback.
10 Control of TRPV4 and Its Effect on the Lung 245

10.1.4 Segmental Vascular Effects of TRPV4


Figure 10.4 shows segmental and total Kf data in isolated rat lungs ventilated
at high peak inflation pressures (PIP) in untreated lungs (solid bars) and lungs
treated (shaded bars) with gadolinium chloride (Parker and Yoshikawa, 2002).
The largest impact of ventilator-induced lung injury was on the lung microvas-
cular compartment. Notably, segmental Kf values in high-pressure ventilated,
gadolinium-treated lungs were not significantly different from baseline measure-
ments. Although TRPV4 is expressed in endothelial cells in both alveolar and
extra-alveolar vessels, activation of TRPV4 has the greatest effect on permeability
in alveolar capillaries. Using electron microscopy, Alvarez et al. (2006) observed
preferential separation of endothelial and epithelial layers as well as breaks in these
layers in the alveolar septal wall in rat or mouse lung after TRPV4 activation. In
contrast, activation of store-operated Ca2+ channels with thapsigargin preferentially
disrupted inter-endothelial junctions of extra-alveolar vessels. Activation of store-
operated channels in cultured alveolar capillary endothelial cells results in only
a small Ca2+ response with no concomitant permeability response (Cioffi et al.,
2009). In contrast, pulmonary extra-alveolar vessels and pulmonary artery endothe-
lial cells in culture which express TRPC1, 3, 4, and 6 display robust Ca2+ currents
during store depletion and significant permeability increases (Townsley et al., 2006;
Chetham et al., 1999).
The impact of such heterogenous responses to activation of TRPV4 vs store-
operated channels on lung function has been investigated in a series of recent
studies. In lungs treated with doses of either 4PDD or thapsigargin sufficient
to increase Kf by threefold, markedly different distributions of edema formation
resulted. Alvarez et al. (2006) reported that only activation of TRPV4 was asso-
ciated with alveolar flooding, as measured by the alveolar fluid volume fraction.
Subsequently, Lowe et al. (2007) confirmed the impact of TRPV4 activation on

Fig. 10.4 Segmental and


total filtration coefficients in
isolated rat lungs after
ventilation with high peak
inflation pressures (PIP),
showing attenuation of the
injury by pretreatment with
gadolinium (Parker and
Yoshikawa, 2002,
Reproduced with permission
from the American
Physiological Society)
246 J.C. Parker and M.I. Townsley

Fig. 10.5 Photomicrographs of toluidine blue-stained sections of rat lung showing preferential
distribution of lung edema in perivascular cuffs induced by thapsigargin (a) and in alveoli after
activation of TRPV4 with 14,15-EET (b). Pa, pulmonary artery; AF, alveolar fluid

alveolar flooding and further noted that TRPV4-mediated lung injury was associ-
ated with little perivascular cuff formation. In contrast, thapsigargin- treated lungs
had cuff formation without alveolar flooding. Although the lungs in both groups
gained approximately the same amount of edema, the thapsigargin-treated lungs
had a threefold greater decrease in dynamic lung compliance compared to 4PDD-
treated lungs. Perivascular cuff formation presumably facilitated a collapse of the
small airways to reduce compliance. Figure 10.5 contrasts the cuff formation in
isolated rat lungs treated with thapsigargin (A) with the alveolar flooding result-
ing from TRPV4 activation via 14,15-EET (B), even when the increase in Kf was
similar in these lungs. In addition to specificity for segmental vascular permeabil-
ity, the TRPV4 channel also exhibits functional specificity within the alveolar septal
endothelium. Wu et al. (2009) observed that TRPV4 activation increased endothelial
cytosolic Ca2+ and Kf in isolated mouse lungs, without causing P-selectin expres-
sion. The same increase in intracellular Ca2+ elicited by a high potassium perfusate
and activation of T-type Ca2+ channels resulted in P-selectin expression without any
increase in permeability.

10.2 TRPV4 Structure and Potential Regulatory Sites


As previously discussed, growing evidence supports a role for EETs as the signal-
ing link between mechanical stress, evoked by hypotonicity, stretch or shear, and
gating of the TRPV4 channel. All of the EET regioisomers have been shown to
activate TRPV4 in vitro. Pinpointing the endogenous EET regioisomer involved is
difficult, since each epoxygenase metabolizes arachidonic acid to produce a dis-
tinct mixture of EETs. Based on in vitro studies with microsomal fractions from
10 Control of TRPV4 and Its Effect on the Lung 247

rat and human lung, 14,15- and 11,12-EET proved to be the predominant products
(Zeldin et al., 1996), though all were present in some amount. Thus, availability
of EETs during mechanotransduction in lung cells will depend upon the epoxyge-
nase expressed in the lung cell exposed to mechanical stress, as well as expression
of the key enzyme responsible for EET degradation soluble epoxide hydrolase
(Spector, 2009). Interestingly, epoxygenase expression is itself up-regulated by
mechanical stress (Fisslthaler et al., 2001). While in vitro studies have identified
a number of regulatory domains in TRPV4, the molecular mechanisms underly-
ing mechanotransduction-initiated and EET-dependent activation of TRPV4 have
not been resolved. The TRPV4 channel consists of 871 amino acids with 6 trans-
membrane spanning segments and cytoplasmic C and N termini. The pore region
lies between transmembrane segments 5 and 6, as shown in Fig. 10.6. EETs could
potentially impact TRPV4 gating via a number of mechanisms.

10.2.1 Oligomerization and Membrane Localization

Crystallography has resolved six ankyrin repeats in TRPV4s N-terminus (reviewed


in (Everaerts et al., 2010), which function in protein recognition. Splice variants
of human TRPV4 lacking exons 5 and/or 7 which code for sequences within this
region failed to produce Ca2+ transients in response to hypotonicity, 4PDD, or
arachidonic acid (Arniges et al., 2006). Arniges et al. reported that these variants
also failed to localize to the plasma membrane due to their inability to oligomerize
in the endoplasmic reticulum (Arniges et al., 2006). More recently, Becker and col-
leagues determined that the most distal C-terminal sequence (amino acids 828871)
of TRPV4 was also required for oligomerization and membrane trafficking (Becker
et al., 2008).

Fig. 10.6 Diagram of


TRPV4 structure showing
potential sites for regulation
of channel trafficking or
gating. See text for more
detail
248 J.C. Parker and M.I. Townsley

In addition to proper assembly, surface expression of TRPV4 is also modulated


by ubiquitination, binding of PACSIN3 or microfilament-associated protein 7, and
glycosylation (Wegierski et al., 2006; Cuajungco et al., 2006; Suzuki et al., 2003; Xu
et al., 2006). PACSIN3 specifically interacts with a proline-rich domain upstream
of the ankyrin repeats (Cuajungco et al., 2006). While these in vitro observations
lend insight into assembly and trafficking of TRPV4, there are no reports which
specifically evaluate whether active shuttling of TRPV4 plays a role in the response
to mechanical stress in the intact lung, that is, whether mechanical stress evokes
insertion of channels into the plasma membrane. Regulated trafficking is recog-
nized for some TRP channels (reviewed in (Niemeyer, 2005; Cayouette and Boulay,
2007). Loot and colleagues (Loot et al., 2008) observed a shift in TRPV4 localiza-
tion from a perinuclear distribution to the plasma membrane in cultured endothelial
cells exposed to shear. This active trafficking required P450 epoxygenase activity.
These observations need to be confirmed in lung cells exposed to mechanical stress
in vivo.

10.2.2 Ligand Binding Pocket

Activation of TRPV4 by 4PDD is independent of the pathways utilized by cell


swelling. The ligand-binding pocket between transmembrane domains 3 and 4 (S3
and S4) binds synthetic ligands such as the phorbol ester 4PDD, while the binding
domain for GSK1016790A has not been defined (Everaerts et al., 2010; Watanabe
et al., 2002; Willette et al., 2008). Mutation of critical amino acids in these two trans-
membrane domains, specifically Y556, L584, and W586, markedly diminished the
Ca2+ response to 4PDD (Vriens et al., 2007). However, this binding pocket is not a
likely site for EET interaction with TRPV4, since binding efficacy appears to require
a dual ring structure (Everaerts et al., 2010) not present in EETs. Further, the Ca2+
response to 5,6-EET remained intact when these S3/S4 mutants were heterologously
expressed. Vriens and colleagues consider the diminution of the Ca2+ response to
hypotonicity, arachidonic acid and 5,6-EET resulting from mutation of more dis-
tal residues in S4 (Y591 and R594) to be due to altered channel gating rather than
altered ligand binding. Phorbol esters other than 4PDD, such as phorbol myristate
acetate (PMA), also activate TRPV4 (Vriens et al., 2007), though in part activation
is secondary to PKC-mediated phosphorylation. Although 4PDD activates TRPV4
directly, heat appears to act through a soluble mediator.

10.2.3 Direct Binding of EETs

Watanabe et al. found that 5,6-EET activated TRPV4 in inside-out membrane


patches prepared from HEK293 cells or endothelial cells expressing heterologous
or endogenous TRPV4, respectively (Watanabe et al., 2003a). This evidence has let
to the conclusion that EETs activate TRPV4 in a membrane-delimited fashion and
10 Control of TRPV4 and Its Effect on the Lung 249

thus by direct binding to the channel. However, based on evidence derived from
studies of EET-dependent regulation of other ion channels, the mechanism of action
could be more complex. In coronary artery smooth muscle cells, EETs activate the
large conductance Ca2+ -activated potassium channel (BKCa) in inside-out patches
via a mechanism which requires GTP and Gs (Li and Campbell, 1997). Further,
EETs appear to elicit responses via binding to G protein-coupled receptors (Wong
et al., 1997, 2000; Yang et al., 2008), suggesting the possibility for outside-in sig-
naling. EETs activate Gs , resulting in synthesis of cAMP and activation of PKA
(Yang et al., 2008; Carroll et al., 2006; Node et al., 2001; Spector and Norris, 2007).
While PKA-mediated phosphorylation has been implicated in TRPV4 activation
with hypotonicity (see below), a role for G protein-coupled receptors, Gs activa-
tion or cAMP synthesis has not been evaluated in lungs response to mechanical
stress.

10.2.4 Phosphorylation
Domain mapping of the TRPV4 sequence has highlighted a number of poten-
tial phosphorylation sites (Nilius et al., 2004; Everaerts et al., 2010; Fan et al.,
2009) for PKC, PKA and Src family tyrosine kinases. Kuebler and colleagues have
recently reported that NO- and cGMP-dependent negative feedback regulation of
TRPV4 gating in lung endothelium exposed to hydrostatic stress (Yin et al., 2008).
However, this feedback control may not be direct, as there does not appear to be a
PKG phosphorylation site in TRPV4. Most relevant to mechanical stress-induced
EET-dependent regulation of TRPV4 are PKA and the Src kinases. EET-induced
activation of Gs leads to recruitment of cAMP-PKA signaling (Yang et al., 2008;
Carroll et al., 2006; Node et al., 2001; Spector and Norris, 2007; Imig et al., 2008).
While EETs are known to activate a number of tyrosine kinase cascades via Src
kinases, MAPK or PI3 kinase (Spector and Norris, 2007), a role for tyrosine phos-
phorylation of TRPV4 in response to mechanical stress remains controversial. Xu
et al. surveyed a number of Src kinase family members, investigating a role for tyro-
sine kinase-mediated phosphorylation of TRPV4 in the response to hypotonicity
(Xu et al., 2003). Using a mutagenesis approach, these investigators mapped the Src
kinase-mediated phosphorylation to Y253, within the first ankyrin repeat domain.
While others found no role for tyrosine phosphorylation in hypotonicity-induced
TRPV4 activation (Vriens et al., 2004), a recent study utilizing a mass spectromet-
ric analysis has implicated Y110 in regulation of TRPV4 function (Wegierski et al.,
2009). While translation of these observations to the in vivo setting is limited, the
available evidence does support a role for tyrosine phosphorylation in the response
to mechanical stress. Parker and colleagues have observed tyrosine phosphoryla-
tion in lung following high pressure mechanical ventilation and have implicated
TRPV4, PI3K, Src kinases and Akt in the resultant ventilator-induced lung injury
(Parker et al., 1998b; Hamanaka et al., 2007; Miyahara et al., 2007). Similarly,
Src tyrosine kinases and TRPV4 have been implicated in mechanical hyperalgesia
(Alessandri-Haber et al., 2008).
250 J.C. Parker and M.I. Townsley

10.2.5 Other Regulatory Domains


TRPV4 contains several domains potentially regulated by protein-protein interac-
tions. In addition to the contribution of PACSIN3 binding to channel trafficking,
PACSIN3 binding to the N-terminal proline-rich domain in TRPV4 also inhibits
the basal activity of the channel and attenuates TRPV4 activation by hypotonicity
and heat (D hoedt et al., 2008). Further, deletion mutants lacking the N-terminal
proline-rich domain were unresponsive to hypotonicity and to 4PDD despite
proper localization of TRPV4 to the plasma membrane (Garcia-Elias et al., 2008).
This in vitro evidence highlights this region as critical for overall channel regulation.
Two additional protein-binding sites exist in the C-terminus: a calmodulin-binding
domain and a PDZ-like domain. Deletion of the PDZ-like domain does not alter
the response to hypotonicity or 4PDD (Garcia-Elias et al., 2008), but could par-
ticipate in interaction of TRPV4 with other PDZ domain scaffolding proteins
(Everaerts et al., 2010). The calmodulin-binding domain appears to participate in
Ca2+ -dependent regulation of TRPV4 (Garcia-Elias et al., 2008; Strotmann et al.,
2003). Interestingly, IP3 potentiates the Ca2+ response to 5,6-EET via IP3 receptor
binding to this calmodulin domain (Fernandes et al., 2008). Activation of TRPV4 is
also tightly controlled by both intracellular and extracellular Ca2+ levels. Strotman
et al. (2003) demonstrated potentiation of TRPV4 Ca2+ entry during the initial
increase in intracellular Ca2+ from baseline followed by a vigorous feedback inhi-
bition at higher Ca2+ levels. Feedback inhibition of the Ca2+ entry was also greatly
facilitated by increased extracellular Ca2+ concentration (Watanabe et al., 2003b).

10.2.6 Homo- or Heteromultimeric Channels

Current evidence for heteromultimeric assembly of TRPV subunits into functional


channels, based upon heterologous expression of FRET-TRPV reporter constructs,
is conflicting. Hellwig et al. concluded that most TRPV channel subunits including
TRPV4 preferentially assemble as homotetramers (Hellwig et al., 2005). In con-
trast, Cheng and colleagues found that TRPV proteins can form heteromultimeric
channels with other proteins in this TRP subfamily (Cheng et al., 2007). TRPV4
has also been reported to form heteromultimers with TRPP2 (Kttgen et al., 2008).
Since channel subunit stoichiometry can impact channel conductance and regula-
tion, more definitive information regarding assembly of TRPV4 alone or with other
potential TRP proteins in cultured lung cells and in the intact lung is needed. If
indeed TRPV4 in lung is expressed as a component of a heterotetrameric chan-
nel, then EET-dependent regulation of the channel with mechanical stress becomes
potentially more complex.

10.3 Conclusion and Perspectives


TRPV4 is ubiquitously present in lung cells and appears to be the major chan-
nel for transduction of mechanical signals in these cells. Calcium influx occurs
10 Control of TRPV4 and Its Effect on the Lung 251

with relatively modest increases in pulmonary vascular or airway pressures, and


the channel is essential for initiating the increased vascular permeability induced
by high vascular and airway pressures. Mechanical gating is mediated by epoxye-
icosatrienoic acids (EETs), whereas heat and phorbol ester activation occur through
different mechanisms. TRPV4 is a major mediator of injury to pulmonary microvas-
cular endothelium, which lacks the store operated TRPC channels of macrovascular
endothelium. Several potential regulatory sites on the TRPV4 molecule have been
identified for phosphorylation by Src family kinases, PKA, and PKC. Altered
membrane trafficking, oligomerization and binding to scaffold proteins have been
proposed as other potential regulatory mechanisms, but details of the mechanism
whereby EETs or phosphorylation control channel opening during mechanical stress
remain to be elucidated.
Acknowledgements Research support by NIH HL066299 and HL092992

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Chapter 11
The Role of Protein-protein Interactions
in Mechanotransduction: Implications
in Ventilator Induced Lung Injury

Matthew Rubacha and Mingyao Liu

Abstract Critically ill patients often require mechanical ventilation to support


their breathing. This is especially true for patients with Acute Respiratory Distress
Syndrome, where therapeutic intervention still remains largely ineffective. A clini-
cal study supported by the National Institutes of Health has indicated that low tidal
volume ventilation is beneficial, which has established mechanical ventilation an
important contributor to lung injury in these patients. Mechanical ventilation can
lead to increased production of cytokines and chemokines related to inflamma-
tion and tissue damage. Further understanding of mechanotransduction may reveal
targeting strategies for therapeutic intervention. It is known that cells can sense
mechanical forces across the plasma membrane through a variety of mechanisms.
In addition, intracellular force sensors have been proposed to play an important role
in conversion of physical forces into biochemical signals through protein-protein
interactions. In this chapter we reviewed this novel mechanism for mechanosensa-
tion and mechanotransduction, and proposed to inhibit Src protein tyrosine kinase
activation as a potential therapy for ventilator induced lung injury.

Keywords AFAP p130Cas Src Acute lung injury ARDS

11.1 Introduction
Like in many other solid organs, physical forces play an important role in the lung
physiology, and are involved in the regulation of fetal lung development (Liu and
Post, 2000), airway branching and alveolarization, and the regulation of pulmonary
physiology (Liu et al., 1999). The lung is unique from other organs in that it is

M. Liu (B)
Faculty of Medicine, University of Toronto, 101 College Street Toronto Medical Discovery Tower,
Room 2-814, Toronto, ON, M5G 1L7 Canada
e-mail: mingyao.liu@utoronto.ca

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 255


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_11,

C Springer Science+Business Media B.V. 2011
256 M. Rubacha and M. Liu

constantly expanding and contracting to facilitate the flow of air into and out of
a complex network of airways. The physical force associated with normal breath-
ing does not lead to aberrant side effects. Abnormal physical forces however, are
known to contribute to the pathogenesis of lung diseases, such as asthma, pulmonary
hypertension, chronic obstructive pulmonary disease, etc. Mechanical ventilation as
a therapeutic modality can be used to provide life support during surgical operation,
or to patients with respiratory distress. This externally applied physical force which
ventilators use to force air into the lungs can lead to tissue damage and is associated
with several side effects known as ventilator-induced lung injury (VILI) (Girard and
Bernard, 2007). Before clinicians can address VILI, we need to recognize and under-
stand more about the mechanisms and pathways of mechanotrasduction initiated
both by normal breathing and by mechanical ventilation.
Acute lung injury (ALI), and its most severe form, acute respiratory distress syn-
drome (ARDS), is defined by The American-European Consensus Conference on
ARDS as severe gas exchange dysfunction and radiographic abnormalities after
a predisposing injury in the absence of heart failure (Bernard et al., 1994). The
age-adjusted incidence of ALI is around 86.2 per 100,000 person-years. There are
approximately 190,600 patients diagnosed with ALI each year in the United States
which is associated with 74,500 deaths and 3.6 million hospital days, represent-
ing a significant cost to the healthcare system (Rubenfeld et al., 2005). It has been
known for some time that physical force associated with high tidal volume ven-
tilation contributes to the progression of ALI/ARDS (Liu, 2007). Since the first
recommendations from the ARDS network were published in 2000 (ARDSNetwork,
2000), the importance of controlled mechanical ventilation conditions has been real-
ized. Physicians are required to perform a balancing act between using mechanical
ventilation to support the lives of critically ill patients and limiting the injurious side
effects that this treatment includes.
The acute inflammatory response associated with ARDS is thought to be one of
the major mechanisms associated with lung injury (Suter, 2006). Injurious mechan-
ical ventilation leading to VILI is a contributor to this inflammatory reaction. There
has been growing interest in the role that mechanotransduction plays in VILI in an
effort to better understand how physical forces are transmitted into biochemical sig-
nals in the lung and how these signals can be controlled to reduce or eliminate the
deleterious effects of mechanical ventilation while keeping the benefit (Liu, 2007;
Oeckler and Hubmayr, 2007).
There are a variety of mechanisms that cells can use to detect mechanical force
including stretch mediated ion channels (Hamill and Martinac, 2001), extracellular
matrix (ECM)-cytoskeleton perturbations (Ingber, 1991), and disruptions in cell-
cell adhesions (Ko et al., 2001). This chapter will focus on the role that protein-
protein interactions play in mechanosensation and mechanotransduction. There is
evidence to show that multiple pathways can be activated by mechanical ventilation
and researchers have begun looking at blocking the signals in the lung initiated
by the mechanical force associated with injurious mechanical ventilation. We will
discuss the possible therapeutic application of blocking Src protein tyrosine kinase
(PTK) pathway to reduce ALI/ARDS associated with VILI.
11 The Role of Protein-protein Interactions in Mechanotransduction 257

11.2 Basic Lung Mechanics vs. Mechanical Ventilation


The lung is considered to be a gravitationally deformed elastic solid organ which
shape matches that of its surroundings, namely, the chest wall and the heart. The
shape conforming nature of the lungs leads to a non-uniform strain during inspira-
tion. Since the weight of the lung itself is negligible when determining this strain,
the movements of diaphragm and chest-wall are actually the main influences on
regional ventilation (Vlahakis et al., 1999). It has been shown that the lung can
expand in volume up to 40% from the functional reserve capacity to the total lung
capacity. Because of the heterogeneity that exists at the micro-scale that cannot be
accounted for by the force of gravity, this predicted expansion of lung tissue is likely
an overestimate of the actual forces experienced at the cellular level. Airways and
blood vessels are known to be more resistant to mechanical forces than the lung
parenchyma itself. Based on observations in fixed tissue, airway micromechanics
are strongly influenced by the role that elastin and collagen fibers play in bearing
much of the mechanical strain experienced during normal ventilation (Vlahakis and
Hubmayr, 2005).
At the alveolar level, the shape of the airway is maintained by surface ten-
sion, and is thought to fold and unfold during the constriction and expansion cycle
of normal respiration (Oldmixon and Hoppin, 1991). Using electron microscopy
observations of the length of the basement membrane surrounding alveoli, it was
estimated that the area of the basement membrane increased approximately 35%
during inspiration, representing a cell stretch of around 15% from the functional
reserve (Tschumperlin and Margulies, 1999). This prediction is limited by the fact
that fixing tissue affects the hydration and surface tension of the specimen, which
could impair accurate observations of the real lung architecture. In the future, it is
hoped that living, unfixed samples can be studied to improve our understanding of
the forces that the airway and alveoli experiences while breathing (Bachofen et al.,
2002).
Often the only life saving therapy for patients with ALI/ARDS, mechanical ven-
tilation can lead to the production of inflammatory mediators. Understanding the
basic concepts of lung mechanics helps to understand why mechanical ventilation
may induce tissue injury. Vlahakis and Hubmayr have summarized the two leading
theories as to why the forces acting on injured lungs leads to VILI while ventila-
tion of normal lungs (i.e. during surgery) typically does not lead any significant side
effects (Vlahakis and Hubmayr, 2005). First, as the injury to the lung progresses,
alveoli become less able to facilitate the exchange of gas across the alveolar-blood
barrier. This results in a smaller number of functional aerated alveoli being preferen-
tially recruited, which end up bearing most of the tidal volume during an inspiratory
maneuver. As a result, there is a regional over-distension in portions of the lung
where airway epithelial cells experience a higher than normal mechanical force ulti-
mately leading to further injury (Maunder et al., 1986). Secondly, because of local
decreases in the ability of certain areas of the lung to expand during inspiration
caused by edema, infection, or restrictive forces, there is heterogeneity in lung com-
pliance. As the expansive force of a tidal volume inspiration leads to a non-uniform
258 M. Rubacha and M. Liu

expansion of the lung there is shear stress between neighboring regions of different
compliance which is not seen in normal lungs (Mead et al., 1970).

11.3 Ventilator Induced Inflammatory Response


A biotrauma hypothesis has been proposed to account for the relationship between
mechanical ventilation and the associated inflammatory response. The injurious
mechanical ventilation may lead to the release of cytokines and pro-inflammatory
mediators, resulting in excessive immune system activation (Tremblay et al., 1997).
These mediators can cause lung damage themselves, or in the case of ventilated
patients, become a contributing factor to their ongoing lung injury by further recruit-
ing inflammatory cells into the lung (Girard and Bernard, 2007). In an isolated
perfused rat lung model, injurious ventilation is associated with the production of
TNF-, IL-1, and IL-6 among other inflammatory cytokines found in BAL fluid
(Tremblay et al., 1997).
In vitro, it has been shown that the stretch of A549 cells, a human alveolar epithe-
lial cancer cell line, can cause the secretion of IL-8, an inflammatory cytokine, into
the culture medium (Vlahakis et al., 1999). The release of both IL-8 and TGF-
from A549 cells in response to mechanical stretch is dependent upon the amount
of physical force applied to the cells as measured by the percentage of elongation
from rest (Yamamoto et al., 2002). The amount of force required to elicit cytokine
response from A549 cells appears to be non-physiological, much more than cells
are predicted to experience in vivo during normal breathing. BEAS-2B cells, a
human normal bronchiole airway epithelial cell line, can also respond to mechanical
stretch by secreting IL-8 (Oudin and Pugin, 2002). This cell line has been used to
uncover the mechanism by which mechanical stretch force leads to the production
of cytokines and chemokines. With 20% elongation, a significant increase in IL-8
release over unstimulated cells was observed. On top of this, paxillin, a compo-
nent of the focal adhesion complex, was translocated from the plasma membrane to
the peri-nuclear area. Under the same stretch conditions, application of Y-27632, a
Rho-activated kinase inhibitor, completely abolished the increase in IL-8 secretion.
Based on these observations, the authors concluded that integrin mediated signalling
is critical for mechanotransduction in airway epithelial cells (Thomas et al., 2006).
It is also believed that mechanical ventilation can inhibit lung repair after injury.
Waters et al. have shown that mechanical stretch, simulating injurious ventilation
can inhibit airway epithelial cell migration (Savla and Waters, 1998), an important
process for repair and regeneration of airway epithelium after injury. It has been
shown that cyclic mechanical stretch impairs airway cell migration via a pathway
that involves FAK (focal adhesion kinase), JIP3 (JNK-interacting protein 3) and
JNK (Jun-N-terminal kinase) (Desai et al., 2009). Mechanical stretch also decreased
migration of primary cultured rat alveolar epithelial cells through inhibition of Rac1
related signalling (Desai et al., 2008).
These studies and others have demonstrated activation of signal transduction
pathways related to inflammatory responses. How physical forces associated with
11 The Role of Protein-protein Interactions in Mechanotransduction 259

mechanical ventilation are sensed by cells in the lung and converted to signalling
event is the focus of this chapter. Several basic mechanisms for mechanosensa-
tion and mechanotransduction have been discussed in this book. We will focus on
one of the novel mechanisms, protein-protein interaction in mechanosensation and
mechanotransduction.

11.4 Protein-Protein Interactions


Signal transduction between cells, within cells, and from the extracellular to intra-
cellular environment is critical for sustaining life. Proteins are responsible for
mediating these signals through their intrinsic dynamic properties. Recently, there
has been a large influx of information regarding protein interaction networks based
on genetic and bio-physical studies (Yu et al., 2008).
Adaptor proteins contain multiple protein-protein interaction domains which
function to link binding partners together, creating a signaling complex. Through
various combinations of protein interaction domains, adaptor proteins can link spe-
cific proteins together in response to cellular signals providing specificity and proper
subcellular localization of signaling partners. In this manner, adaptor proteins are
important for regulating the specificity of downstream cell signaling and regulating
proteins which are important for individual signaling cascades in a spatial and tem-
poral manner (Flynn, 2001). Studies on several different families of signal adaptors
suggest that these proteins are not only used to temporally and spatially couple suc-
cessive signaling proteins but are highly dynamic interactors capable of directing
and regulating downstream signals.
Typically, adaptors contain functional domains which can selectively recognize
activated cellular surface receptors or intracellular signaling molecules and use
one or more of their other domains to couple the receptor with their downstream
effector(s) (Feller, 2001). A simple example of this phenomenon is the SH2/SH3
adaptor Grb2. Grb2 contains an SH2 (Src homology 2) domain which recognizes
tyrosine phosphorylated YXN motifs on activated receptor tyrosine kinases (RTK)
(Fig. 11.2a). Two SH3 (Src homology 3) domains are then able to recruit effector
proteins, such as the Ras guanine nucleotide exchange factor Sos or docking proteins
such as Gab1/2. Recruited proteins become phosphorylated and interact with other
SH2 domains on downstream effectors such as PI3K or Shp2. In this manner, Grb2
is capable of facilitating the downstream signal cascade that links an activated RTK
to Ras-MAPK or PI3K signaling pathways, ultimately having an effect on cellular
function such as cell proliferation, migration, and differentiation (Rozakis-Adcock
et al., 1993).
In depth structural analysis of Grb2 demonstrated that the interaction domains of
this adaptor function almost independently of each other (Maignan et al., 1995).
This observation implies a very simple linker or organizer function for adap-
tor proteins as one binding motif can interact with upstream signaling proteins
while another binds to downstream effectors, propagating a signaling cascade. As
more adaptor proteins are studied, the complexity of their function is becoming
260 M. Rubacha and M. Liu

more evident and the linker/organizer model for adaptor proteins appear to be the
exception rather than the rule (Burns K, 1998). Adaptor proteins themselves can be
dynamically regulated, adding an extra layer of complexity to the signaling cascade
(Pawson, 2007). It has been demonstrated that the function of adaptor proteins can
even be regulated by intracellular signals such as DNA damage (Stucki et al., 2005).
The association of signaling proteins with adaptors can act cooperatively to enhance
signaling or even antagonistically to gate a signal cascade (Hoshi et al., 2005).
The complexity of signal adaptor proteins doesnt end there. A single adaptor
protein can be associated with different proteins depending on the signaling pathway
functioning at any given time. In this way they serve to link different signaling pro-
teins to different downstream effectors depending on the cell type or even subcellu-
lar compartment being investigated. A classic example of this is the intracellular pro-
tein trafficking of signaling proteins. This important cellular function is dependent
on the diversity and specificity of adaptors involved in endocytosis and sorting in
the endosomes (Traub, 2003). The various capabilities of adaptors demonstrate that
the interaction subunits of these proteins are capable of much more than the simple
beads on a string model with which they were originally labeled (Pawson, 2007).
Knowing that protein-protein interaction can mediate and coordinate signal trans-
duction pathways in the cell, we would like to know whether protein-protein
interaction can be the first step to activate signal transduction, especially to con-
vert physical forces to activation of protein tyrosine kinase for signal transduction.
Several mechanosensory complexes have been defined and demonstrated that adap-
tor proteins can act as the actual mechanosensor or play an integral role in linking
the mechanosensor to the downstream signaling pathways.

11.5 Unfolding of p130Cas as a Mechanosensor

Previously it was thought that the force sensing function of cells occurs at the
cytoplasm membrane via mechanisms such as calcium movement and ion chan-
nel gating (Hamill and Martinac, 2001), activation of cell surface receptors by their
ligands (Correa-Meyer et al., 2002; Tschumperlin et al., 2004), ECM-integrin inter-
actions on basolateral side of the cells by mechanical stretch (Chen et al., 1999;
Ingber, 1991), or shear stress on adhesion molecules on the apical membrane of the
cells (Tzima et al., 2005). Through the aforementioned studies it was unclear as to
whether cells can sense mechanical force without intact cellular membranes.
It is difficult to separate the role of ion channel and membrane protein function
from that of the cytoskeleton cells use to bear much of the tension and physical
force. One interesting way to solve this problem is to remove the non-cytoskeletal
components of the cell through Triton X-100 treatment of cells cultured on a col-
lagen coated matrix, leaving behind the Triton insoluble cytoskeleton. This process
removes transmembrane ion channels as well as minimizes the influence of ion flux
on mechanotransduction. In this manner, any measurable changes in protein status
(i.e. protein phosphorylation) resulting from mechanical stretch can only result from
interactions between proteins associated with the cytoskeleton. Sawada and Sheetz
11 The Role of Protein-protein Interactions in Mechanotransduction 261

used this method to remove the cellular membrane and soluble proteins from the
cytoplasm, and stretched the remaining cytoskeleton attached to the extracellular
matrix. Detergent soluble proteins were then replaced for a short period of time,
during which some were found to undergo tyrosine phosphorylation (Sawada and
Sheetz, 2002). This interesting study suggests that in the absence of membrane
related proteins and local changes in ion concentration mechanical force still can
be sensed by cells.
It has been reported that small GTPases can be involved in the mechanical
force induced signal cascade (Sawada et al., 2001). Biaxial stretch was found to
inhibit Ras activity, decrease lamellipodia formation, and activate Rap1. C3G (Rap1
guanine nucleotide exchange factor) is known to contain multiple SH3 domain
binding motifs that are able to associate with the Crk family of adaptor proteins
(Knudsen et al., 1994; Tanaka et al., 1994). It is believed that the association of
the C3G-Crk complex with other proteins containing a phosphotyrosine residue
allows for the release of cis-acting negative regulatory domain of C3G, facilitat-
ing its activation (Ichiba et al., 1999). Interestingly, Crk, C3G, and Rap1 have
all been shown to be associated with Src PTK in the regulation of cell adhesion
(Li et al., 2002), prompting researchers to suspect that this pathway may be
associated with mechanotransduction.
Using the model of cytoskeletal stretch, Tamada et al. showed that the Crk-C3G-
Rap1 signaling pathway was activated. In response to stretch, Crk was recruited to
ECM-cell contacts where it is believed to sense force applied to the cytoskeleton
(via ECM stretch) and facilitates the activation of this signaling pathway. In addi-
tion, they showed that Src PTK was associated with the cytoskeleton after stretch,
confirming the hypothesis that mechanical force can recruit Src for local activation
by protein-protein interactions (Tamada et al., 2004).
These studies showed that protein-protein interactions are able to facilitate
mechanotransduction in the absence of transmembrane ion channels or cytoplasmic
ion flux; however the question of what is the actual mechanosensor? remained
unanswered. It has been reported that p130Cas, when tyrosine phosphorylated on
its multiple internal YXXP repeats, can bind to the N-terminal SH2 domain of
Crk. Interestingly, under normal conditions, the YXXP repeats of p130Cas are
hidden through its folded tertiary structure and can be exposed only under cer-
tain conditions. p130Cas contains an N-terminal SH3 domain that can bind FAK
as well as a C-terminal binding site for Src PTK. These two binding domains
help to link p130Cas to focal adhesions where it is believed to play a role in
mechanotransduction. When cells are mechanically stretched, p130Cas underwent
extensive tyrosine phosphorylation at its multiple internal YXXP repeats (Sawada
et al., 2006). Phosphorylation of p130Cas allows for Crk binding, C3G activation,
and increased GTP-loading of Rap1. It was also shown that this effect was not the
result of an increase of Src PTK activity, implying the possibility that under mechan-
ical stretch, p130Cas is unfolded exposing internal tyrosine phosphorylation targets
for Src that are not apparent under non-stretch conditions (Sawada et al., 2006).
In this way, p130Cas can be considered a dynamic mechanosensor that can detect
a cell stretch force, and facilitate the beginning of a signal cascade through the
aforementioned protein-protein interactions (Pawson, 2007) (Fig. 11.1).
262 M. Rubacha and M. Liu

Fig. 11.1 Mechanical stretch-induced unfolding of p130Cas as a mechanosensor. p130Cas is


represented as an accordion-like structure with an N-terminal SH3 domain and a C-terminal Src
binding region (top). The SH3 domain binds to FAK while the Src binding motif binds to a Src
PTK. These interactions localize p130Cas to focal adhesion sites. Under stretch conditions, the
accordion-like structure extends, and reveals multiple internal YXXP domains to Src PTK for
phosphorylation (middle). The phosphorylation of YXXP motifs allows for the binding of the Crk-
C3G complex. This interaction leads to the activation of C3G and subsequent activation of the
small GTPase Rap1. Reproduced from Pawson (2007) with permission from Elsevier

11.6 AFAP as an Activator of Src PTK


In the above p130Cas model of mechanotransduction, the adaptor protein was
stretched and unfolded by mechanical force to expose its internal tyrosine phospho-
rylation sites to Src PTK. However, it has been seen that the activities of Src PTK
are increased by mechanical forces in multiple cell types (Franchini et al., 2000;
Han et al., 2004; Naruse et al., 1998; Sai et al., 1999). This raises the interesting
question: can adaptor proteins function as activators of protein kinases in order to
convert physical force to biochemical reaction for signal transduction?
Liu et al. found that actin filament associated protein (AFAP) could be a good
candidate for protein-protein interaction related mechanotransduction in fetal rat
lung cells (Liu et al., 1996). They used a model of cell culture where fetal rat
lung cells were grown in a gelfoam matrix, an artificial 3-dimensional structure
which provides a scaffold to support cells. Interestingly, under this condition, mixed
fetal lung cells develop into alveolar like structures that mimic the architecture
of the lung parenchyma (Liu et al., 1992). Cells were subjected to intermittent
stretch that is believed to closely mimic the physiological forces experienced by
alveolar epithelial cells during fetal breathing movements. It was shown that the
stretch force could enhance cell proliferation (Liu et al., 1992, 1995b, 1993) through
activation of c-Src (Liu et al., 1996), which can subsequently induce tyrosine phos-
phorylation of phospholipase C, generation of secondary messengers IP3 and DAG,
increase in intracellular free calcium, and activation of protein kinase C (Liu et al.,
11 The Role of Protein-protein Interactions in Mechanotransduction 263

1995a). Activation of c-Src also resulted in increased tyrosine phosphorylation of


multiple proteins related to cytoskeleton, including FAK, p130Cas, cortactin, and
paxillin among others, however, only the binding of c-Src to AFAP was increased
by mechanical stretch, as determined by co-immunoprecipitation (Liu et al., 1996).
AFAP is distributed along stress fibers and is closely associated with F-actin
(Flynn et al., 1993). This puts AFAP in an excellent position to respond to small
perturbations affecting the cytoskeleton. Indeed AFAP is unique in that its position
changes with stress fibers in response to mechanical stretch. This may increase inter-
action of AFAP with other cytoplasmic proteins, including c-Src. Because AFAP
contains high affinity binding sites for c-Src, it is possible that the interaction
between AFAP and c-Src may lead to binding of these two proteins, and subse-
quently alter the confirmation of c-Src, resulting in its activation. This could be one
of the initial steps of mechanotransduction in a well defined signal pathway (Liu
and Post, 2000; Liu et al., 1999).
In order to fully understand this hypothesis, more must be known about c-Src
itself (Fig. 11.2b). When inactive, c-Src exists in a closed confirmation bound by
two intra-protein interactions. Firstly, in the closed state the c-terminal tail is tyro-
sine phosphorylated and folds in on itself to interact with the SH2 domain. Secondly,
the SH3 domain binds to the linker region between the SH2 and the kinase domain
(Fig.11.3). c-Src can be activated either by dephosphorylation of tyrosine in its
C-terminal tail, or by high affinity binding of its SH2 and/or SH3 domain(s) with
another protein, which can be considered a Src activator. When active, c-Src unfolds
to expose Y416 , located in the kinase domain. Once phosphorylated at Y416 , c-Src

Fig. 11.2 Proteins can be considered as molecules built with functional modules. (a). The multiple
modular domain structure of Grb2 is typical for that of an adaptor protein. (b). Molecular struc-
ture of c-Src protein tyrosine kinase. (c). Molecular structure of actin filament associated protein
(AFAP)
264 M. Rubacha and M. Liu

Fig. 11.3 Mechanical stretch-induced Src activation via competitive binding with AFAP. In the
inactive state, phosphorylation at Y527 in the C-terminus of c-Src binds to its own SH2 domain and
the linker region between the SH2 and kinase domains binds to its own SH3 region. This intra-
protein folding locks c-Src in a closed confirmation (left). When cells are stretched, deformation
of F-actin stress fiber presents AFAP to c-Src. The SH3 and SH2 domain binding motifs of AFAP
bind c-Src with high affinity, disrupting the intra-protein inhibitory interactions of c-Src (middle).
While stabilized in the open confirmation by interactions with AFAP, c-Src can be phosphorylated
at the Y416 residue, locking it in the open and active confirmation (right). Reproduced from J Biol
Chem 2004;279:5479354801. Permission granted from the Journal of Biological Chemistry

goes on to be a potent tyrosine kinase, involved in many different intracellular


signaling processes (Xu et al., 1999).
AFAP is a complex signal adaptor protein in that it contains a putative SH2
domain binding motif and a putative SH3 domain binding motif in its N-terminus.
This is followed by a WW binding motif, then two PH domains which flank mul-
tiple internal Ser/Thr phosphorylation sites which in turn are flanked by two SH2
domain binding motifs finally followed by a C-terminal leucine zipper/actin bind-
ing domain (Baisden et al., 2001b) (Fig. 11.2c). With multiple protein interaction
domains, it is easy to hypothesize that AFAP could be involved in many different
biological functions which are linked to the actin cytoskeleton. Through the analy-
sis of the modular domains of AFAP, it can be seen that the actin binding domain
in the C-terminus allows it to associate with actin filaments, and the special struc-
ture of N-terminus of AFAP could bind the SH2 and/or SH3 domains of Src with
high affinity. Mechanical stretch-induced deformation of cytoskeleton may present
AFAP (along the stress fiber) to c-Src (in the cytoplasm). This interaction has the
potential to lead to the translocation of Src to the actin cytoskeleton and activation of
c-Src. Subsequent downstream tyrosine phosphorylation of other substrate proteins
completes the conversion of the physical force to a biochemical signal (Han et al.,
2004).
11 The Role of Protein-protein Interactions in Mechanotransduction 265

AFAP was first discovered through research directed at discovering the mech-
anism by which constitutively activated v-Src could induce morphological trans-
formations in cells. AFAP was found to form a stable complex with v-Src through
its SH2 or SH3 domain binding motifs. The observation that AFAP was normally
associated with stress fibers and with rosette-like structures in v-Src transformed
chicken embryonic fibroblast cells indicated that AFAP may be involved in Src
activity-dependent alteration of actin filaments (Baisden et al., 2001a; Kanner et al.,
1991).
The avian form of AFAP was first cloned by Flynn et al. who reported the
sequence (Flynn et al., 1993). The mammalian form was first cloned in rats (Lodyga
et al., 2002) and later in humans (Han et al., 2004). The mammalian sequences
have a >80% homology with the avian form (Han et al., 2004; Lodyga et al., 2002).
Importantly, the putative Src binding and activation motifs and actin binding domain
described above are well conserved from avian to mammalian forms (Han et al.,
2004). It has been shown that AFAP can interact with c-Src through both its SH2
and SH3 domain binding motifs. Mutation of five putative tyrosine phosphorylation
sites or a single amino acid mutation from proline to alanine in the SH3 domain bid-
ing motif significantly reduced the ability of AFAP to bind to c-Src and to lead to its
activation. Han et al. further over-expressed these mutant forms of AFAP as dom-
inant negative inhibitors and demonstrated these mutants blocked stretch-induced
c-Src activation in multiple cell types (Han et al., 2004). Based on these results, Han
et al. have proposed that deformation of the cytoskeleton increases the competitive
binding between AFAP and c-Src SH2 and SH3 domains. The interaction between
AFAP and c-Src leads to a conformational change in Src, facilitating its activation.
Activation of Src allows for the initiation of a downstream signaling cascade (Han
et al., 2005) (Fig. 11.3).

11.7 Src PKT Activation by Multiple Physical Forces

In multiple models of mechanotransduction, Src activation has been found to be one


of the early upstream signals (Han et al., 2005). Whether using models of cell stretch
in fibroblasts (Sai et al., 1999) or endothelial cells (Naruse et al., 1998), pressure
overload in vivo (Franchini et al., 2000), or sheer stress (Okuda et al., 1999), Src
tyrosine kinase activation has been detected by the applied physical force.
Src PTK activation is associated with mechanotransduction in lung tissue (Liu
et al., unpublished observation). Parker et al. found that when they inhibited pro-
tein tyrosine phosphotase activity, there was an increase in susceptibility to VILI
resulting from a high peak airway inflation pressure in mechanically ventilated rat
lungs, whereas inhibition of protein tyrosine kinase reduced ventilation-induced
lung injury (Parker et al., 1998).
One of the particularly damaging features of VILI is the breakdown of epithelial
and endothelial barriers. Barrier disruption leads to an increase in vascular perme-
ability allowing proteins and fluid to leak from circulation into the alveolar space
266 M. Rubacha and M. Liu

ultimately leading to edema formation and the interruption of gas exchange across
the air-blood barrier. It has been shown that after only 20 min of injurious venti-
lation, vascular permeability is significantly increased in isolated, perfused mouse
lungs, much sooner than expected if this was the result of cytokine influence alone.
These results implied a rapid mechanism for mechanotransduction leading to lung
injury independent of cytokine response (Miyahara et al., 2007).
The force associated with mechanical ventilation is not limited to the airways of
the lung. This force is also transmitted to the vasculature, where a different type of
mechanical force is experienced. During normal breathing the airway and alveolar
pressure remains within an accepted range, however during mechanical ventilation,
the airway and alveolar pressure increase during inspiration to facilitate air flow
(von Bethmann et al., 1998). As the airway and alveolar pressure increases from
mechanical ventilation, the lung vasculature is stretched causing vessel narrowing
and an increase in pulmonary circulation pressure. This increase in pressure may
increase shear stress, a force parallel to the plane of reference, which is known
inducer of mechanotransduction in endothelial cells (Uhlig, 2002).
Observations from studies directed at determining the role of -catenin in the
development of increases in vascular permeability have implicated Src PTKs as
upstream modifiers of this process. Normally found at adherens junctions, -catenin
can be tyrosine phosphorylated by Src, a process leading to the re-distribution of
-catenin to the cytoplasm where it is degraded, resulting in an increase in vascular
permeability, possibly thorough the destabilization of the adherens junction (Mehta
and Malik, 2006).
The PI3K-Akt signaling pathway has also been shown to act on -catenin.
Activation of this signaling pathway can lead to the negative regulation of GSK3,
which when active, can phosphorylate -catenin on serine residues, tagging it for
degradation. PI3K signaling, however, can also activate Src. Depending on the
strength of these signaling pathways, PI3K signaling could be both a positive and
negative regulator of vascular permeability (Miyahara et al., 2007). PI3K is a diverse
kinase that is activated in response to many different stimuli (Uhlig and Uhlig,
2004). Mechanical ventilation of isolated, perfused mouse lungs leads to the activa-
tion of PI3K. Inhibition of PI3K during mechanical ventilation leads to a decrease
in vascular permeability and a similar effect is seen when Src is inhibited directly.
Interestingly, inhibition of Akt, a downstream kinase of PI3K, lead to the augmenta-
tion of VILI induced vascular permeability. Further study revealed that mechanical
ventilation induced activation of PI3K signaling may lead to a two pronged sig-
naling cascade. One prong activates Src leading to the removal of -catenin from
adherens junctions and an increase in vascular permeability. The other prong acti-
vates the Akt-GSK3 signaling pathway leading to the preservation of -catenin at
the adherens junction and the maintenance of endothelial barrier function (Miyahara
et al., 2007). It appears that during high pressure or injurious mechanical ventilation,
activation of Src is stronger than Akt signaling leading to VILI.
A recent paper by Tzima et al. provides a very comprehensive overview of
endothelial mechanotransduction (Tzima et al., 2005). They focused on investigat-
ing how integrins became activated in response to shear stress. Through a series of
11 The Role of Protein-protein Interactions in Mechanotransduction 267

inhibitor experiments, they were able to show that the Src-PI3K-integrin pathway
was responsible for the intracellular changes associated with shear force. It is known
that shear stress induces a conformational change in integrins and cell alignment
with the direction of flow, as well as the activation of NF-B (Tzima et al., 2002;
Tzima et al., 2001). The addition of a PI3K inhibitor (LY294002 or wortmannin) is
able to block integrin activation. PI3K itself has been found to become rapidly acti-
vated in response to mechanical force (as measured through phosphorylation of the
p85 regulatory subunit of PI3K, the increase of PtdIns(3,4,5)P3 and kinase activity
(Tzima et al., 2005). Src is also known to become active within seconds of the onset
of shear stress (Okuda et al., 1999; Pampori et al., 1999), even faster than PI3K. Src
inhibition with PP2 or SU6656 prevented p85 phosphorylation as well as integrin
activation (Tzima et al., 2005).
To determine what exactly is the mechanosensor in shear stress-induced
mechanotransduction, endothelial cells lacking VE-cadherin or PECAM-1
(VE-cadherin/ and PECAM-1/ respectively) were tested for their ability to
respond to shear stress. Both types of cells were unable to undergo integrin activa-
tion in response to shear stress. When VE-cadherin and PECAM-1 were ectopically
reexpressed in these cells, normal functionality was restored. Further studies demon-
strated that PECAM-1 is upstream of VE-cadherin and is required for Src activation,
and VE-cadherin acts as an adaptor protein to link Src to PI3K allowing for the Src
mediated activation of the PI3K signal cascade (Tzima et al., 2005).

11.8 Blocking Src PTK as a Potential Therapy for VILI

By investigating the role of protein-protein interactions in mechanotransduction, we


hope to find some novel strategies to treat or prevent VILI. This is an important
goal to strive for because with the limitations of technology available to us today,
mechanical ventilation is unavoidable for patients with ALI/ARDS, and sometimes
a reduction in tidal volume to reduce mechanical strain while maintaining accept-
able blood gas parameters is unattainable (Villar et al., 2004). Activation of Src PTK
has been implicated in acute inflammatory responses and tissue injuries in multiple
organs, including the lung (Okutani et al., 2006). Therefore, although mechanotrans-
duction in the lung may be activated by multiple mechanisms, the mechanical forces
induced Src PTK activation may represent a common pathway that leads to acute
lung injury.
It has been shown that Src PTK activity can be involved in many of the processes
that we associate with lung injury including cytokine and chemokine production,
immune cell recruitment, and increased vascular permeability (Okutani et al., 2006).
Indeed Src PTK inhibition has been shown to be protective in animal models
of stroke (Paul et al., 2001), myocardial infarction (Weis et al., 2004), and ALI
(Khadaroo et al., 2004; Severgnini et al., 2005). In multiple models of mechan-
otransduction, Src activity was found to be one of the upstream events in a defined
signal cascade that responds to a mechanical force (Han et al., 2005; Pawson,
268 M. Rubacha and M. Liu

2007; Sawada et al., 2006). As mentioned above, Src is believed to be directly acti-
vated by AFAP in response to mechanical stretch (Han et al., 2004) and is thought
to phosphorylate p130Cas on its YXXP repeats when it is unfolded in response
to mechanical force (Pawson, 2007; Sawada et al., 2006), it is also one of the
first signaling proteins activated in shear stress induced mechanostransduction in
endothelial cells (Tzima et al., 2005). Therefore, Src PTKs are an attractive target
for the treatment of VILI.
The role of protein-protein interactions in mechanotransduction is likely not lim-
ited to adaptor proteins. Interactions between proteins and other molecules, such as
membrane lipids, calcium and other ions, RNA and DNA molecules may also be
affected by physical forces. At every level of the signal cascade, there is a complex
and largely unknown mechanism for the interaction of signaling molecules. These
interactions may occur locally in a particular sub-cellular compartment to deter-
mine a specific cellular function; they may also be affected further by other factors
cells are exposed, such as hyperoxia, hypoxia, inflammatory mediators and environ-
mental conditions, etc. To further explore the role of protein-protein interaction and
interaction between proteins and other molecules will improve our understanding of
mechanosensitivity and mechanotransduction under physiological and pathophys-
iological conditions and reveal new targets for clinical therapies, such as VILI in
ALI/ARDS.

11.9 Conclusions and Perspective


VILI is a complex problem that will likely require a multifaceted approach to solve
including work from basic scientists, clinicians, and even engineers. Interestingly
there are research groups currently investigating ways to remove or limit the use of
mechanical ventilation in the treatment of patients with ALI/ARDS and replacing
it with a modified extracorporeal membranous oxygenator (Fischer et al., 2006). If
successful, this technology will eliminate the need to apply external force to injured
lungs, as all the blood gas exchange could occur outside of the body. Unfortunately,
early trials of such treatment have indicated several critical limitations on this tech-
nology making it unsuitable for some of the most critically ill patients. Based on
the current standard of this treatment, it does not appear that the technology to com-
pletely remove mechanical ventilation from the treatment of critically ill patients
will be available in the near future. With this in mind, it is as important as ever
continue to strive to understand how mechanical ventilation leads to lung injury
through various mechanotransduction mechanisms and look for novel strategies to
manipulate these pathways to benefit patients.
Based on the information in this chapter, we propose that protein-protein inter-
actions play an important role in intracellular mechanotransduction. We believe that
the mechanisms presented are physiologically relevant and are part of an ongo-
ing process that contributes to lung injury in mechanically ventilated patients.
It should be noted that missing from this review are several other types of
11 The Role of Protein-protein Interactions in Mechanotransduction 269

mechanotransduction mechanisms which may be involved in the development of


VILI such as ion flux or lateral intercellular space compression (Tschumperlin et al.,
2004) that were omitted to maintain a focus on what we believe to be a relatively
novel and understudied mechanism. The idea that an adaptor protein can respond to
physical force and initiate a signal cascade is an exciting one. It allows for the iden-
tification of the initial step in the mechanotransduction pathway that is interesting
not only from a bioengineering standpoint, but will hopefully reveal therapeutic tar-
gets for future treatment of VILI. We have emphasized that Src activation appears to
be a common theme in the mechanotransduction pathways that have been described
and propose that intervention directed at dampening Src activity could be therapeu-
tically beneficial. Although this is an exciting thought, the body of literature to draw
upon in the field of protein-protein interactions in VILI limited, meaning that there
is still much work to be done before we are able to fully understand this process
lead alone make the transition from the bench to the bedside.

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Part V
Mechanosensing
and Mechanotransduction in Bone
and Joint Tissues
Chapter 12
Cellular Mechanisms of Mechanotransduction
in Bone

Suzanne R.L. Young and Fredrick M. Pavalko

Abstract Bone is a dynamic tissue that adjusts its structure over time to adapt to
changes in mechanical load. This adaptive ability is critical to skeletal development
and maintenance of optimal skeletal health throughout life. Imbalances in the abil-
ity of bone to keep pace with demands placed on it by mechanical loading results
in bone that is fragile and susceptible to fracture. The cells within bone, i.e. osteo-
cytes and osteoblasts, are responsible for detecting and responding to mechanical
loading. Understanding how bone cells sense and respond to mechanical signals
(the process known as mechanotransduction) has been the focus of considerable
research. Despite excellent progress over the past two decades, the precise mech-
anisms by which bone cells perceive and mediate proper responses to mechanical
loading are not fully understood. When bone is loaded, movement of interstitial
fluid is generated within the small spaces that surround bone cells. This movement
generates fluid shear stress (FSS) that stimulates bone cells, resulting in enhanced
anabolic activity. Most research on cellular mechanisms of mechanotransduction is
now focused on understanding the mechanisms through which bone cells sense FSS
and generate a proper biochemical response. Mechanisms of cellular mechanotrans-
duction likely involve the adhesive junctions, integrins, mechanically-sensitive ion
channels, purinergic receptors, gap junctions and primary cilia. The integrin fam-
ily of cell adhesion molecules may, as their name implies, integrate mechanical
signals across the cell surface. It is also likely that ion channels, particularly cal-
cium changes, play a critical role in mediating mechanical responses to FSS. This
review will focus on recent progress in understanding how bone cells detect and
respond to mechanical stimuli using novel multi-protein signaling complexes called
mechanosomes.

Keywords Fluid shear stress Integrins Focal adhesion Primary


cilia Mechanosome

F.M. Pavalko (B)


Department of Cellular and Integrative Physiology, Indiana University School of Medicine,
635 Barnhill Drive, MS 346A, Indianapolis, IN 46202, USA
e-mail: fpavalko@iupui.edu

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 277


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_12,

C Springer Science+Business Media B.V. 2011
278 S.R.L. Yong and F.M. Pavalko

12.1 Introduction
Cellular mechanotransduction is the process by which cells sense and respond to
physical cues from their surrounding environment. This mechanical sensitivity is
evident from the simplest isolated cells on two-dimensional substrates to cells inte-
grated within the most complex tissues. Understanding the biological processes that
mediate cellular mechanotransduction has been a growing area of interest during
the past 20 years and is particularly important in understanding how bone responds
to mechanical load (Allori et al., 2008; Chen et al., 2010; Huang et al., 2004;
Papachristou et al., 2009; Papachroni et al., 2009; Robling and Turner, 2009a).
Bone is an extremely dynamic tissue that normally undergoes constant turnover,
a process that defines bone remodeling (Turner and Pavalko, 1998). The mechan-
ical responsiveness of the cells that reside in bone, including osteocytes, the most
abundant cells, and osteoblasts, the cells that secrete new osteoid, has been a pri-
mary focus in efforts to understand how bone turnover is regulated in response to
the mechanical demands placed on the skeleton (Bonewald, 2006; Bonewald and
Johnson, 2008; Turner et al., 1994, 2009). This article will discuss recent progress in
the identification and characterization of molecules at the bone cell membrane that
are responsible for detecting mechanical signals. We then discuss progress in iden-
tifying intracellular signaling pathways that are responsible for propagating those
signals inside the cell and regulate bone cell function.

12.2 Detection of Mechanical Stimuli

12.2.1 Focal Adhesions and the Mechanosome Hypothesis

Focal adhesions are sites of attachment between the cell and the extracellular
matrix and have been proposed as cell mechanosensors. Focal adhesions appear
to be integral for the detection and propagation of mechanical signals across the
plasma membrane and into the cytoplasm and nucleus. Direct and indirect connec-
tions between the extracellular matrix (ECM), integrin cell adhesion molecules, and
focal adhesion-associated signal transduction molecules play an important role in
mechanotransduction in many types of cells, including bone cells. Each of these
three key elements (matrix, integrins and intracellular signaling molecules) con-
verges at these sites of structural attachment between cells and the ECM. Although
there is strong evidence that focal adhesions are involved in mechanical signaling,
the molecular mechanisms through which mechanical signaling via focal adhesions
occurs is not completely understood. Focal adhesions are characterized by the pres-
ence of integrin cell adhesion molecules that link the ECM outside the cell to the
cytoskeleton and signaling molecules inside the cell (Burridge and Chrzanowska-
Wodnicka, 1996). Focal adhesions serve as important sites of structural attachment
between cells and the ECM (Burridge and Chrzanowska-Wodnicka, 1996), how-
ever, focal adhesions also function as important organizing centers for signal
12 Cellular Mechanisms of Mechanotransduction in Bone 279

transduction activity (Alahari et al., 2002; Burridge and Chrzanowska-Wodnicka,


1996). Importantly, signaling proteins associated with focal adhesions are also
found in the nucleus (Carvalho et al., 2003; Cattaruzza et al., 2004; Ogawa et al.,
2003; Yi et al., 2003). This has led us to propose the concept of the mechanosome
(Pavalko et al., 2003) in which proteins that can associate with focal adhesions
respond to mechanical stimuli by participating in signaling events leading to
alteration of transcriptional activity in the nucleus. The mechanosome hypothesis
states that the release of molecules from adhesion complexes following mechan-
ical loading alters transcriptional activity regulating genes that control osteoblast
function. The mechanosome should be considered as a multi-protein complex com-
prised of adhesion-associated and DNA-binding proteins that, upon translocation
to the nucleus in response to mechanical loading, transfers mechanical information
from adhesion complexes to target genes. The protein complexes we describe as
mechanosomes are not specific to osteoblasts, or even bone, but may be particularly
well-suited to mediate mechanotransduction in this tissue. Additional reports show-
ing that proteins associated with focal adhesions can also be present in the nucleus
include focal adhesion kinase (FAK) (Lobo and Zachary, 2000; Stewart et al., 2002),
NMP-4/CIZ (Feister et al., 2000; Nakamoto et al., 2000), p130cas and zyxin (Nix
et al., 2001).
Evidence that integrins are activated by fluid shear stress (FSS) is compelling
and comes both from direct demonstration of conformational changes in integrin
structure (Tzima et al., 2001) and from inhibition studies using integrin-specific
inhibitory antibodies and Arg-Gly-Asp (RGD) peptides (Ponik and Pavalko, 2004).
Using antibodies that recognize conformational changes in integrin structure asso-
ciated with integrin-ligand affinity changes, Tzima et al. (2001) showed increased
immunostaining of endothelial cells subjected to FSS indicating a modulation of
v3 integrin affinity by shear. Similarly, Jalali et al. (2001) using antibodies that
specifically recognize the high affinity binding state of 1 and 3 containing integrin
heterodimers have shown that FSS also causes conformational changes of these inte-
grins in endothelial cells. In complimentary studies in endothelial cells, others have
inhibited integrin function using blocking antibodies and RGD peptides to inhibit
FSS induced activation of intracellular signaling pathways and cell motility (Urbich
et al., 2002). These include blockade of shear induced signaling by extracellular
signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and the IB complex
(Labrador et al., 2003). Similarly, Liu et al. (2002) used integrin function blocking
strategies to inhibit shear induced activation of key transcription factors that regu-
late sterol and lipid homeostasis called sterol regulatory element-binding proteins
(SREBPs). Shear induced secretion of basic fibroblast growth factor (bFGF) was
also blocked by anti-integrin antibodies (Gloe et al., 2002) as were the anti-apoptotic
effects of fluid shear (Urbich et al., 2000).
Data from our laboratory demonstrates that inhibition of integrin-ECM
interactions using RGD peptides inhibits both shear induced up-regulation of
cyclooxygenase-2 (COX-2) protein and shear induced release of PGE2 from cells
(Ponik and Pavalko, 2004). Previously we found that osteoblasts rapidly (<1 h)
respond to FSS by recruiting 1 integrins into focal adhesions at the cell periphery
280 S.R.L. Yong and F.M. Pavalko

(Pavalko et al., 1998). With longer periods of shear, osteoblasts increase formation
of fibrillar adhesions which contain 5/1 integrins localized more toward the inte-
rior of the cell (Ponik and Pavalko, 2004). Consistent with results in osteoblasts,
studies using endothelial cells demonstrate increased mRNA and protein levels of
5 and 1 integrins in response to FSS (Urbich et al., 2000). Thus, considerable
evidence supports the hypothesis that focal adhesions function as mechanosensors
that are critical in regulating the response of mechanically sensitive cells to FSS
(reviewed in, (Geiger and Bershadsky, 2002)).

12.2.2 Ion Channel and Purinergic Signaling in Bone

When bone cells are subjected to mechanical stimulation, either by fluid shear stress
or by strain, one of the earliest responses that can be detected experimentally is a
rapid increase in intracellular Ca2+ that occurs within 30 s of exposure to shear stress
or strain (Hung et al., 1995; Jones et al., 1991). This mechanically-induced cytoplas-
mic Ca2+ spike involves both entries of extracellular Ca2+ via surface membrane ion
channels and intracellular Ca2+ stores (Hung et al., 1996). This rapid Ca2+ response
to mechanical loading has led investigators to focus on the role of ion channels in
mechanotransduction. Duncan and colleagues found that intracellular Ca2+ release
from internal stores affects gene expression through activation of phospholipase C
(PLC) and the resulting production of IP3 (Chen et al., 2003). These studies sup-
port a role for ion channel-mediated mechanical signaling in bone and highlight the
important question of how fluid shear stress results in such a rapid Ca2+ spike and
activation of PLC. Specifically, this work suggests that fluid shear-induced changes
in gene expression in osteoblasts are dependent on Ca2+ release from intracellu-
lar stores that is mediated by IP3 . In addition, numerous studies suggest that Ca2+
entry through ion channels plays an important role in mechanically-induced Ca2+
increases. A number of different ion channels have been described in osteoblasts
(for review, see (Duncan et al., 1998)). Two of these channels have been directly
linked to the mechanical response of bone cells. One is the L-type voltage-sensitive
Ca2+ channel (L-VSCC) (Li et al., 2002a) and the other is the mechanosensitive,
cation-selective channel (MSCC) (Ryder and Duncan, 2001; Zhang et al., 2006).
Experimental evidence supporting an important role for MSCCs during mechan-
otransduction includes the demonstration that inhibition of MSCCs blocks FSS-
induced release of prostaglandins in osteocytes (Ajubi et al., 1999), of TGF-1 in
osteoblastic cells (Sakai et al., 1998) and of nitric oxide (NO) in organ cultures
(Rawlinson et al., 1996). Inhibition of the L-VSCC, which has been proposed to
control growth and development of bone (Duriez et al., 1993) and affects prolifer-
ation of osteoblasts (Loza et al., 1994), significantly reduces mechanically-induced
bone formation in rodents (Li et al., 2002a). The increase in mechanically-induced
intracellular Ca2+ mediated by MSCCs and L-VSCCs is thought to promote activa-
tion of several important signaling pathways in bone cells including ERK, JNK and
p38 MAP kinases (Liu et al., 2008). In general, MAP kinases, which are activated
by phosphorylation of tyrosine and threonine residues, play a ubiquitous role in
12 Cellular Mechanisms of Mechanotransduction in Bone 281

regulation of cell proliferation, differentiation and apoptosis in a variety of cell


types, including bone cells (Jessop et al., 2002; Matsuda et al., 1998).
Purinergic signaling may also play a key role in mechanotransduction in bone.
Recently ATP was found to be released very rapidly (<1 min) from osteoblasts
in response to mechanical stimuli, and depends upon entry of extracellular Ca2+
through both MSCCs and L-VSCCs (Genetos et al., 2005). These findings have
suggested a possible mechanistic link between Ca2+ dependent responses, PLC acti-
vation, and ATP in osteoblasts (Genetos et al., 2005). It appears that ATP acts as an
autocrine/paracrine signaling molecule by binding to either of two purinergic recep-
tors, P2Y or P2X. P2X receptors are ion channels that are gated by ATP, while the
P2Y receptors are G-protein coupled receptors (North, 2002). In bone cells, consid-
erable interest has focused on the role of a couple of P2 receptor isoforms, P2Y2
(You et al., 2002) and P2X7 (Ke et al., 2003; Li et al., 2005) that may play important
roles in mechanically-induced bone regulation. Recently, Liu et al. (2008) evaluated
the role of intracellular Ca2+ and ATP release on the activation of ERK following
exposure of MC3T3-E1 cells to fluid shear stress. They found that extracellular Ca2+
entry through both MSCCs and L-VSCCs, but not release of intracellular calcium
from store, was required for ERK activation. Protein kinase C activation was also
shown to contribute to mechanically-induced Ca2+ -dependent ERK activation. The
finding that ERK activation is dependent on release of ATP in response to fluid shear
stress represents the identification of a potentially important mediator of mechan-
ical regulation in bone. The answers to several important unanswered questions,
including the role of mechanically regulated ion channels and purinergic receptors
on mechanically-induced bone formation in vivo, will be important for evaluating
their role in bone. Recently, Li et al. (2009) reported the exciting observation that
mice lacking P2X7 have osteopenia in load bearing bones and are less sensitive to
mechanical loading-induced bone formation than are wild type mice. The loss of
P2X7 also resulted in the inability of bone cells to release PGE2 in response to fluid
shear suggesting a link between this important mechanically induced prostaglandin
in bone and ATP signaling. This finding is among the first in vivo reports suggest-
ing that purinergic receptors play a key role in the skeletal response to mechanical
loading.

12.2.3 Primary Cilia

Many cells express a single cilium that extends from the cell surface and is gen-
erally referred to as the primary cilium. Unlike other cilia, the primary cilium is
not motile but has been proposed to play a unique function as a mechanosensor
and/or chemosensor. (Wheatley et al., 1996). Primary cilia have been suggested
to be involved in several signaling pathways that are important in development
(Christensen et al., 2007, 2008) and are implicated in a number of human diseases,
such as polycystic kidney disease (Tobin and Beales, 2007; Yoder, 2007). Over the
past several years, primary cilia have gained significant attention for their potential
282 S.R.L. Yong and F.M. Pavalko

role in development and maintenance of the skeleton (Koyama et al., 2007; Malone
et al., 2007a; McGlashan et al., 2007; Whitfield, 2008; Xiao et al., 2006).
Primary cilia were shown to function as sensors of fluid flow in kidney epithelial
cells (Liu et al., 2005). Movement of fluid has been suggested to physically bend the
primary cilium leading directly to an increase in the level of intracellular calcium
(Schwartz et al., 1997) but primary cilia also appear to function independently of
calcium in bone (Malone et al., 2007b). Primary cilia have also been visualized in
bone cells, including osteocytes and osteoblasts (Matthews and Martin, 1971; Xiao
et al., 2006). Future studies using mice in which the function of primary cilia are
targeted specifically in bone cells are likely to provide more definitive evidence on
the role of primary cilia on regulation of mechanically-induced bone formation in
vivo.

12.3 Propagation of Mechanical Signals in Bone Cells

12.3.1 Focal Adhesion Kinase (FAK)

Because integrins do not possess intrinsic kinase activity, signals from various
sources including mechanical stimulation must be transduced indirectly into the
cytoplasm, and eventually the nucleus. Focal adhesion kinase (FAK) is one of many
proteins recruited as part of a multiprotein complex in focal adhesions and functions
as a key intermediate in various integrin-dependent signal transduction pathways
(reviewed in, (Abbi and Guan, 2002; Hauck et al., 2002; Parsons et al., 2000)).
FAK has been shown to play a role in mediating the downstream activation of
mitogen activated protein kinase (MAPK) in response to FSS in endothelial cells
(Li et al., 1997; Takahashi et al., 1997) and our group has evidence of FAK activation
in response to FSS in osteoblasts.
Upon activation, FAK is autophosphorylated at tyrosine 397 (Tyr397). Activated
FAK can associate with various other signaling molecules including the tyrosine
kinase c-Src. As a result of this association between FAK and c-Src, c-Src becomes
capable of phosphorylating two additional focal adhesion proteins, paxillin and
p130cas . The association of phosphorylated FAK, paxillin, and p130cas serves as a
scaffold for recruitment of adaptors and signaling intermediates including the adap-
tor protein Crk and the guanine nucleotide exchange for Rap1, C3G, which can lead
to the activation of EFK (reviewed recently in, (Panetti, 2002; Turner, 2000). Thus,
FAK plays a key role in signaling through focal adhesions.
The dominant negative FAK mutant, FAK-Related Non-Kinase, or FRNK, con-
tains only the C-terminal domain of FAK and lacks kinase activity (Cooley et al.,
2000). FRNK can localize to focal adhesions, inhibit autophosphorylation of and
displace endogenous FAK from focal adhesions, thereby negatively regulating the
function of endogenous FAK (Lin et al., 1997). There is significant experimen-
tal evidence that FAK mediates integrin-dependent fluid shear induced signals via
focal adhesions from several laboratories including our own. First, in addition
to activation of FAK by FSS (Li et al., 1997), other focal adhesion components
12 Cellular Mechanisms of Mechanotransduction in Bone 283

including c-Src and p130cas which are substrates of FAK, are also rapidly acti-
vated by FSS in endothelial cells (Berk et al., 1995; Chen et al., 1999; Okuda
et al., 1999) . Second, expression of dominant negative FAK and c-Src mutants in
endothelial cells inhibits shear induced activation of ERK (Li et al., 1997). Third, a
role for FAK has been implicated in the shear induced mechanotaxis of endothe-
lial cells based on studies using GFP-tagged FAK which revealed that FAK is
recruited to newly formed focal adhesions at the leading edge of cells migrating
in the direction of fluid flow (Li et al., 2002b). p130cas is rapidly phosphorylated
in response to FSS. Determining whether shear induced p130 cas phosphoryla-
tion is dependent on the activity of FAK remains to be determined. FAK plays an
essential function in vivo as evidenced by the fact the FAK / mouse is embryonic
lethal (Ilic et al., 1995). Studies using fibroblasts isolated from FAK / embryos
support a role for FAK in several cellular processes related to cell adhesion, per-
haps via FAK-mediated inhibition of Rho GTPase activity (Chen et al., 2002; Ilic
et al., 2003; Sieg et al., 2000; Sokabe et al., 1997). Alteration of cell motility in
FAK / fibroblasts (Sieg et al., 1999), another focal adhesion regulated process,
further suggests a critical role for FAK in signaling pathways mediated through
focal adhesions. This hypothesis is supported by a study showing that mechanical
stimulation of trabecular bone tissue activates FAK in vivo (Moalli et al., 2001).
Recently the role of FAK in bone differentiation and bone regeneration was
examined using a conditional fak/ mouse. It was determined that FAK is not
required for osteoblast differentiation in vivo, but FAK is needed for appropriate
bone regeneration in adult mice (Kim et al., 2007). Although these data implicate a
role for FAK in bone biology, there is very little known regarding the role of FAK
during mechanotransduction. We have investigated the role of FAK as an important
mechanosensory component for FSS-induced signaling in osteoblasts (Young et al.,
2009). We examined both early and late FSS-induced signaling pathways as mea-
sured by activation of extracellular signal-related kinases (ERK) phosphorylation,
COX-2 up-regulation, PGE2 release, c-fos up-regulation, and osteopontin (OPN)
upregulation. Furthermore, we used osteoblasts from multiple sources and three
different methods to disrupt FAK activity to test our hypothesis. Using siRNA tech-
nology to target FAK expression, we found that reduced FAK expression resulted in
a decrease in both the early and late FSS-induced signaling pathways in osteoblasts.
In addition, we used dominant negative FRNK, which is endogenously expressed
in osteoblasts, to inhibit FAK activity. Over expression of FRNK disrupted FAK
activation, which also resulted in a reduced response to FSS in osteoblasts. FAK/
osteoblasts were also examined and they exhibited a diminished response to FSS.
Importantly, re-expression of FAK in the FAK/ osteoblasts rescued FSS-induced
mechanotransduction as measured by COX-2 up-regulation and OPN expression.
Nuclear factor-kappa B (NF-B) activity also plays a key role in post-natal bone
remodeling by inhibiting bone formation in mice (Chang et al., 2009), and NF-B
nuclear translocation is activated by FSS in osteoblasts (Chen et al., 2003). However
it has also been reported that oscillatory fluid shear stress inhibited TNF- induced
NF-B activation in osteoblast-like cells (Kurokouchi et al., 2001). Our laboratory
has also evaluated the role of FAK in regulation of fluid shear-induced activation
284 S.R.L. Yong and F.M. Pavalko

of NF-B signaling in osteoblasts (Young et al., 2010). Immortalized FAK+/+ and


FAK/ osteoblasts were exposed to periods of oscillatory fluid shear stress and
NF-B activation was analyzed. We determined that FAK is required for shear-
induced nuclear translocation and activation of NF-B in osteoblasts. In addition
we saw that shear-induced phosphorylation of the IB kinases in both FAK+/+ and
FAK/ osteoblasts, but only FAK+/+ osteoblasts demonstrated the resulting degra-
dation of NF-B inhibitors IB and IB. Oscillatory fluid shear stress (OFSS)
did not induce the degradation of IB or the processing of p105 in either FAK+/+
and FAK/ osteoblasts. These data indicate a novel relationship between FAK and
NF-B activation in osteoblast mechanotransduction.

12.3.2 Wnt/-Catenin/Sclerostin

Fluid shear stress and parathyroid hormone (PTH) both promote anabolic bone
formation. Interestingly, both fluid shear and PTH also promote translocation of
-catenin from the cytoplasm to the nucleus, a critical aspect of mechanosome activ-
ity (Kulkarni et al., 2005; Norvell et al., 2004; Robinson et al., 2006; Santos et al.,
2010; Tobimatsu et al., 2006). Because -catenin plays a role in some load-induced
changes in osteoblast gene expression (Case et al., 2008) and is sensitive to mechani-
cal load and PTH (Wang et al., 2008), it may play a key role in mechanically-induced
bone formation, reviewed in (Williams and Insogna, 2009). A breakthrough in
understanding of the role of -catenin signaling in bone occurred when it was real-
ized that the low-density lipoprotein receptor-related protein 5 (LRP5) played a key
role in regulating the skeleton (Gong et al., 2001; Little et al., 2002). Patients with
loss of function mutations in LRP5 have severely reduced bone mass (Gong et al.,
2001). Turner and colleagues investigated the role of LRP5 on bone strength using
mice engineered with a loss-of-function mutation in Lrp5 (Sawakami et al., 2006).
They found that that Lrp5 is critical for mechanotransduction in osteoblasts. These
and other studies, reviewed recently in (Robling and Turner, 2009b), strongly sug-
gest that LRP5 plays a key role in transduction of mechanical signals into a proper
skeletal response.
Lrp5 appears to play a critical role in the Wnt/-catenin signaling pathway.
Secreted Wnt proteins bind a complex of proteins at the cell surface including Lrp5
and an associated transmembrane co-receptor called Frizzled. Activation of this
complex at the cell surface leads to the translocation of -catenin to the nucleus,
which complexes with members of the TCF/LEF transcription factor family to
regulate gene transcription. Several cytoplasmic proteins control the accumulation
of -catenin in the cytoplasm including glycogen synthase 3 (GSK3), casein
kinase 1 (Ck1), the protein disheveled (Dsh), Axin, and adenomatous polyposis coli
gene product (Apc). When Wnt signaling activity is low, a complex of proteins
including axin, Ck1 and Gsk3i interact with -catenin resulting in the phospho-
rylation and ubiquitination of -catenin, which targets the -catenin protein for
proteosomal degradation. Increased Wnt signaling via Lrp5 disrupts this complex
causing stabilization and accumulation of -catenin in the cytoplasm and allows
12 Cellular Mechanisms of Mechanotransduction in Bone 285

for its accumulation in the nucleus. The observation that mechanical loading pro-
motes -catenin nuclear translocation and activity in bone cells strongly supports
a link between loading and Wnt/-catenin signaling in bone (Norvell et al., 2004;
Robinson et al., 2006).
Although there is considerable evidence to suggest mechanical loading of bone
involves Wnt signaling and regulation of -catenin activity via Lrp5, less clear is
precisely how mechanical stimulation alters this pathway. Most thought in this area
considers whether a decrease in antagonistic signals (Wnt inhibitors) or an increase
in agonist activity (Wnt secretion or stimulation of Wnt signaling) is involved. A key
molecule receiving a great deal of attention is the protein product of the SOST gene,
sclerostin, reviewed recently in detail (Robling and Turner, 2009b). Sclerostin has
been identified as a modulator of Lrp5 activity in response to mechanical loading
in bone. Sclerostin is expressed exclusively in osteocytes, the most abundant cells
in bone and the cells most uniquely positioned to detect and respond to changes
in mechanical loading in bone. This protein is a potent inhibitor of bone forma-
tion and several mutations in the SOST gene have been associated with high bone
mass (Balemans et al., 2001, 2002). Sclerostin binds Lrp5 and mechanical load-
ing of bone results in a dramatic decrease in sclerostin protein levels and in SOST
transcripts (Robling et al., 2008). Significantly, regions of the bone that showed the
most dramatic decreases in sclerostin were those that experienced the highest levels
of strain and exhibited the most new bone formation in response to loading. Thus,
the level of expressed sclerostin by osteocytes appears to tightly control the bal-
ance between mechanical load and bone formation, potentially through inhibition
of Wnt/Lrp5/-catenin signaling.

12.3.3 Gap Junctions

Gap junctions, formed by the association of connexin molecules within cell mem-
branes, also clearly play a role in the propagation of mechanical signals between
bone cells in response to loading (Bonewald, 2006; Donahue, 2000; Jiang et al.,
2007). The role of gap junctions in oscillatory fluid flow-induced changes in cal-
cium and prostaglandin production was demonstrated in osteoblast-like MC3T3-E1
cells with normal or defective gap junctional communication (Saunders et al., 2001).
Connexin 43 (Cx43) plays a particularly important role in bone and expression of a
dominant-negative Cx43 disrupts gap junction communication in and demonstrated
that prostaglandin production in bone cells required functional gap junctions, while
shear-induced cytosolic calcium spikes did not. A recent study investigated whether
inhibition of gap junction communication between bone cells affected the response
to fluid flow (Jekir and Donahue, 2009). These studies showed that in the pres-
ence of the gap junction inhibitor 18 beta-glycyrrhetinic acid (BGA), MC3T3-E1
cells exposed to oscillatory fluid flow failed to upregulate expression of osteopontin
mRNA 24 h after flow, in contrast to flowed cells that did not receive the inhibitor.
Surprisingly, however, osteopontin protein secretion was still increased 48 h after
flow despite the inhibition of gap junctions. Gap junction hemichannels which are
286 S.R.L. Yong and F.M. Pavalko

formed by connexins on a cell that are not in contact with connexins on an adjacent
cell also mediate the response of bone cells to loading (Cherian et al., 2005; Siller-
Jackson et al., 2008). For example, fluid flow induces translocation of Cx43 to the
membrane surface where unapposed hemichannels are formed. These hemichan-
nels function to promote mechanically-induced prostaglandin secretion (Cherian
et al., 2005). Release of prostaglandins by fluid shear was inhibited by an antibody
that specifically blocks Cx43-hemichannels, but not gap junctions or other channels
(Burra and Jiang, 2009).

12.3.4 NFAT
Recent studies suggest a novel mechanism through which mechanical signals may
be propagated in bone. Nuclear factor of activated T cells (NFAT), a transcription
factor well known in inflammatory signaling pathways, was shown to be activated in
response to mechanical stimulation and mediated cyclooxygenase-2 (Cox-2) expres-
sion (Celil Aydemir et al., 2010; Riddle et al., 2007). Application of fluid shear
stress or tensile strain resulted in translocation of NFAT from the cytoplasm to
the nucleus in bone cells. This pathway could be blocked by a peptide inhibitor of
NFAT signaling. It has been suggested that ATP is required for fluid flow-induced
increases in intracellular calcium concentration, activation of calcineurin, and the
nuclear translocation of NFAT (Riddle et al., 2007). Together, these findings sup-
port a novel role for NFAT in mechanotransduction in bone and are consistent with
the tenants of the mechanosome hypothesis which suggests that mechanical load-
ing propagates signals from the cell membrane, through the cytoplasm, and into the
nucleus to regulate transcription of mechanically-sensitive genes.

12.3.5 Nitric Oxide cGMP-Dependent Kinases


Load-induced production of nitric oxide (NO) appears to be another important medi-
ator of mechanotransduction in bone cells (Johnson et al., 1996; McAllister and
Frangos, 1999; McGarry et al., 2008). The most abundant cells in bone, osteocytes,
seem to be particularly capable of efficiently producing significant amounts of NO
in response to loading (Zaman et al., 1999). There is evidence that very short bouts
of mechanical loading of bone tissue can result in significant increases in release of
NO. Explants of bone from juvenile rats responded to mechanical loading in vitro
by increasing NO release by 50% after less than 10 min of loading, compared to
unloaded explants (Pitsillides et al., 1995; Rawlinson et al., 1996). In vivo, knock
out of a key enzyme in NO production, inducible nitric oxide synthase (iNOS),
resulted in a loss of load-induced bone formation (Watanuki et al., 2002). Future
studies will most certainly be aimed at trying to understand the cellular target of NO
produced in response to mechanical loading.
Recently, a potential mediator of NO-regulated responses in bone has been
identified. Using cultured human osteoblasts and a mouse immortalized cell line,
12 Cellular Mechanisms of Mechanotransduction in Bone 287

MC3T3-E1 cells, Rangaswami et al. (2009) have reported that cyclic GMP-
dependent (cGMP) protein kinase (PKG II) was activated in response to fluid shear
stress via a NO-dependent mechanism. PKG II was shown to play a role in fluid
shear stress induced expression of several genes important in skeletal regulation
including c-fos, fra-1, fra-2, and fosB. When cells were treated with pharmacologi-
cal inhibitors of the NO/cGMP/PKG pathway, shear-induced expression of each of
these genes was blocked. Furthermore, shear-induced MAP kinase activation was
shown to be dependent on this NO/cGMP/PKG pathway. Interestingly, the effects
of fluid shear stress on gene expression and MAP activity could be mimicked by
treating cells with a membrane-permeable cGMP analog.

12.3.6 Nmp4/CIZ

Nmp4 was initially characterized as a PTH-responsive nuclear matrix architec-


tural transcription factor, i.e. a protein that alters gene activity by bending DNA
(Alvarez et al., 1997, 1998; Thunyakitpisal et al., 2001). We have hypothesized that
the PTH- or load-induced changes in osteoblast adhesion and shape were trans-
duced by Nmp4 into alterations in target gene DNA conformation and ultimately
activity (Bidwell et al., 1998; Pavalko et al., 2003). CIZ was independently identi-
fied as a nucleocytoplasmic shuttling Cys2 His2 zinc finger transcription factor that
interacts with the focal adhesion protein p130cas (Nakamoto et al., 2000). The asso-
ciation of NMP4/CIZ with p130cas , a force sensor/transducer of the cell (Sawada
et al., 2006), supports the hypothesis that this association mediates communication
between integrins and the nucleus (Nakamoto et al., 2000) and may be particu-
larly relevant to changes in adhesion signaling that occur during bone cell response
to PTH or load (Childress et al., 2010; Pavalko et al., 2003). Nmp4/CIZ has been
shown to regulate the expression of Mmp-13 in rat bone cells in response to fluid
shear stress (Charoonpatrapong-Panyayong et al., 2007). In fact, the expression of
Nmp4/CIZ itself is sensitive to fluid shear stress, consistent with a role for this pro-
tein in bone mechanotransduction. Nmp4/CIZ is a general repressor of the anabolic
bone response. The independently prepared Nmp4 knockout (KO) and CIZ-KO
mice both exhibit a modestly enhanced skeletal phenotype including elevated
bone mineral density and content compared to wild-type (WT) mice (Morinobu
et al., 2005; Robling et al., 2009). The CIZ-KO mice show a greater increase
in bone formation in response to BMP2 as compared to WT mice (Morinobu
et al., 2005) and the Nmp4-KO mice exhibit an enhanced increase in bone for-
mation in response to intermittent PTH as compared to their WT counterparts
(Robling et al., 2009). Remarkably Nmp4/CIZ mediates disuse-induced bone loss
in mice since the CIZ-nulls are impervious to hind limb suspension (Hino et al.,
2007). Therefore, Nmp4/CIZ may suppress pathways common to both PTH- and
mechanical load-induced anabolic response (Childress et al., 2010).
The p130cas /Nmp4/CIZ and -catenin/Lef1 signaling pathways share several
similar features (reviewed in, (Childress et al., 2010)). Both Nmp4/CIZ and
Lef1 are nucleocytoplasmic shuttling high mobility group (HMG) architectural
288 S.R.L. Yong and F.M. Pavalko

transcription factors that associate with adhesion complex proteins (p130cas and
-catenin, respectively) and appear to translocate between adhesion sites at the
membrane and the nucleus. In a recent study we examined the relationship between
Nmp4/CIZ and -catenin signaling in response to mechanical stimulation. We
found that translocation of -catenin to the nucleus in osteoblasts that is normally
induced by oscillatory fluid shear stress (OFSS) is attenuated in the presence of
Nmp4/CIZ. Furthermore, we found that other aspects of OFSS-induced mechan-
otransduction that are associated with the -catenin pathway, including ERK, Akt
and GSK3 activity, as well as expression of the -catenin-responsive protein cyclin
D1 (Tetsu and McCormick, 1999) are enhanced in cells lacking Nmp4/CIZ. Finally,
we found that in the absence of Nmp4/CIZ, OFSS-induced reorganization of the
actin cytoskeleton and formation of focal adhesions, as has been described previ-
ously (Ponik and Pavalko, 2004) is qualitatively enhanced, further suggesting that
Nmp4/CIZ may reduce the sensitivity of bone cells to mechanical stimuli. Together,
these results support the concept that Nmp4 normally plays an inhibitory role in
bone cell mechanotransduction.
It is unclear how Nmp4/CIZ inhibits nuclear translocation of -catenin. One
potential convergence point between the p130cas /Nmp4/CIZ and Lef1/-catenin
pathways may be the SMAD proteins (Childress et al., 2010). R-SMAD activity
is upregulated by both PTH and load, and these proteins are important components
of the anabolic pathways in bone (Li, 2008; Ogita et al., 2008; Sowa et al., 2003;
Tobimatsu et al., 2006). The R-SMADs physically interact with the Lef1/-catenin
proteins and together synergistically enhance osteoblast gene expression (Guo et al.,
2008; Labbe et al., 2000; Sato et al., 2009). Nmp4/CIZ attenuates R-SMAD activ-
ity (Shen et al., 2002) but it is not yet clear whether this occurs in the cytoplasm,
the nucleus or both. Additionally, p130cas , a major binding partner of Nmp4/CIZ,
suppresses R-SMAD activation (Kim et al., 2008). Elucidating the components and
interactions comprising this putative mechanosome network is necessary for under-
standing the cellular and molecular basis of the skeletons response to PTH and
loading.

12.4 Conclusions and Perspectives

Mechanical loading clearly plays a key role in the regulation of the mammalian
skeleton. This review has discussed several of the key mechanisms used by bone
cells to convert mechanical signals into altered biochemical responses. Recent
research has resulted in significant progress toward the identification of the key
molecular components used by cells to detect mechanical stimuli and to propagate
those signals through the cytoplasm and into the nucleus. Future work will undoubt-
edly focus on gaining a better understanding of how these individual molecular
pathways are integrated into functional tissue responses to mechanical loading.
Acknowledgements This work was supported by NIH AR052682 and AR056188
12 Cellular Mechanisms of Mechanotransduction in Bone 289

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Chapter 13
The Mechanosensitivity of Cells in Joint Tissues:
Role in the Pathogenesis of Joint Diseases

Christelle Sanchez, Marianne Mathy-Hartert, and Yves Henrotin

Abstract Joint tissues, including cartilage, bone, meniscus, tendon, ligament and
synovial membrane, are exposed to high mechanical stimulation. The response of
these tissues to mechanical strains is a key factor of the OA onset and progression.
Any little failure in this mechanical function may lead to cell phenotype alteration
and tissue damages. While mechanical responses of cartilage are well known, very
few data are available on the effects of mechanical strains on other articular tis-
sues. Mechanical loading plays a dual role in the homeostasis of joint tissues: when
applied at moderate and physiological range, mechanical stimuli are beneficial to
the good health of joint tissues whereas excessive loading initiate abnormal tis-
sue remodeling and structural changes. Appropriate mechanical load could play an
important role in the homeostasis of joint tissues. This raises the question of the role
played by exercises in the prevention and treatment of OA. This paper is a narrative
review based on selected recent literature in this field.

Keywords Osteoarthritis Bone Cartilage Mechanical stimuli

13.1 Introduction
Osteoarthritis (OA) is the most common form of arthritic disease, and it is a major
cause of disability and impaired quality of life in the elderly. A hallmark of the
disease is the progressive degeneration of articular cartilage and subsequent joint
space narrowing. However, OA is a global disease affecting not only the cartilage,
but also synovial membrane, subchondral bone, tendons, ligaments and menisci. OA
is characterized by a progressive loss a cartilage, meniscus tears and clefts, synovial

Y. Henrotin (B)
Bone and Cartilage Research Unit, University of Lige, Institute of Pathology Level 5, CHU Sart-
Tilman, 4000 Lige, Belgium
e-mail: yhenrotin@ulg.ac.be

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 297


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_13,

C Springer Science+Business Media B.V. 2011
298 C. Sanchez et al.

membrane inflammation and an increase of subchondral bone remodelling leading


to subchondral bone plate sclerosis. Mechanical factors play a key role in the onset
and progression of these features.
This paper aims to summarize joint cell responses to mechanical stimuli in
physiologic and pathologic conditions.

13.2 Mechanical Stimuli and Chondrocyte Metabolism

13.2.1 Mechanical Stimuli and Cartilage Matrix Remodeling

Cartilage is composed of sparsely chondrocyte residing in a highly hydrated extra-


cellular matrix (ECM) consisting of a tension-resistant fibrilar type II collagen (Col
II) network and polyanionic proteoglycan aggregates named aggrecans (Agg). The
high negative-charges density of the glycosaminoglycan (GAG) chains on proteo-
glycan molecule leads to a high osmotic pressure, resulting in an influx of water.
This association of Agg and Col network gives to the cartilage its mechanical
properties such as elasticity and compressibility. Due to its unique location at
joint surfaces, articular cartilage experiences a range of static and dynamic forces
that include shear, compression and tension. These physical forces induce cells
deformation, flow of fluids within the tissue and streaming potentials and currents
induced by fluid convection of counter-ions through the negatively charged extra-
cellular matrix. In addition, local changes in tissue volume caused by compression
also lead to alterations in matrix water content, extracellular matrix fixed charge
density, mobile ion concentrations and osmotic pressure. Any of these mechan-
ical and physicochemical phenomena in the microenvironment of chondrocytes
may affect cellular metabolism. Chondrocytes sense and convert the mechani-
cal signals they receive into biochemical signals, which subsequently mediate
both anabolic and catabolic process. The nature of the response depends on the
nature (static, dynamic, compression, shear or tensile stress. . .) of the mechani-
cal stimuli. Whereas specific components of certain mechanotransduction pathways
have been identified, the exact mechanisms by which mechanical forces influence
the biologic activity of chondrocytes are not yet fully understood.
However, the general consensus is that static loads (0.013 MPa) result in an
decrease of cell proliferation, Col II synthesis (evaluated by 3 H proline incor-
poration, Col II gene expression) and proteoglycan synthesis (evaluated by 35 S
incorporation, Agg gene expression) whereas cyclic dynamic loads (0.15 MPa,
0.11 Hz) are found to increase these parameters (references (Palmoski and Brandt,
1984; Sah et al., 1989; Wong et al., 1997; Fitzgerald et al., 2006) for compression
of cartilage explants, (Buschmann et al., 1995; Lee and Bader, 1997; Hunter et al.,
2002) for compression of 3D-scaffold chondrocytes culture, (Smith et al., 2004;
Fitzgerald et al., 2006) for shear stress and (Hall et al., 1991; Jortikka et al., 2000)
for hydrostatic pressure). These responses are strongly dependent on the magni-
tude and the frequency of the applied load. Mechanical forces also influence the
13 The Mechanosensitivity of Cells in Joint Tissues 299

homeostasis of cartilage by regulating expression and activities of matrix metal-


loproteases (MMP) and A Disintegrin and Metalloprotease with Thrombospondin
motifs (ADAMTS) 4 and 5 which are responsible for the degradation of ECM.
In cyclically loaded-injured cartilage explants, cell death, proteoglycan loss and
Col damages are associated with an increase of MMP-3 immunostaining (Lin
et al., 2004). The compression of cartilage explants at physiological magnitude
(0.5 MPa, 1 Hz), conditions leading to an increase of proteoglycan synthesis,
results in an up-regulation of MMP-2 and 9 activities indicating that mechanical
load can affect remodeling of ECM (Blain et al., 2001). Dynamic compression
(510% of deformation, 0.10.3 Hz) of 3D-scaffold chondrocytes culture stimu-
lates proteoglycan synthesis evaluated by 35 S incorporation, but also increases 35 S-
GAG release from the 3D-scaffold indicating a degradation of the proteoglycans
(Buschmann et al., 1995; Lee et al., 2003a; Kisiday et al., 2009). MMP-3 and
-9 activities evaluated by zymography and expression of the genes coding for MMP-
3, 9 13, ADAMTS-4 and 5 were higher in the compressed 3D-scaffold chondrocytes
cultures (2.5% of deformation, 0.3 Hz) than in the controls (Kisiday et al., 2009).
These findings suggest that physiological mechanical stimuli contribute to main-
tain cartilage matrix homeostasis by regulating both the anabolic and the catabolic
functions of the chondrocytes.
Traumatic joint injury has been linked to an increase risk of developing OA.
Abnormal loading of cartilage was studied in vitro by applying strong impact load
on cartilage explants. Single static compression (1420 MPa) of cartilage explants
has been reported to induce chondrocyte apoptosis (DNA fragmentation, activa-
tion of caspase-3) and apoptosis is reversed in the presence of specific inhibitors
of casapase-3 and 9 (DLima et al., 2001; Huser et al., 2006). These deleterious
effects are accompanied with ECM damages (GAG release into culture medium,
Col fibril disruption, decrease of Agg and Col II gene expression) (Loening et al.,
2000; Chen et al., 2001; DLima et al., 2001; Huser et al., 2006; Wheeler et al.,
2009) and with induction of MMP-3 and ADAMTS-5 gene expression (Lee et al.,
2005). Immunohistochemistry analysis of injured cartilage shows an increase of
ADAMTS-5 protein expression (Lee et al., 2009).
Continuously applied shear stress (1.6 MPa) on chondrocyte is associated with
a decreased synthesis of anti-apoptotic factor bcl-2 and with nucleosomal degrada-
tion, characteristics for apoptotic process (Lee et al., 2003b). Chondrocyte apoptosis
evaluated by cleavage of caspases and DNA fragmentation is activated by shear
stress (Hashimoto et al., 2009) and by hydrostatic pressure (Islam et al., 2002).
These induction of apoptosis is accompanied by expression of tumor suppressor
protein p53 (Islam et al., 2002; Hashimoto et al., 2009) and is suppressed by p53
small interfering RNA and by p53 specific inhibitor (Hashimoto et al., 2009).
Beside the production of MMPs, mechanical stimuli may also regulate the
production of inflammatory mediators by chondrocytes.
Links between mechanical induced break down of cartilage and inflammation
of the joint are showed by in vivo studies. Repetitive impacts loading of rabbit
knee joint induce cartilage breakdown which is followed by synovial inflammation
(Lukoschek et al., 1986). An increase of immunostaining of interleukin (IL)-1beta
300 C. Sanchez et al.

and tumor necrosis factor (TNF) alpha in cartilage is observed after joint impact
of canine patellae (Pickvance et al., 1993). In vitro studies show that exposure of
cartilage explants and chondrocytes to mechanical strain of high amplitude leads to
the synthesis of inflammatory mediators. Intermittent compression (0.10.5 MPa,
0.5 Hz) and shear stress (1.6 MPa) stimulates nitric oxide (NO), prostaglandin (PG)
E2 and IL-6 productions through the activation of respectively inducible NO syn-
thase (iNOS), cyclooxygenase (COX)-2 and IL-6 gene expression (Mohtai et al.,
1996; Das et al., 1997; Fermor et al., 2002).
In contrast, biochemical signals could have anti-inflammatory effects. Cyclic ten-
sile stress (CTS) counteracts the IL-1 dependent chondrocyte catabolic response.
CTS (10% of deformation, 1 Hz) reduce the IL-1beta dependent synthesis of IL-6
and PGE2 (Mathy-Hartert M, 2008) in chondrocytes. The IL-1beta dependent stimu-
lation of NO/iNOS, PGE2 /COX-2 protein and gene expression is decreased by CTS
application (48% of deformation, 0.05 Hz) (Gassner et al., 1999; Agarwal et al.,
2004). The inhibition of Agg synthesis due to IL-1 is also reversed. These effects are
mediated by Nuclear Factor (NF)-B: its translocation to the nucleus is prevented by
CTS. When magnitude of the strain is increased to 15% of deformation, these anti-
inflammatory effects are nullified (Agarwal et al., 2004). IL-1 dependent mRNA
expression of MMP-3, 7, 8, 9, 13, 16, 17 and 19 of chondrocytes are also decreased
by CTS. Expression of MMP-2, 11, 14 and Tissue Inhibitor of Metalloproteinases
(TIMP) -1, 2 and 3 are not affected (Deschner et al., 2006).
Similar results were obtained in chondrocyte/agarose construct submitted to
dynamic compression (15%, 1 Hz). Load inhibited IL-1 stimulated NO/iNOS and
PGE2 /COX-2 synthesis and gene expression (Chowdhury et al., 2003; Chowdhury
et al., 2006) by a mechanism implicating Mitogen Activated Protein Kinase
(MAPK) and NF-B pathway.

13.2.2 Chondrocyte Mechanotransduction

The mechanisms by which mechanical stimuli alter the cellular metabolic functions
are known as mechanotransduction. When applied to cartilage, load causes a lot
of complex physiological modifications such as deformation of cells and matrix,
gradient in hydrostatic pressure, alteration in ionic, osmotic and pH composition,
intra-tissue fluid flow and streaming potentials. Biomechanics forces can be trans-
mitted into the cell by a variety of structures: deformation of the cell, strain on
the cytoskeleton, deformation of the nucleus and direct signaling pathways via
mechanoreceptors of the extracellular matrix.
Members of the integrin family play an important role in chondrocyte mechan-
otransduction as ECM-receptors (Loeser, 2002). The cytoplasmic domain of inte-
grin interacts with cytoskeletal proteins and mediates change in cell shape enabling
various cellular responses such as proliferation, ECM matrix synthesis and degra-
dation. In response to cyclic tension (10% of deformation, 1 Hz), chondrocytes
in a 3D fibrin construct undergo modification in their cytoskeletal organization:
loaded cells exhibit projection containing F-actin, vimentin and vinculin filaments
13 The Mechanosensitivity of Cells in Joint Tissues 301

(Vanderploeg et al., 2004). Alpha5/beta1 integrin (receptor for fibronectin) is a


major mechanoreceptor in chondrocyte. Its expression in OA cartilage is higher
than those observed in normal cartilage (Loeser et al., 1995). When adult bovine
cartilage explants are submitted to cyclic compression (1 MPa, 0.5 Hz), the content
of alpha5 and beta1 integrin subunit of compressed chondrocytes is increased com-
pared to free loading chondrocytes (Lucchinetti et al., 2004). Integrins-mediated
mecanotransduction include a lot of protein adapters such as protein kinase (PK)
A (Fitzgerald et al., 2004), PKC (Lee et al., 2002), paxilin (Lee et al., 2000), focal
adhesion kinase (FAK) (Lee et al., 2000) and members of the MAPK family (extra-
cellular signal-regulated kinase (ERK), and p38) (Hung et al., 2000; Fanning et al.,
2003; Li et al., 2003). These cascades of events reach to regulation of the transcrip-
tion of gene implicated in the maintenance and the integrity of ECM by transcription
factors such as cAMP response element (CRE) binding protein (CREB), activator
protein (AP)-1 and NF-B. The use of ERK1/2 and p38 inhibitors demonstrates
their requirement in mechano-induced Agg, Col II, MMP-3 and ADAMTS5 gene
expression (Fitzgerald et al., 2008).
Application of compression on cartilage explants results in membrane hyper-
polarisation and ion (Ca2+ , K+ ) channels activation such as stretch activated ions
channels (SAC), small calcium-activated potassium channels (SK). Many differ-
ent cascades of signaling molecules are activated by the entry of Ca2+ into the
cell including calmodulin, tyrosine protein kinase and PKC, focal adhesion kinase
(FAK). Inhibitors of signaling molecules such as calmodulin (Valhmu and Raia,
2002; Shimazaki et al., 2006), phosphoinositol (Valhmu and Raia, 2002), integrin
(Holledge et al., 2008; Chai et al., 2010), actin cytoskeleton (Wright et al., 1997),
SAC channel (Lee et al., 2000) abolish the increase of gene and protein expression of
Agg, Col II and MMP-3 by mechanical stimulation. The cascades G-protein/Adenyl
cyclase/cAMP/pKA is also involved in the stimulation of mRNA expression of
Agg and Col II in cartilage explants submitted to continuous compression (50%
of deformation) during 1 or 8 h (Fitzgerald et al., 2004).
ERK1/2 and p38 activation is implicated in the chondrocyte response to dynamic
shear stress (3% of deformation, 0.3 Hz) (Fitzgerald et al., 2008).
The Fig. 13.1 shows the signaling events reported in chondrocyte mechanotrans-
duction.

13.3 Mechanical Stimuli and Subchondral Bone

The subchondral bone is a global term which includes the subchondral bone plate
(cortical bone) and the underlying trabecular bone and bone marrow space. The
subchondral bone plate consists of cortical bone, which is relatively nonporous and
poorly vascularised. It is separated from the overlying articular cartilage by a zone
of calcified cartilage. The so-called tide-mark, which can be distinguished based
on its metachromatic staining pattern, provides a line of demarcation between the
hyaline cartilage and the calcified cartilage (Goldring, 2009). In OA, there is an
increase of the osteochondral bone plate thickness, a process likely affecting the
302 C. Sanchez et al.

Fig. 13.1 Mechanotransduction signal cascade activated in chondrocyte. ECM: extracellular


matrix, SAC: stretch activated ions channel, FAK: focal adhesion kinase, PLC: phospholipase
C, DAG: diacylglycerol, PK: protein kinase, MAPK: mitogen activated protein kinase, CREB:
cAMP response element binding protein, AP-1: activator protein 1, NF-B: nuclear factor B,
Agg: aggrecan, Coll II: type II collagen, MMP: matrix metalloprotease, ADAMTS: A disintegrin
and metalloprotease with thrombospondin motifs, iNOS: inducible nitric oxide synthase, COX-2:
cyclooxygenase, IL-6: interleukin 6

biomechanical properties of the overlying cartilage (Fig. 13.2a). The tide-mark


is duplicated with advancement of the calcified cartilage into the hyaline cartilage
further contributing to thinning of the cartilage lining (Fig. 13.2b) and subchondral
bone plate becomes sclerotic.
Subchondral bone sclerosis is associated with an increase of osteoid substance
deposition (sclerosis) and an abnormally low mineralization pattern. Thus subchon-
dral bone stiffness is due to an increase in material density, not mineral density.
It is now established that some osteoblasts of OA subchondral bone are phe-
notypically different, and may produce increased levels of alkaline phosphatase
(AP), osteocalcin, osteopontin, IL-6, -8, Transforming Growth Factor (TGF) -beta1,
Insulin-like Growth Factor-1 (IGF-1), urokinase plasminogen activator (uPA) and
PGE2 . Simultaneously, levels of IGF binding proteins 3, 4 and 5 are lower and
plasminogen activator inhibitor (PAI)-1 and IL-1beta levels remain unchanged
13 The Mechanosensitivity of Cells in Joint Tissues 303

Fig. 13.2 Osteoarthritic osteochondral junction (a) Subchondral bone sclerosis, Safranin-O/Light
green staining, 20x (b): Tidemark duplication, Hematoxylin/Eosin, 20x

(Sanchez et al., 2008). Due to it being a potent stimulator of bone matrix for-
mation by osteoblasts, the local accumulation of free IGF-1 is presented as a key
feature of subchondral bone plate sclerosis in OA. Additionally, OA osteoblasts are
resistant to parathyroid hormone (PTH) stimulation, a finding that contributes to
explain the abnormal bone remodeling in OA. OA osteoblasts produce an abnormal
homotrimeric type I collagen with a low affinity for calcium, which is responsible
for the low mineralization of the collagen matrix of OA subchondral bone. These
findings suggest that abnormal osteoblasts play a critical role in subchondral bone
sclerosis. Conversely, IL-6, PGE2 and Receptor Activator for Nuclear Factor B
Ligand (RANKL) may also be responsible for the increased number of active osteo-
clasts in OA subchondral bone and bone resorption observed in the early phase of
experimental OA (Liu et al., 2006b).
The bone tissue remodeling is the result of coordinated and balanced activities
of osteoblasts and osteoclasts. Osteoblast function is intimately linked to osteoclast
activity via the production of cytokines, growth factors and prostaglandins (PGs)
by osteoblasts. The production of some of these factors is controlled by mechani-
cal stimuli. Recently, a numbers of in vitro models attempted to screen genes and
signalling pathways involved in this mechanism, mainly by stretching osteoblasts
or by submitting them to a fluid shear stress. Osteoblasts possess mechanosen-
sors which activate intracellular signals including ion channels, integrins, calveolar
membrane structure and cytoskeleton. Nevertheless, response to physical signals
may be quite different according to the type of mechanical stress applied. Fluid
shear stress applied on osteoblasts in monolayer have been shown to elicit multiple
intracellular signalling pathways involving intracellular calcium rise, ERK1/2 acti-
vation of c-Fos and nuclear NF-B translocation (Chen et al., 2003; Inoue et al.,
2004). Downstream of such signalling events, various gene expression are induced,
including type I collagen (COL1), osteopontin (OPN), insulin-like growth factor-I
(IGF-1) and COX-2 (Chen et al., 2003).
304 C. Sanchez et al.

Dynamic cyclic tensile stresses are also potent activator of the signalling cascade
formed by ERK/c-fos/NF-B (Liu et al., 2006a). Stretching increases the production
of vascular endothelial growth factor (VEGF), TGF-beta1 (Singh et al., 2007), alka-
line phosphatase activity (AP), osteocalcin (OC), osteoprotegerin (OPG), MMP-1
and -3 (Kanno et al., 2007), COX-1 and -2, prostaglandin (PG)D2 synthase, per-
oxisome proliferator-activated receptor (PPAR) gamma-1 (Siddhivarn et al., 2006),
but decreases the release of the soluble receptor activator of nuclear factor ligand
(sRANKL) by osteoblasts (Tang et al., 2006). In contrast, no significant effect has
been reported on MMP-2, tissue inhibitor of metalloproteinases (TIMP)-1 and -2,
and PPARgamma-2 synthesis (Siddhivarn et al., 2006; Sasaki et al., 2007).
One major barrier to understanding bone physiology at a cellular level is the
lack of models for studying cells in their native environment. Usually, compres-
sion is generated by a bending system (Liu et al., 2006a) or a glass cylinder and
is applied on osteoblasts cultured in monolayer on flat surfaces (Mitsui et al.,
2006). We have developed an original model of 3D-osteoblast culture, allow-
ing the study of compression on osteoblasts embedded in their own produced
extracellular matrix (Sanchez et al., 2009). In this model, cell/matrix interactions
are conserved and fluid flow through a three dimensional extracellular matrix is
allowed.
In our study, loading was applied at large amplitude (11.67 MPa) and at a fre-
quency of 1 Hz. These loading conditions are included in the physiological range
of amplitude and frequency of mechanical strains applied on bone during locomo-
tion. In our experimental conditions, we have observed a strong release of PGE2
in the culture medium of loaded 3D-osteoblasts. This result confirms previous
studies demonstrating that fluid flow, compression and stretching stimulate PGE2
production by osteoblasts (Bakker et al., 2006; Grimston et al., 2006). Further,
we demonstrated that PGE2 release probably results from an imbalance between
PGE2 synthesis and degradation. Indeed, in our experimental conditions, compres-
sion increased COX-2 expression but decreased 15-PGDH expression. 15-PGDH
is a cytosolic enzyme which catalyzes the first step in the catabolic pathway of
prostaglandins, and believed to be the key enzyme responsible for the biological
inactivation of this biologically potent eicosanoid (Cho et al., 2006). Another impor-
tant finding was that compression had no significant effects on COX-1 and mPGES
gene expression. This contrast with previously reported data showing that compres-
sion of cartilage explants increased mPGES1 expression by chondrocytes (Gosset
et al., 2006).
PGE2 is also a mediator involved in IL-6-induced osteoclast formation and bone
resorption (Liu et al., 2006b). Recently, we have shown for the first time that IL-6
is a highly mechanosensitive gene. IL-6 expression was early increased (1 h) and
IL-6 protein secretion was highly stimulated (up to 32-fold) by 4 h 1 Hz 1.67 MPa
compression. The role of IL-6 on bone physiology is complex. IL-6 clearly stimu-
lates osteoclast activation and bone resorption in vivo and in vitro (Palmqvist et al.,
2002; De Benedetti et al., 2006). Data on the in vivo and in vitro effects of IL-6 on
osteoblasts are still conflicting, and several models have shown contradictory results
(Franchimont et al., 2005). In a recent model of transgenic mice overexpressing
13 The Mechanosensitivity of Cells in Joint Tissues 305

IL-6, a marked decrease in osteoblast activity, proliferation and expression of gene


of bone matrix protein was found (De Benedetti et al., 2006).
Interestingly, IL-6 neutralization significantly reduced PGE2 production and the
full inhibition of PGE2 synthesis by piroxicam drastically inhibited IL-6 produc-
tion. These findings provide evidence for cross-talk between PGE2 and IL-6. We
speculate that this interaction contributes to amplify the response of osteoblasts
to compression. Previous studies have demonstrated that the COX-2/PGE2 sys-
tem stimulates osteoclasts differentiation via an up-regulation of IL-6 secretion
by osteoblasts. Therefore, PGE2 secretion could enhance IL-6 production by
osteoblasts in response to a mechanical stress and then, induce osteoclast differen-
tiation and activation. This cross-talk results in increasing osteoclast differentiation
and activation via an effect on the RANK/RANKL/OPG system in bone cells (Liu
et al., 2005).
Interestingly, we have observed that compression (1.67 MPa at 1 Hz) decreased
OPG gene expression. OPG is a decoy receptor for RANKL which inhibits
osteoclast activation. IL-6 is the mediator of PGE2 induced suppression of OPG
production by osteoblasts. Therefore, we can hypothesize that physiological com-
pression induced the activation of osteoclasts via reciprocal interactions of IL-6 and
PGE2 produced by osteoblasts.
Matrix metalloproteinases have been reported to play a role in the physiologi-
cal bone remodeling. This study shows that compressive stress stimulate MMP-2,
MMP-3 and MMP-13 gene expressions and MMP-3 synthesis, suggesting that
osteoblasts may contribute to bone remodeling. One possible role for MMPs is to
prepare recruitment sites for osteoclasts and its progenitors by degrading collage-
nous extracellular matrix covering the mineralized bone surface, and then to expose
RGD (Arg-Gly-Asp) sequences which allow osteoclasts adhesion via alpha v/beta
3 integrin receptor (Helfrich et al., 1996). Thus, degradation of collagen on bone sur-
face not only allows osteoclasts attachment, but may also stimulate them to proceed
to activation and resorption phases (Fig. 13.3). MMP-3 contributes to the resorption
of osteoid matrix through activation of collagenases.
These findings also contribute to explain subchondral bone sclerosis in OA. We
have demonstrated that compression increased IL-6, PGE2 and MMP-3 produc-
tion by osteoblasts coming non sclerotic area to the level of sclerotic osteoblasts
(Sanchez et al., 2008). Therefore, we hypothesize that mechanical stress could be
responsible for these alterations in subchondral bone osteoblasts phenotype.
Finally, subchondral bone sclerosis could affect the overlying cartilage not only
by increasing the strain applied on it, but also through mediators produced by
osteoblasts. Indeed, mechanical stress (overloading or trauma) may generate micro-
cracks and fissures at the bone/cartilage interface allowing an increased exchange of
mediators between the two tissues (Fig. 13.2). Indeed, the osteoblasts secrete a num-
ber of biochemical factors that are involved in the remodeling of bone tissue, which
could also contribute to the remodeling of the overlying cartilage in weight bear-
ing joints after seeping through microcracks or blood vessels in the calcified layer
of articular cartilage. Three elements support this hypothesis: (1) in OA, microc-
racks have been identified at the bone/cartilage junction allowing exchange between
306 C. Sanchez et al.

Fig. 13.3 Hypothetic schema of effect of compression on bone remodelling. Osteocytes and
osteoblasts respond to mechanical stimuli and produce NO, PGE2 and IL-6. These mediators
modify the OPG/RANKL balance in favour to RANKL and to osteoclasts activation and matrix
degradation. In parallel, osteoblasts produce also MMPs which degrade the bone matrix, allow-
ing osteoclasts attachment and OPN, OC, TGFs release. VEGF is also produced by osteoblasts,
promoting the arrival of new blood vessel and new osteoclasts precursors. IL-6: Interleukin-6,
MMP: matrix metalloproteinase, NO: nitric oxide, PGE2 : prostaglandin E2, OPG: osteoprote-
gerin, RANKL: receptor activator of NF-B ligand, VEGF: vascular endothelial growth factor;
OPN: osteopontin; OC osteoocalcin; TGF- transforming growth factors

the two tissues (Fig. 13.3); (2) blood vessels have been observed in the calcified
cartilage; (3) hepatocyte growth factor, which is secreted by osteoblasts (not chon-
drocytes), are detected in the deep layers of OA cartilage; (4) co-culture models
have shown that conditioned media from primary osteoblasts of OA patients sig-
nificantly increases GAG release from normal cartilage explants whereas culture
medium conditioned by normal osteoblasts had no effects (Westacott et al., 1997),
and that osteoblasts from sclerotic (SC) zones of subchondral OA, but not non-
sclerotic (NSC) osteoblasts, downregulated AGG synthesis and upregulated MMPs
expression by chondrocytes (Sanchez et al., 2005). These effects were driven by
IL-6, a cytokine which is overexpressed by sclerotic osteoblasts.

13.4 Others Joint Tissues Mechanosensitivity

Other joint tissues, including meniscus, tendon, ligament and synovial membrane,
are exposed to mechanical stimuli. However, the influence of mechanical stimuli on
these tissues remains unexplored.
Two different aspects of biomechanical involvement of tendons and ligaments
have to be considered in articular pathologies. On the one hand the importance of
13 The Mechanosensitivity of Cells in Joint Tissues 307

mechanical stimuli on the integrity of tendons and ligaments, and the other side
the importance of these tissues in maintaining the stability of the joint, and there-
fore the optimal distribution of mechanical stimuli on cartilage. Tendons connect
muscle to bone and are designed to transmit forces and withstand tension during
muscle contraction. They contain few cells, mostly represented by tenoblasts along
with endothelial cells and some chondrocytes-like cells. Tendons are dense connec-
tive tissues composed of a composite of collagens (mainly type I), proteoglycans
and other noncollagenous proteins (Cartilage Oligomeric Matrix Protein, decorin
and fibromodulin) (Kannus, 2000). Age-related tendon failure is due to cumulative
damage resulting from a combination of diminished matrix repair and fragmenta-
tion of extracellular matrix proteins induced by prolonged cyclical tensile strength
(Dudhia et al., 2007). Physiological cyclic tensile (5% strain at 1 Hz during 24 h)
induces the release of MMPs and degraded COMP by tendon cells or explants.
These changes are more pronounced in old than in young tendon (Dudhia et al.,
2007). Low dynamic tensile stimulation of tendons cells up-regulates MMP-13
expression (Lavagnino andArnoczky, 2005) whereas static loading down-regulates
the expression of this protease. (Arnoczky et al., 2008). These findings suggest that
tendon requires a tensile strain threshold to prevent the tendon extracellular matrix
resorption and its weakening (Kjaer et al., 2006).
Ligaments are bands of dense connective tissue which courses from bone to bone,
across joint. They play a crucial role in joint stability. The most studied is the ante-
rior cruciate ligament (ACL) which crosses joint cavity from the femur to the tibia.
It is composed of three zones: the proximal part, which is less solid, is highly cel-
lular, rich in round and ovoid cells, containing some fusiform fibroblasts, collagen
type II and glycoproteins such as fibronectin and laminin; the middle part, contain-
ing fusiform and spindle-shape fibroblasts, is a high density of collagen fibers, a
special zone of cartilage and fibrocartilage, and elastic, and oxytalan fibers; and the
distal part, which is the most solid, is rich in chondroblasts and ovoid fibroblasts,
and with a low density of collagen bundles (Duthon et al., 2006). Mechanical stim-
uli are important for ligament homeostasis. They have stress-oriented structures of
collagen bundles. Uni-axial cyclic stretch increases the gene expressions of type I
and III collagens by mediating the autocrine secretion of TGF-1 (Kim et al., 2002),
and by modulating the integrin V3-dependant focal adhesion in human ACL cells
(Tetsunaga et al., 2009).
The menisci are wedge-shaped semi-lunar fibrocartilaginous structures that
correct the incongruence of the femoral and tibial articular surfaces (Hellio Le
Graverand et al., 2001). Menisci provide important biomechanical functions to the
knee joint such as load bearing, load distribution, shock absorption and joint sta-
bility (Fithian et al., 1990). Meniscal tissue is composed mainly of type I collagen
and contain considerably less proteoglycan (<1%) than hyaline cartilage. ACL sec-
tion causes mechanical damage in meniscus. At 3 and 8 weeks following the ACL
section, histological examination demonstrated extensive extracellular matrix dete-
rioration. Altered cell distribution, areas depleted of cells, and areas of cell clusters
were found within the medial but not in the lateral meniscus (Hellio Le Graverand
et al., 2001). Type I and III collagen immunostaining was increased in both lateral
308 C. Sanchez et al.

and medial menisci. In contrast, type II collagen staining was overtly increased only
in the medial meniscus (Hellio Le Graverand et al., 2001).
In OA, synovial membrane is inflamed secondary to the release of degradation
products and inflammatory mediators from cartilage. Synovial inflammation plays
a key role in the clinical and structural changes occurring in cartilage degradation
through the release by synovial cells (macrophages, synoviocyte, lymphocyte T).
Mechanical tensile stress down-regulates the expression and the release of MMP-1
and -13, but doesnt change MMP-2 production by synovial fibroblasts (Wang et al.,
2009). In contrast, MMP-3 is up-regulated by cyclic tensile load (Raif el, 2008).

13.5 Conclusion and Perspectives

Mechanical loading plays a dual role in the homeostasis of joint tissues: when
applied at moderate and physiological range, mechanical stimuli are beneficial to
the good health of joint tissues whereas excessive loading initiate abnormal tis-
sue remodeling and structural changes. Appropriate mechanical load could play an
important role in the homeostasis of joint tissues. This raises the question of the
role played by exercises in the prevention and treatment of OA. Recent recommen-
dations, clearly indicate that patients with hip and knee OA should be encouraged
to undertake, regular aerobic, muscle strengthening and range of motion exercises
(Zhang et al., 2008). Future clinical trials are required to demonstrate the chon-
droprotective effects of regular exercises on cartilage metabolism to sustain these
expert consensus-based guidelines.
Acknowledgements Christelle Sanchez is a post-doctoral researcher of the National Fund for
Scientific Research (FNRS, Belgium)

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Part VI
Mechanotransduction of Sensor System
Chapter 14
Primary Cilia are Mechanosensory Organelles
in Vestibular Tissues

Surya M. Nauli, Hanan S. Haymour, Wissam A. Aboualaiwi, Shao T. Lo,


and Andromeda M. Nauli

Abstract Primary cilia have been observed for over a century, but their sensory
roles have only been revealed within the past decade. In this chapter, we will
describe cilia as newly recognized mechanosensory organelles. Cilia can sense bod-
ily fluid movement in all vestibular organs. These include nodal flow in Hensens
node, urine in the renal nephron, bile in the hepatic biliary system, digestive fluid
in the pancreatic duct, dentin in dental pulp, lacunocanalicular fluid in bone and
cartilage, and blood in vasculature. To exert their sensory functions, cilia require
both structural and functional proteins. Cells without ciliary function or structure
are unable to sense fluid-shear stress, but their sensitivity toward other mechanical
or pharmacological stimuli remains intact. The functional machineries found in the
cilia include mechanosensory polycystin-1, mechano-calcium channel polycystin-2,
and other interacting proteins. The roles of cilia as fluid sensors in Hensens node
as well as in the kidney, liver, pancreas, bone, and cardiovascular system will be
discussed.

Keywords Fluid flow Mechanosensing Primary cilium Shear stress Signal


transduction

14.1 Introduction

Biologists have long been studying the complexity of cellular functions of a sin-
gle cell, which signifies a fundamental unit of life. Cilia are among the organelles
possessed by cells that have been discussed extensively. Structurally, cilia can be
classified into two types, based on their microtubule arrangements of either 9+0
or 9+2 (Fig. 14.1). However, based on their motility, cilia can be functionally

S.M. Nauli (B)


Department of Pharmacology, MS 1015; The University of Toledo; Health Science Campus, HEB
274; 3000 Arlington Ave., Toledo, OH 43614, USA
e-mail: Surya.Nauli@UToledo.Edu

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 317


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_14,

C Springer Science+Business Media B.V. 2011
318 S.M. Nauli et al.

Fig. 14.1 A brief introduction of a cilium. (a) A cilium is an organelle projected at the apical
membrane of many cell types. It is projected from the basal body. (b) A cilium is considered a cel-
lular organelle, which is composed of a membrane domain, soluble compartment, axoneme, and
basal body. The membrane domain contains various sensory proteins. The soluble compartment
has been shown to contain many regulatory proteins that are involved in signal transduction sys-
tems. The axoneme is surrounded by the nine pairs of microtubules that are thought to control the
length of a cilium. The basal body is the anchoring point for axonemal microtubules. (c) Based
on the central pair of the microtubules in the axoneme, a cilium can be categorized into 9+2
and 9+0 structures. It was once thought that a cilium with 9+0 axoneme was always immotile.
Classification of cilia becomes more complex, particularly since the presence of 9+4 cilia has
also been reported (Feistel and Blum, 2006)

classified as motile or non-motile organelles. Like any organelles in the cells, cilia
have many important and specialized cellular functions (Table 14.1). Classification
of cilia can thus provide a broad spectrum of understanding about their cellular
functions.
Primary cilia are usually classified as 9+0 non-motile organelles (Table 14.1).
Although they were first described as early as 1898 (Wheatley, 2005), primary cilia
were thought to be vestigial organelles. It was assumed that cilia were remnants
from flagella of a single-cell ancestral organism and no longer had any particular
function. A primary cilium is a microtubule-based, antenna-like structure and is
found in a single copy on the apical surface of fully differentiated mammalian cells.
The diameter of a cilium is approximately 0.25 m, and its length can vary from
Table 14.1 Ciliary classification, function and disease relevance in mammals
14

Axoneme Motility Function Disease relevance Reference

9+0 Motile Nodal flow generation Situs inversus; Situs ambiguus; Nonaka et al. (1998, 2002), and Essner et al.
Situs isomerism (2002)
Non-motile Nodal flow sensor Situs inversus; Situs ambiguus; McGrath et al. (2003) and Karcher et al. (2005)
Situs isomerism
Mechanosensor Polycystic kidney, liver, pancreas Nauli et al. (2003), Cano et al. (2006), and
Masyuk et al. (2006)
Shear-stress sensor Hypertension; Arthrosclerosis Nauli et al. (2008), Van der Heiden et al. (2008),
and AbouAlaiwi et al. (2009b)
Osmo sensor Respiratory diseases; Infertility Andrade et al. (2005) and Teilmann et al. (2005)
Gravitational sensor Osteoporosis; Chondroporosis Malone et al. (2007), McGlashan et al. (2007)
and Moorman and Shorr (2008)
Smell sensor Anosmia; Hyposmia Kulaga et al. (2004), Layman et al. (2009)
Light sensor Retinitis pigmentosa, Blindness Nishimura et al. (2004a), Moore et al. (2006),
Ghosh et al. (2010)
Chemo sensor Nephrocystin; Diabetes; Obesity Hearn et al. (2005), Winkelbauer et al. (2005),
and Davenport et al. (2007)
Developmental Abnormal development; Cancer (Christensen et al. (2008), Han et al. (2009), and
regulator Wong et al. (2009)
9+2 Motile Chemo sensor Chronic obstructive pulmonary Shah et al. (2009)
disease
Fluid clearance device Chronic obstructive pulmonary Salathe (2007), Zariwala et al. (2007), and Mall
Primary Cilia are Mechanosensory Organelles in Vestibular Tissues

disease (2008)
Oocyte transport Infertility Eddy and Pauerstein (1980) and Lyons et al.
(2006)
Sperm motility Infertility Brunner et al. (2008), Lee et al. (2008), and Imai
et al. (2009)
Fluid transport Hydrocephalus; Cell migration Ibanez-Tallon et al. (2004), Sawamoto et al.
(2006), and Wodarczyk et al. (2009)
Non-motile Sound wave sensor Hearing loss Littlewood Evans and Muller (2000), Grillet
et al., (2009a, b)
319
320 S.M. Nauli et al.

2 to 50 m. A cilium is projected from a mother centriole (Sorokin, 1962). A dif-


ferentiated cell usually has mother and daughter centrioles that are arranged in
such orientation to form a centrosome (Paintrand et al., 1992; Kenney et al., 1997).
The centrosome itself is a major microtubule-organizing center of the cell (Rose
et al., 1993; Archer and Solomon, 1994). Abnormal primary cilia function and/or
ciliary proteins are now linked to various developmental issues and other disorders
known as ciliopathies. These include left-right asymmetry defect, nephronophthisis,
Bardet Biedl Syndrome, Oral Facial Syndrome, polycystic kidney disease, obesity,
and hypertension, among others.

14.1.1 Building Blocks of Cilia


The primary cilium extending from the cell membrane has nine parallel doublet
microtubules and the absence of the central pair of microtubule within the central
sheath (Fig. 14.1). The cilium also possesses radial spokes linked to the micro-
tubules. The presence of inner and outer arm dynein motors and dynein-regulatory
proteins with ATPase activity was once thought to be responsible for cilia motil-
ity. However, the dynein arms have also been found in 9+0 non-motile cilium
(Lungarella et al., 1984; Yamamoto and Kataoka, 1986). Centrioles are required for
cilia formation (Marshall, 2009). During the G0 stage of the cell cycle, the mature
centriole becomes the basal body, where a cilium is projected and extended toward
the apical cell membrane (Satir and Christensen, 2007). The extended cilium is
enclosed by the membrane and filled with microtubule to form the ciliary axoneme
(Pan et al., 2005).
As a cellular organelle, a cilium structurally has at least five domains (Fig. 14.1).
The first domain is the ciliary membrane. This domain has lipid composition that
is different from the rest of plasma membrane. Many sensory receptors and ion
channels are localized in this domain to support the mechanosensory roles of cilia.
Ligand-activated receptors have also been localized in this domain to support the
chemical-sensing functions of cilia. The second domain is the soluble compartment,
which is also called the matrix compartment or cilioplasm. The cilioplasm is com-
posed of fluid material to support various signaling proteins. The third domain is
the axoneme, which is composed of nine pairs of microtubules. These microtubules
are built from - and -tubulin subunits, which form a heterodimer structure. The
microtubules are posttranslationally modified to support the long ciliary structure.
The axoneme plays important roles for intraflagellar transport proteins to deliver
cellular components into and out of the ciliary shaft. Proper axonemal complex is
needed to support assembly and maintenance of the long ciliary structure. The fourth
domain of cilia is the tip or the distal part of cilia. The ciliary tip contains specialized
protein complexes whose roles are still to be explored further. The fifth domain is the
basal body, which is a mature or mother centriole from which the primary cil-
ium is projected. Centriole duplication during cell division remains an unknown and
appealing phenomenon to be pursued. Because centrosomes will become anchoring
points for mitotic spindles during mitosis, many centriole proteins have thus been
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 321

associated with cell division. In the fruit fly, interestingly, centriole formation does
not seem to be a prerequisite of cell divisions, although centrioles are still essential
for the formation of cilia (Basto et al., 2006).
It is estimated that a cilium has over a thousand proteins (Dutcher, 1995; Pazour
et al., 2005; Gherman et al., 2006), making this organelle very complex to study. To
date, a handful of proteins have been characterized and localized to different ciliary
domains (Table 14.2). The centrosomal proteins tend to be more convoluted because
of the recruitments of other proteins into centrosomes during cell division. As such,
knowledge about centrosomal proteins is expected to expand in the near future as
their relationships to ciliary localization and/or function are ascertained.
The assembly of primary cilia was first discovered through the unicellular green
alga Chlamydomonas. Cilia are assembled from proteins that are synthesized in the
cell body and transported to the ciliary tips by a complex process involving intraflag-
ellar transport (Rosenbaum and Witman, 2002; Pan and Snell, 2007). This process
involves bidirectional movements of nonmembrane-bound macro-molecular pro-
tein complexes or multi-meric protein particles along the axoneme (Scholey, 2003;
Scholey and Anderson, 2006). Thus, a successful cilia assembly depends on three
key components: the intraflagellar transport particles, the anterograde motors and
the retrograde motors. The anterograde proteins, mediated by complex B raft com-
ponents, carry axonemal components from the cell body to the growing tip of
cilia. Retrograde proteins, mediated by complex A components, carry the axone-
mal machineries back from the tip of the cilium to the cell body. Whereas kinesin-II
is the anterograde motor, dynein-2 is the retrograde motor. Both of these anterograde
and retrograde proteins are powered by microtubule motors.

14.1.2 Cilia and Vestibular Organs

Almost all human cell types have either a bundle of motile cilia or a solitary
immotile cilium. In general, only circulating cells or cells under suspension do not
develop cilia. Such cells include circulating red and white blood cells. As will be
described in the next section, mechano-fluid sensing is the most understood role
among the major functions of primary cilia. As such, it is not surprising that pri-
mary cilia as mechano-fluid sensors can be found in all organs that support and
sense fluid perfusion in the human body. By definition, the body tissues that have
small canal or cavity spaces (vestibules) to support perfusion of body fluid are called
vestibular organs.
The mechano-fluid sensing role of primary cilia has been demonstrated in many
vestibular organs. Such organs depend on mechanosensing cilia to sense and trans-
mit extracellular signals to intracellular biochemical reactions. Cilia can thus sense a
variety of fluid movements in the body, including blood in vasculature (AbouAlaiwi
et al., 2009a), interstitial fluid in the bone matrix (Turner et al., 1994; Whitfield,
2008), urine in kidney nephrons (Nauli and Zhou, 2004; Kolb and Nauli, 2008), bile
in the hepatic biliary system (Masyuk et al., 2008, 2009), digestive fluid in the pan-
creatic duct (Aughsteen, 2001; Kaestner, 2006), cerebral spinal fluid in the neuronal
322

Table 14.2 Structural and functional ciliary proteins based on their ciliary domains

Ciliary tip Ciliary soluble compartment

EB1 Pedersen et al. (2003) and Schroder et al. (2007) 14-3-3 Fan et al. (2004a)
Gli Haycraft et al. (2005) and Liem et al. (2009) Adenylyl cyclase Menco (2005) and Bishop et al. (2007)
KIF7 Endoh-Yamagami et al. (2009) and Liem et al. Arl13b Cantagrel et al. (2008) and Hori et al. (2008)
(2009)
Smo Corbit et al. (2005), Haycraft et al. (2005) and Arl2l1 Sun et al. (2004)
Liem et al. (2009)
Sufu Jia et al. (2009) ATP synthase Hu and Barr (2005)
-arrestin-2 Menco (2005)

Ciliary axoneme CaM Kinase II [16374707]


DNAH11 Bartoloni et al. (2002) CAML Nagano et al. (2005)
DNAH5 Ibanez-Tallon et al. (2004) CRB1 Fan et al. (2004a)
DNAH7 Zhang et al. (2002) CRB3 Fan et al. (2004a), Omori and Malicki (2006), and
Fan et al. (2007)
DNAI1 Pennarun et al. (1999) Cystin Hou et al. (2002) and Yoder et al. (2002a)
Dyf-1 Ou et al. (2005a) and Dave et al. (2009) GRK3 Menco (2005)
Dyf-3 Murayama et al. (2005) and Ou et al. (2005b) GSK3 Etienne-Manneville and Hall (2003) and Thoma
et al. (2007)
DYNC2H1 May et al. (2005) Importin Fan et al. (2007)
DYNC2LI1 Rana et al. (2004) Mek1/2 Schneider et al. (2005)
Hydin Davy and Robinson (2003) and Pazour et al. OSEG family Avidor-Reiss et al. (2004)
(2005)
IFT140 Tsujikawa and Malicki (2004) Par3 Fan et al. (2004a), Nishimura et al. (2004b), and
Sfakianos et al. (2007)
IFT172 Huangfu et al. (2003), Sun et al. (2004), Par6 Fan et al. (2004a)
Pedersen et al. (2005), Gorivodsky et al.
(2009), and Lunt et al. (2009)
S.M. Nauli et al.
14

Table 14.2 (continued)

Ciliary tip Ciliary soluble compartment

IFT20 Follit et al. (2006) and Jonassen et al. (2008) Phosphodiesterase Menco (2005)
IFT46 Gouttenoire et al. (2007) PKC Etienne-Manneville and Hall (2003) and Fan
et al. (2004a)
IFT52 Tsujikawa and Malicki (2004) and Liu et al. (2005a) pVHL Okuda et al. (1999), Lolkema et al. (2007), and
Thoma et al. (2007)
IFT57/curly Krock and Perkins (2008) and Lunt et al. (2009) STAT6 Low et al. (2006)
IFT57/hippi Tsujikawa and Malicki (2004) and Houde et al. Tubby Mukhopadhyay et al. (2005) and Mak et al.
(2006) (2006)
IFT80 Beales et al. (2007) TULP2 Stolc et al. (2005)
IFT81 Sun et al. (2004) and Lucker et al. (2005)

IFT88 Murcia et al. (2000), Pazour et al. (2000), Haycraft Ciliary membrane
et al. (2001), Qin et al. (2001), and Yoder et al.
(2002b)
Kif17 Jenkins et al. (2006) EGFR Ma et al. (2005)
Kif3A/B Marszalek et al. (1999), Takeda et al. (1999), Fibrocystin Ward et al. (2003), Wang et al. (2007)
Marszalek et al. (2000), and Lin et al. (2003)
MDHC7 Neesen et al. (2001) and Vernon et al. (2005) Mchr1 Berbari et al. (2008)
PACRG Lorenzetti et al. (2004) and Dawe et al. (2005) PDGFR Schneider et al. (2005)
PF13 Omran et al. (2008) Polycystin-1 Barr and Sternberg (1999) and Yoder et al.
Primary Cilia are Mechanosensory Organelles in Vestibular Tissues

(2002a)
PF16 Sapiro et al. (2002) and Zhang et al. (2005) Polycystin-2 Barr and Sternberg (1999), Pazour et al. (2002),
and Yoder et al. (2002a)
PF2 Rupp and Porter (2003) Somatostatin-3 receptor Schulz et al. (2000)
PF20 Zhang et al. (2004) Serotonin-6 receptor Brailov et al. (2000)
Tektin Tanaka et al. (2004) Tie-1,Tie-2 receptors Teilmann and Christensen (2005)
TRPN1 Kim et al. (2003) and Shin et al. (2005)
TRPV4 Qin et al. (2005) and Teilmann et al. (2005)
323
Table 14.2 (continued)

Ciliary tip Ciliary soluble compartment


324

Ciliary base (centrosome)


ALMS1 Hearn et al. (2005), Graser et al. (2007), Li et al. Nek1 Mahjoub et al. (2005) and White and Quarmby (2008)
(2007), and Mikule et al. (2007)
BBS1 Oliveira and Goodell (2003), Davis et al. (2007), Nek2 Bahe et al. (2005)
and Oeffner et al. (2008)
BBS2 Nishimura et al. (2004a), Nachury et al. (2007), Nek7 Yissachar et al. (2006) and Kim et al. (2007)
and Oeffner et al. (2008)
BBS3 Fan et al. (2004b) Nek8 Mahjoub et al. (2005) and Otto et al. (2008)
BBS4 Kim et al. (2004), Gerdes et al. (2007), and NPHP-1 Otto et al. (2003) and Winkelbauer et al. (2005)
Oeffner et al. (2008)
BBS5 Li et al. (2004) and Yen et al. (2006) NPHP-2 Otto et al. (2003)
BBS6 Kim et al. (2005) NPHP-3 Olbrich et al. (2003) and Bergmann et al. (2008)
BBS7 Oliveira and Goodell (2003) and Blacque et al. NPHP-4 Mollet et al. (2005) and Winkelbauer et al. (2005)
(2004)
BBS8 Ansley et al. (2003) and Blacque et al. (2004) NPHP-5 Otto et al. (2005)
CC2D2A Gorden et al. (2008) NPHP-6 Sayer et al. (2006)
Cep164 Graser et al. (2007) ODF2 Donkor et al. (2004) and Ishikawa et al. (2005)
CEP290 Gorden et al. (2008) and Kim et al. (2008) OFD1 Romio et al. (2004) and Ferrante et al. (2006)
EBI Askham et al. (2002), Piehl et al. (2004), and p-150 Askham et al. (2002)
Schroder et al. (2007)
Fa2p Mahjoub et al. (2004) PCM-1 Kim et al. (2004), Graser et al. (2007), Mikule et al.
(2007), and Kim et al. (2008)
FAPP2 Vieira et al. (2006) Pericentrin Jurczyk et al. (2004), Graser et al. (2007), and Mikule
et al. (2007)
Fin1 Grallert and Hagan (2002) POC12/MKS1 Kyttala et al. (2006), Dawe et al. (2007), and
Weatherbee et al. (2009)
Fleer Pathak et al. (2007) Rab8 Kim et al. (2008)
Jouberin Eley et al. (2008) Rootletin Yang et al. (2002, 2005) and Bahe et al. (2005)
MKS-1 Kyttala et al. (2006) and Dawe et al. (2007) RPGR Shu et al. (2005)
MKS-3 Smith et al. (2006), Dawe et al. (2007), and Seahorse Morgan et al. (2005) and Kishimoto et al. (2008)
Tammachote et al. (2009)
S.M. Nauli et al.

UNC Baker et al. (2004)


14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 325

tube (Lang et al., 2006; Wodarczyk et al., 2009), fluid pressure in the inner ears
(Kernan, 2007; Petit and Richardson, 2009), and so on. The inability to sense fluid-
shear in these vestibular organs may thus contribute to multiple organ pathogenesis,
from hypertension to hydrocephalus and from deafness to cystic organ formation.

14.2 Cilia as Fluid Sensors


The idea of primary cilia as sensory organelles was probably developed in 1950s
(Fawcett and Porter, 1954; De Robertis, 1956). It was realized that cilia in the
mammalian photoreceptors are immotile and have a 9+0 axonemal structure.
Since then, hypotheses about primary cilia as sensory organelles were developed
(Poole et al., 1985; Roth et al., 1988; Wheatley, 1995). It was further shown that
primary cilia can be bent when cells were superfused with fluid (Schwartz et al.,
1997). At an optimal fluid shear stress, primary cilia can respond by bending
at their axonemal structures (Fig. 14.2). Dr. Springs laboratory and our labora-
tory have further confirmed the hypothesis that primary cilia are mechanosensory
organelles and are responsive to fluid shear-stress (Nauli et al., 2003; Praetorius and
Spring, 2003).
Mechanosensory studies on primary cilia of kidney epithelial cells showed and
confirmed that cilia are responsive to fluid and mechanical stress. Cilia can be acti-
vated by bending with either suction through a micropipette (Praetorius and Spring,
2001) or apical fluid perfusion through changing the flow rate (Nauli et al., 2003;
Praetorius and Spring, 2003). Because cilia are micro-sensory compartments, the
role of cilia depends on mechano-proteins such as polycystin-1 (Nauli et al., 2008),
polycystin-2 (AbouAlaiwi et al., 2009b), fibrocystin (Wang et al., 2007), transient
receptor potential-4 (Kottgen et al., 2008), and probably many others, yet to be dis-
covered. Thus, the overall functions of the sensory cilia compartments depend on
both functional and structural axonemal proteins.

Fig. 14.2 Cilia are mechanosensory organelles. Cilia are sensory organelles that sense fluid-shear
stress on the apical membrane of the cells. Fluid flow that produces enough drag-force on the cells
will bend sensory cilia. These biomechanics play very crucial roles in vestibular organs to support
bodily fluid perfusion. Figure is reproduced from AbouAlaiwi, et al. with permission (AbouAlaiwi
et al., 2009a)
326 S.M. Nauli et al.

14.2.1 Structural Proteins


Structurally, cilia length is supported by heterodimers of and tubulin sub-
units. Upon polymerization at the (+) end, the axonemal structure is assembled and
extended toward the distal end of the ciliary tip. Unlike cytoskeletal tubulin, how-
ever, the ciliary tubulin is highly modified. The ciliary tubulin is post-translationally
acetylated, detyrosinated, polyglycylated and polyglutamylated (Redeker et al.,
2005; Gaertig and Wloga, 2008). Although tubulin is by far the most abundant struc-
tural protein, the overall ciliary structure is more complex than previously thought.
The assembly of ciliary structure depends on complex transport machinery known
as interflagellar transport (IFT) protein. Mutations in any of these transport proteins
will result in abnormal ciliary structure (Table 14.2).
The basal body has a primary function for anchoring and organizing microtubules
that assemble the axoneme. The basal body and centriole serve as a microtubule-
organizing center, which functions as the base for cilia to facilitate movement of
IFT up and down the ciliary shaft. The microtubule-organizing center also provides
unidirectional movement of microtubules and other transport molecules (vesicles).
The centriole is composed of -tubulin subunits. The -tubulin is the most abundant
protein in the centriole and centrosome. In addition to being the base for cilia, the
centrosome is also involved in the organization of the mitotic and meiotic spindle
apparatus (Rieder et al., 2001; Doxsey et al., 2005). Many other proteins within the
centrosome may thus have additional functions during cell division (Table 14.2).
Accordingly, many abnormal ciliary proteins would eventually result in abnormal
cell division.
The microtubule-organizing center also appears to integrate cilia with the cells
cytoskeleton and integrin. A communication system in the cytoskeletal network
seems to begin from a cilium to a centrosome. Once it reaches the centrosome, the
microtubule-organizing center converges the cytoskeletal network to the cystosol
and cell membrane. Any interruption of this communication network would result
in cilia dysfunction. For example, when cytosolic actin or tubulin microfilaments
are not fully functional, sensory cilia become unable to initiate a sensing mecha-
nism in response to extracellular mechanical signal (Alenghat et al., 2004; Hierck
et al., 2008).

14.2.2 Sensory Proteins

The polycystins are probably the most studied mechanosensory proteins in cilia.
The mechanosensory functions of polycystins have been independently described
in the mouse and human kidney epithelia (Chauvet et al., 2004; Nauli et al., 2006;
Xu et al., 2007, 2009), vascular endothelia (Nauli et al., 2008; AbouAlaiwi et al.,
2009b), osteochondrocytes (Xiao et al., 2006; Hou et al., 2009), cholangiocytes
(Masyuk et al., 2006) and developing nodes (McGrath et al., 2003). Polycystin-1
is an 11-transmembrane protein with an extracellular domain that is long, flexi-
ble, elastic, and of remarkable mechanical strength (Forman et al., 2005; Qian et al.,
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 327

2005), whereas polycystin-2 is a cation channel with 6-transmembrane domains and


belongs to a superfamily of transient receptor potential (TRP ) ion channels.
Polycystin-1 and polycystin-2 can form a multi-protein complex at the primary
cilium. This complex may also be involved with and interact with other ciliary pro-
teins such as fibrocystin (Wu et al., 2006; Wang et al., 2007). It has been shown that
the indirect interaction of fibrocystin with polycystin-2 requires Kinesin-II Family
member (Kif)-3a/b as the adaptor protein (Fig. 14.3). Furthermore, the interaction
of polycystin-2 and fibrocystin is necessary in regulating polycystin-2 activity as

Fig. 14.3 Complex of mechanosensory proteins in the cilia. Polycystin-1 and polycystin-2 interact
with each other at the COOH termini, forming a polycystin complex. Kif, as an adaptor protein,
bridges the interaction of between fibrocystin and polycystin complex. Figure is reproduced from
Kolb, et al. with permission (Kolb and Nauli, 2008)
328 S.M. Nauli et al.

a mechanosensory calcium channel. This complex therefore has been proposed to


have a mechanosensory transduction role (Kolb and Nauli, 2008).
Ciliary polycystin-1 is regulated by proteolytic cleavage upon cilia activation
(Chauvet et al., 2004; Low et al., 2006; Nauli et al., 2008). This implies that the
polycystin-1 function can be inactivated by proteolytic cleavage, especially after
exposure to high fluid-shear stress. Polycystin-1 undergoes proteolytic cleavage
through a regulated intramembrane proteolysis mechanism. The cleaved product
translocates to the nucleus to activate transcription factors. The proteolytic cleavage
and translocation of polycystin-1 require polycytin-2 for the initial cilia activation.
Overall, this indicates that cilia function can also be regulated through modification
of polycystin-1.
Like polycystin-1, ciliary fibrocystin is also regulated by proteolytic cleavage
upon cilia activation (Hiesberger et al., 2006; Kaimori et al., 2007; Hogan et al.,
2009). Unlike polycystin-1, however, the cleavage product of fibrocystin is released
extracellularly. A functional polycystin-2 channel may be required to elicit the pro-
teolytic cleavage of fibrocystin. Increases in intracellular calcium and subsequent
activation of protein kinase C are required to initiate the cleavage process. Thus,
while fibrocystin is required for ciliary mechanosensing, the cleaved product may
function in a paracrine fashion to maintain cell direction and polarity.

14.3 Mechanosensory Cilia Function


Primary cilia, serving as mechanosensory organelles, have been described in various
vestigial organs. In this section, we will briefly describe the mechanics and func-
tionalities of sensory cilia in Hensens node as well as the kidney, liver, pancreas,
chondrocyte, osteocyte and blood vessel.

14.3.1 Hensens Node

The earliest mechanosensory role of primary cilia is required in the asymmetric


morphogenesis during embryogenesis. Evidence that cilia are involved during early
symmetry breakage started to emerge when the absence of dynein arms was reported
in Kartagener patients presented with reversal of the internal organs (Afzelius,
1976). During gastrulation, primitive streak and pit are formed where the upper
layer of embryonic cells (epiblast) invaginates. In Hensens node at the end of the
primitive streak, two types of cilia can be found (Table 14.1). The motile 9+0 cilia
function by generating a leftward flow in the embryos (Nonaka et al., 1998; Essner
et al., 2002; Nonaka et al., 2002), whereas the immotile 9+0 cilia have impor-
tant roles as chemosensors (Tanaka et al., 2005) and mechanosensors (McGrath
et al., 2003; Karcher et al., 2005). Because of their positions at Hensens node,
these motile cilia have been referred as nodal cilia. Immotile cilia at Hensens node
therefore are primary cilia known to have the earliest physiological role during early
embryonic development.
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 329

To function properly, primary cilia as mechanosensory organelles depend on


the mechanosensory polycystin-2 calcium channels. Abnormal or dysfunctional
polycystin-2 would result in dysfunction of primary cilia, followed by situs inversus,
situs ambiguus, or situs isomerism (Pennekamp et al., 2002; McGrath et al., 2003).
Unlike situs solitus, where visceral organs are generally located within the chest
and abdomen, aberrant nodal or primary cilia would result in randomized positions
of the internal organs. The mechanosensory cilia at Hensens node are therefore
flow-sensing organelles that transmit the leftward mechanical signal of nodal flow.
Responding to the nodal flow, only the left-side margin of the node would show
an increase in intracellular calcium, an early contributing factor to determining
left-right body asymmetry.

14.3.2 Kidney

The earliest evidence demonstrating primary cilia as mechanosensory organelles


came from the renal epithelial cells. A pharmacological agent, chloral hydrate, was
used to remove primary cilia from the renal epithelial cells. Cells with removed pri-
mary cilia were demonstrated to be unresponsive to fluid-shear stress (Praetorius and
Spring, 2003). Using a genetic knockout mouse model, expression of polycystin-
1 as mechanosensory protein in the cilia was blocked. Removing polycystin-1
genetically provided similar outcomes in which renal epithelia became unrespon-
sive to fluid-shear stress (Nauli et al., 2003). Further inhibition of polycystin-2
calcium channel with polycystin-2 antibody also abolished the sensitivity of
renal epithelial cells to mechano-fluid sensing. In addition, isolation of renal
epithelial cells from patients with mutation in polycystins further indicate the
dysfunction of cilia in response to fluid flow (Nauli et al., 2006; Xu et al.,
2007, 2009).
To further examine the role of cilia as mechanosensory organelles, transgenic
renal epithelial cells that did not express the IFT88 molecule, and thus did not have
cilia, also demonstrate the loss of fluid sensing (Siroky et al., 2006; Hovater et al.,
2008). Rescued IFT88 cells, which showed well-developed cilia, were responsive
to fluid-shear stress. In an ex vivo perfusion study, renal nephrons obtained from
one-week-old transgenic IFT88 mice also showed an expected blunted response to
the luminal microperfusion (Liu et al., 2005b).
Because fibrocystin is co-localized with both polycystin-1 and polycystin-2 in
the cilia, a possible mechanosensory role of fibrocystin was analyzed (Wang et al.,
2007). Overall inhibition of fibrocystin blocked the renal epithelial response to flow
stimulation. Interestingly, a similar effect was not seen in isolated kidney cells from
a patient with abnormal fibrocystin (Rohatgi et al., 2008). Although ciliary response
to fluid flow was not inhibited in patient cells, compared to the age-matched human
kidney cells, there was an alteration in the patient cells with abnormal fibrocystin.
It was noted that cilia length was about 20% shorter in the cells derived from these
patients. In particular, it has been suggested that abnormality in fibrocystin could
alter ciliary structure and morphology (Woollard et al., 2007). In kidney cells where
330 S.M. Nauli et al.

fibrocystin protein was repressed by 90%, the primarly cilia failed to develop (Mai
et al., 2005).
Polycystin-1, polycystin-2, and fibrocystin are localized to the primary cilia
in renal epithelial cells (Fig. 14. 3). This complex is involved in ciliary medi-
ated response to flow by initiating an increase of intracellular calcium. Interaction
of fibrocystin with calcium modulating cyclophilin ligand has also been shown,
suggesting an additional calcium protein might modulate intracellular calcium in
response to fluid-shear stress (Nagano et al., 2005). It is therefore not surprising that
mutation in genes responsible for polycystin-1, polycystin-2 and fibrocystin would
result in polycystic kidney disease. This further suggests that dysfunction of renal
cilia in response to urine flow would result in polycystic kidney disease.
The involvement of primary cilia in polycystic kidney disease has further pro-
vided that dysfunction in cilia would result in the abnormal planar cell polarity
(Fischer et al., 2006; Patel et al., 2008; Saburi et al., 2008). Within the kidney
anatomy, planar cell polarity is defined as an organized arrangement of cells in a
plane of tissue perpendicular to the apical-basal axis as a direction for the orienta-
tion of cell division. Using different mouse models of cystic kidney disease, defects
in any ciliary protein lead to abnormal orientation during cell division. It is there-
fore thought that inactivation of ciliary protein would result in abnormal planar cell
polarity, which in turn triggers tubular diameter in the kidney (Fig. 14.4). The net
result is cystic formation in the kidney.

14.3.3 Liver

The liver is an organ that secretes an aqueous solution called bile. Bile that is ini-
tially secreted by hepatocytes is called primary bile. Primary bile is further modified
within the interlobular bile ducts through secretory and absorptive processes. This
modified primary bile is called ductal bile. The ductal bile travels along the inter-
lobular bile ducts, which are composed of cuboidal and mucus-secreting epithelia
known as cholangiocytes.
Hepatocytes do not have primary cilia, whereas the primary cilia in cholangio-
cytes are extended from the apical plasma membrane into the lumens of interlobular
bile ducts (Huang et al., 2006). The primary cilia in the bile duct sense bile flow,
bile composition and bile formation. The primary cilia are also involved in regu-
lating bile duct formation and the size of the lumen. Similar to primary cilia in the
renal tubules (Brown and Murcia, 2003), the length of cholangiocyte cilia are het-
erogeneous (Huang et al., 2006). Cilia are longer in larger lumens and shorter in
lumens with smaller diameters.
To function as sensory organelles, cholangiocyte cilia express polycystin-1,
polycystin-2, and fibrocystin, among other sensory proteins (Masyuk et al., 2003,
2006). The activation of primary cilia by luminal fluid flow in microperfused intra-
hepatic bile ducts depends on the polycystin-1 and polycystin-2 mechanosensory
complex (Masyuk et al., 2006). Abnormalities in fibrocystin cause structurally
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 331

Fig. 14.4 Abnormal cilia function, planar cell polarity and cystic kidney disease. The illustra-
tion depicts mechanosensory function of renal tubular epithelial cilium. (a) Each cilium plays an
important role in transmitting extracellular information, such as urine flow, into renal epithelial
cells. This message may provide critical signals to the cell regarding the direction of cell division
along the tubule. (b) Insults, such as genetic disorder or random mutation, will result in abnormal
ciliary function for sensing fluid movement. (c) The functional abnormality in ciliary sensing may
result in loss of planar cell polarity. (d) Direction of cell division becomes randomized, result-
ing in increasing tubular diameter rather than tubular elongation. (e) Budding of a cyst from the
renal tubule and abnormal localizations of epidermal growth factor receptor (EGFR) and Na+ /K+
ATPase pump are typical characteristics of polycystic kidneys. (f) The cyst is eventually enlarged
and isolated. Multiple cysts from the neighboring nephrons are illustrated on the bottom left corner.
Figure is reproduced from Kolb et al. with permission (Kolb and Nauli, 2008)

shorter cilia in the bile ducts (Masyuk et al., 2003, 2004; Woollard et al., 2007).
The primary cilia are shown to be shorter, dysmorphic, and malformed, with
bulbous extensions of the ciliary tip. Mutations in cilia-associated genes in cholan-
giocytes have been referred to as cholangiociliopathies (Masyuk et al., 2008).
Cholangiociliopathies are characterized by cystic and fibrotic liver phenotypes.
332 S.M. Nauli et al.

14.3.4 Pancreas
The pancreas is a gland that produces hormones and digestive enzymes. This pan-
creatic fluid is transported to the gut via a system of ducts that is composed of
pancreatic epithelial cells. Each pancreatic epithelial cell has one primary cilium.
The primary cilia in the pancreatic ductal cells function to sense luminal flow to
maintain appropriate luminal dimensions (Cano et al., 2004).
To function as mechanosensory organelles, the pancreatic primary cilia have been
shown to express mechanosensory polycystin-2 channel (Cano et al., 2004). When
polycystin-2 is mislocalized or abnormally expressed, pancreatic cysts are formed.
Mutations in either functional or structural ciliary genes would block the relay of
the mechanosensory signals from extracellular to intracellular compartments (Cano
et al., 2004, 2006). This, in turn, results in uninhibited cell proliferation and pro-
gressive duct dilation. Primary cilia function and structure are thus required for
maturation and maintenance of proper tissue organization in the pancreas (Cano
et al., 2004, 2006). In addition, inability to regulate flow within the pancreatic duct
may lead to ductal dilation and acinar cell death (Scoggins et al., 2000).
Defects due to a reduction in cilia, aberrant ciliary architecture, and abnormal
ciliary function would provide gross pancreatic phenotypes, which include massive
acinar cell loss due to acinar-to-ductal metaplasia, fibrosis, lipomatosis, formation
of abnormal tubular structures, and appearance of endocrine cells in ducts (Cano
et al., 2004, 2006). All these phenotypes are similar to those found in patients with
chronic pancreatitis or pancreatic cystic fibrosis. Moreover, pancreatic primary cilia
may also be involved in pancreatic cancer and pancreatic intraepithelial neoplasia
lesions in human pancreatic ductal adenocarcinoma. Unlike cells in normal ducts,
intraepithelial neoplasia or ductal carcinoma cells do not have cilia (Seeley et al.,
2009).

14.3.5 Bone

Technically, bone has a very stiff structure. The structure, however, is compressible
due to its fluid-filled calcareous sponge. Osteocytes are the most abundant cell type
found in a compact bone. Cartilage, on the other hand, is a more flexible connective
tissue that joins bones together. Cartilage is primarily composed of chondrocytes
that produce a large amount of extracellular matrix.
Both osteocytes and chondrocytes possess primary cilia (Xiao et al., 2006;
Malone et al., 2007). The sensory cilia deflect during fluid flow and are required
for osteogenic and bone resorption in response to dynamic fluid flow. Although
osteocytes are more sensitive to fluid flow than osteoblasts (Klein-Nulend et al.,
1995), both osteocytes and chondrocytes respond to the strains on bones and joints
through primary cilia. Primary cilia in bone and cartilage are responsive to the
compression and cyclical pulses of lacunocanalicular fluid. Fluid flow through the
osteocyte canalicular network provides the physical stimulus for mechanosensa-
tion in bone (Klein-Nulend et al., 1995). Upon activation, primary cilia induce
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 333

intracellular calcium surges, which activate genes that maintain and strengthen bone
and cartilage.
To function as mechanosensory organelles, primary cilia in bone and cartilage
also enclose the mechanosensory polycystin-1 and polycystin-2 complex (Xiao
et al., 2006). Abnormality in these polycystin proteins results in abnormal bone and
cartilage formation (Boulter et al., 2001; Lu et al., 2001; Xiao et al., 2006, 2008;
Kolpakova-Hart et al., 2008; Hou et al., 2009). Mice with such mutations in ciliary
proteins display a decrease in gene products that maintain and strengthen bone and
cartilage. Thus, abnormalities in mechanosensory primary cilia in bone and cartilage
would result in osteoporosis or other bone related disorders (Malone et al., 2007).
The sensory primary cilia are also present in odontoblasts (Carmona, 1990;
Magloire et al., 2004; Maurin et al., 2009). Odontoblasts are cells that are located on
the outer surface of dental pulp. Activation of primary cilia in odontoblasts would
induce secretion of dentin required to form a calcified tissue under the tooth enamel.
Primary cilia are aligned parallel to the dentin walls, with the top part oriented
toward the pulp core. The cilia are positioned in very close proximity to the nerve
fibers. Thus, the role of cilia in sensing the microenvironment in odontoblasts is
thought to be crucial both for dentin formation and tooth pain transmission.
To function as mechanosensory organelles, primary cilia of odontoblasts express
mechanosensory polycystin-1 and polycystin-2 complex, including other ciliary
proteins such as OFD1 (Carmona, 1990; Maurin et al., 2009). Defects in molars
from Ofd1 knockout mice show the dysfunction of odontoblasts in the formation of
dentin. Thus, proper ciliary function is also a prerequisite during tooth development
(Ohazama et al., 2009).

14.3.6 Cardiovascular

Although still not widely accepted by many cardiovascular physiologists, primary


cilia are known to function as mechanosensory organelles in the cardiovascu-
lar system. Mechanosensory function of cilia has been independently studied in
human (Iomini et al., 2004; AbouAlaiwi et al., 2009b), mouse (Nauli et al., 2008;
AbouAlaiwi et al., 2009b), and chicken (Van der Heiden et al., 2006; Hierck et al.,
2008) models. More importantly, mechanosensory cilia can be found throughout the
cardiovascular system (Fig. 14.5). Primary cilia can be found in endocardia (Van
der Heiden et al., 2006; Slough et al., 2008; Van der Heiden et al., 2008), arterial
endothelia (Nauli et al., 2008; AbouAlaiwi et al., 2009b), venous endothelia (Iomini
et al., 2004), corneal endothelia (Gallagher, 1980; Doughty, 1998), arterial smooth
muscle cells (Poole et al., 1997; Lu et al., 2008), airway smooth muscle cells (Wu
et al., 2009), and many others.
Primary cilia in endothelial cells are mechanosensory organelles, which had
eluded researchers for decades in their search for fluid-shear stress sensors.
Endothelial cells isolated from embryonic mouse aortas and adult human inter-
lobar arteries require mechanosensory polycystin-1 and polycystin-2 complex to
334 S.M. Nauli et al.

Fig. 14.5 Mechanosensory cilia in blood vessel. Electron micrograph confirms the presence of
cilia in the lumen of embryonic aorta in vivo. Cilia as sensory organelles are projected from the
apical sides of cells into the lumen of blood vessels to sense fluid (blood) flow. Arrows indicate
the presence of cilia. Figure is reproduced from AbouAlaiwi et al. with permission (AbouAlaiwi
et al., 2009a)

be localized to the cilia. Relying on the functionality of primary cilia, endothelial


cells are able to respond to fluid-shear stress that mimics blood flow in the blood
vessels. Endothelial cells that do not have cilia cannot sense fluid-shear stress.
Likewise, endothelial cilia that do not have mechanosensory machineries cannot
transmit extracellular shear stress to intracellular biochemical responses, including
nitric oxide (NO) biosynthesis (Nauli et al., 2008; AbouAlaiwi et al., 2009b).
The mechanism of cilia-mediated fluid sensing requires a complex biochem-
ical cascade that involves calcium, calmodulin, Akt/PKB and protein kinase C
(Fig. 14.6). The release and production of NO participate in controlling vascu-
lar tone and systemic blood pressure. Patients without proper ciliary function may
therefore exhibit an enhanced propensity for hypertension, which may further result
in other potential complications. These complications may increase susceptibility
to localized blood vessel injury, aneurysm, hemorrhage, edema, atherosclerosis,
vascular ectasia, dissection, and other abnormalities.
14 Primary Cilia are Mechanosensory Organelles in Vestibular Tissues 335

Fig. 14.6 Mechanosensory cilia and nitric oxide production. The presence of cilia in vasculature
plays an important role in the biochemical production of a potent vasodilator, nitric oxide (NO).
The figure depicts production of NO in an artery. Increases in blood pressure, which are translated
to higher vascular shear stress, will be sensed by mechanosensory cilia. Bending or activation of
the cilia involves mechanosensory polycystin-1 and polycystin-2 complex and a cascade of bio-
chemical synthesis of NO. The cascade will further involve extracellular calcium influx (Ca2+ ),
followed by activation of various calcium-dependent proteins, including calmodulin (CaM) and
protein kinase C (PKC). Together with PKB, CaM and PKC are important downstream molecu-
lar components to activate endothelial nitric oxide synthase (eNOS). Figure is reproduced from
AbouAlaiwi, et al. with permission (AbouAlaiwi et al., 2009a)

14.4 Conclusion and Perspective


Primary cilia, as newly-acclaimed mechanosensory organelles, have been referred to
as cellular cybernetic probes (Poole et al., 1985), cellular global positioning systems
(Benzing and Walz, 2006), and antennae (Marshall and Nonaka, 2006; Singla and
Reiter, 2006). Our knowledge on primary cilia, however, is still relatively limited
compared to our knowledge of other cellular organelles. The importance of sensory
cilia in other organ systems is yet to be discovered, and many more cilia-related
336 S.M. Nauli et al.

diseases are still to be identified. There is no doubt that the physiological roles of
primary cilia will continue to be debated in years to come.
Acknowledgement Due to restricted space, we apologize to those whose work is not described
in this review. Authors are grateful for stimulating discussion about primary cilia given by research
assistants, graduates, undergraduates and pharmacy students in our laboratory. Authors thank
Drs. Robert Kolb, Stefan Somlo, Bradley Yoder, and Jing Zhou for valuable insights and use of
their laboratory reagents. Authors also thank Charisse Montgomery for her editorial review of the
manuscript. Work from our laboratory that is cited in this review has been supported by grants from
the NIH (DK080640), AHA (0630257 N), and University of Toledo research programs, including
deArce Memorial Endowment Fund. A. M. Nauli is supported by AHA (0825195F).

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Part VII
Mechanosensitivity
and Mechanotransduction
in Blood Cells
Chapter 15
Mechanosensitive K+ Channels in Mouse
B Lymphocytes: PLC-Mediated Release
of TREK-2 from Inhibition by PIP2

Sung Joon Kim and Joo Hyun Nam

Abstract Blood cells can encounter significant mechanical stimuli in variable flow.
In mouse B lymphocytes and their cell line WEHI-231, we found large-conductance
background K+ channels (LKbg ) that show significant mechanosensitivity. The
biophysical characteristics of LKbg were similar to those of TREK-2, a member
of the mechanosensitive two-pore domain K+ channels, and the genetic knock-
down of TREK-2 largely suppressed the LKbg activity in B lymphocytes. However,
there have been disputes regarding the sensitivities of LKbg and TREK-2 to phos-
phatidylinositol 4,5-bisphosphate (PIP2 ) and their mechanism of stretch-dependent
activation. Our recent results suggest a mechano-biochemical signalling mechanism
for the stretch-dependent activation of TREK-2; the mechanosensitive-activation of
LKbg is mediated by phospholipase C (PLC)-dependent degradation of PIP2 , which
relieves TREKs and LKbg from the intrinsic inhibition by PIP2 . Here we review the
characteristics of LKbg and permanently overexpressed TREKs and compare them
with previous reports about TREKs.

Keywords Background K+ channel TREK-2 PIP2 B lymphocyte


Phospholipase C Stretch

15.1 Introduction

15.1.1 K+ Channels Modulating the Ca2+ Signal in Lymphocytes

Immune cells are electrically non-excitable and their ion channels are not as diverse
as those in neuronal and muscular cells (Panyi et al., 2004). However, the ion
channels in immunocytes have recently drawn attention because they are potential

S.J. Kim (B)


Department of Physiology, Ischemic/Hypoxic Disease Institute, Kidney Research Institute, Seoul
National University College of Medicine, 103 Daehangno, Jongno-gu, Seoul 110-799, Korea
e-mail: sjoonkim@snu.ac.kr; physiolksj@gmail.com

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 353


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8_15,

C Springer Science+Business Media B.V. 2011
354 S.J. Kim and J.H. Nam

pharmacological targets for the regulation of immune responses and inflammatory


diseases (Chandy et al., 2004; Panyi et al., 2006; Roselli et al., 2006; Wulff et al.,
2004; Wulff and Pennington 2007). One of the initial intracellular signalling medi-
ators after antigenic stimulation of lymphocytes is the change in cytosolic Ca2+
concentration ([Ca2+ ]c ); the stimulation of T (TCR) or B cell receptors (BCR)
triggers phospholipase C (PLC)/IP3 -dependent release of stored Ca2+ from endo-
plasmic reticulum (ER). However, Ca2+ influx through the plasma membrane is also
critical for sustained Ca2+ signalling such as Ca2+ -oscillation (Vig and Kinet 2009).
In lymphocytes, the activation of Ca2+ channels triggered by ER depletion is
described as CRAC (Ca2+ -release activated Ca2+ channel). The key molecular iden-
tity of CRAC was recently elucidated as Orai (CRACM) (Cahalan et al., 2007;
Feske et al., 2006). The activation of Orai depends on the interaction with clus-
tered STIM1, an ER transmembrane protein (Fahrner et al., 2009; Roos et al.,
2005). The clinical importance of CRACs is dramatically proven by the congenital
immune-deficiency and autoimmunity in patients having mutated Orai1 and STIM1
(Feske et al., 2006; Picard et al., 2009), and by deficient mast cell functions in Orai1
knock-out mice (Vig et al., 2008).
The intensity of Ca2+ influx in lymphocytes can be modulated by the membrane
potential, which is largely set by K+ channel activity. In this respect, the regulation
of K+ channels is a key issue in immune cell electrophysiology. In both T and B
lymphocytes, voltage-gated K+ channels (Kv1.3) and Ca2+ -activated K+ channels
(SK4) are commonly expressed, whereas their relative levels of expression vary
widely depending on the subset of lymphocytes and their activation states (Cahalan
et al., 2001; Panyi et al., 2004). Specific blockers of Kv1.3 and SK4 have garnered
attention as therapeutics for autoimmune diseases such as multiple sclerosis (Beeton
and Chandy 2005; Chandy et al., 2004; Madsen et al., 2005; Panyi et al., 2006).

15.1.2 Mechanical Stress and Ion Channels in Lymphocytes


Apart from the electrical driving force for cation influx, another important role of
K+ channels is the regulation of cell volume; the hyperpolarized membrane potential
is critical for Cl efflux that provides resistance to the tendency for cell swelling.
Together with the ubiquitously expressed volume-regulated anion channels (Lewis
et al., 1993), K+ channels sensitive to osmotic stress and/or membrane stretch are
required for the regulatory volume decrease (RVD). On the other hand, excessive
efflux of K+ and Cl with concomitant cell volume decrease (apoptotic volume
decrease, AVD) is regarded as a hallmark of apoptosis (Bortner and Cidlowski,
2007). Because lymphocytes undergo extensive apoptotic cell death in physiologi-
cal as well as in pathophysiological conditions, the K+ channels activated by death
signals are interesting targets of investigation.
Previous studies of the modulation of the K+ channels in lymphocytes have
been limited to biochemical signalling mechanisms. However, immune cells in
blood circulation are expected to undergo substantial mechanical stress depending
on situations such as migration across the capillary wall, cell to cell adhesion via
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 355

immunological synapses, and exposure to osmotic stress in inflamed tissues. The


immunological synapse between antigen presenting cells and lymphocytes is not a
simple ligand-receptor binding, but rather complex molecular interactions between
plasma membranes in dynamic motion. Interestingly, the nano-scale mechanical
forces in the immunological synapse have been suggested as a critical factor acti-
vating the intracellular signalling cascades in lymphocytes (Ma and Finkel, 2009).
In addition, although mostly observed in vitro, the extension of plasma membrane
forming the immunological synapse can be very long and thin and is called an
immunological nano-tube (Onfelt et al., 2004, 2005). Such thread-like extensions
of the plasma membrane could be much more sensitive to mechanical stress in vivo.
Despite the intriguing possibility of ion channel regulation by membrane stretch,
investigations of mechanosensitive cation channels in lymphocytes are extremely
rare (Achard et al., 1996; Liu et al., 2005).

15.2 Mechanosensitive Background-Type K+ Channels


in B Lymphocytes

15.2.1 Discovery of Background-Type K+ Channels


in B Lymphocytes
In conventional whole-cell mode patch clamp studies using standard pipette solu-
tion (e.g., high KCl with 3 mM MgATP), the voltage-dependent K+ current (Kv1.3)
is almost the only type of K+ channel that can be found in B lymphocytes. The
clotrimzol- or charybtotoxin-sensitive K+ channel (SK4/IKCa1) are activated by
raising the intracellular Ca2+ activity and show weakly inward-rectifying voltage-
dependence (Zheng 2006; Zheng et al., 2009). Apart from the well-known Kv and
KCa channels, in the whole-cell clamp studies of mouse B lymphocytes (WEHI-
231), we accidently found that voltage-independent K+ channels are spontaneously
induced simply by omitting ATP from the pipette solution (Nam et al., 2004). Lately,
the same type of current was confirmed in freshly isolated mouse splenic B lym-
phocytes (Nam et al., 2007; Zheng et al., 2008). Accompanying single channel
recordings revealed that mouse B lymphocytes express at least two types of voltage-
independent K+ channels: Large-conductance background K+ channels (LKbg ) with
slope conductance around 300 pS, and Medium-conductance background K+ chan-
nels (MKbg ) with slope conductance around 100 pS. The 300 pS channel was ini-
tially called BKbg (big-conductance background K+ channel), but was later renamed
LKbg to avoid confusion with the Ca2+ -activated K+ channel (BKCa , maxi-K).
Furthermore, the activity of LKbg is not affected by changing cytoplasmic Ca2+
concentration ([Ca2+ ]c ).
Among the two types of background K+ channels, LKbg channels show spon-
taneous activation in the inside-out (i-o) configuration in the absence of ATP
(Fig. 15.1a), which is consistent with the phenomenon of spontaneous increase
of K+ conductance observed in the whole-cell clamp study with ATP-free pipette
356 S.J. Kim and J.H. Nam

Fig. 15.1 Inhibition of LKbg activity by cytoplasmic application of MgATP. (a). A representa-
tive case of cell-attached (c-a) and inside-out (i-o) patch clamp recording with KCl (140 mM)
pipette solution in B lymphocytes freshly isolated from mouse spleen. A huge increase of large-
conductance channel activity was observed after making membrane excision (i-o) into MgATP-free
KCl solution (60 mV holding). (b). A representative case of i-o patch clamp in WEHI-231 cells.
The recordings were obtained under the i-o patch clamp conditions at 60 mV with symmetrical
KCl (145 mM) solutions as shown above. The application of MgATP (2 mM) to the cytoplasmic
side of the membrane greatly reduced the activity of LKbg

solution. Furthermore, the LKbg activity is very low in cell-attached (c-a) record-
ings, while the relative activity of LKbg is enormously high in the absence of
cytoplasmic ATP (Nam et al., 2004). In contrast, the spontaneously activated LKbg
is effectively inhibited by MgATP applied to the cytoplasmic side (Fig. 15.1b).
Because the activity of LKbg is very low in the resting B cells, it was initially
questioned to classify them as background K+ channels. Albeit this confusion,
here we regard LKbg as a member of background-type K+ channels due to the
voltage-independence and Ca2+ -insensitivity (Nam et al., 2004).

15.2.2 Stretch-Dependent Activation of LKbg in B Lymphocytes

In the i-o recording with MgATP or in the c-a conditions, LKbg is activated by
membrane-stretch induced by applying negative pressure through the patch pipette
(Fig. 15.2). The activation of LKbg in c-a condition is also induced by osmotic
swelling (Nam et al., 2007). The stretch effects on LKbg are basically reversible
upon relieving the mechanical stress. However, when the application of stretch is
prolonged beyond a certain duration (>35 min) in i-o patch recording, then the
activation of LKbg is irreversible, suggesting that a putative depletion of regulatory
molecules might occur (Nam et al., 2004) (see also Fig. 15.3c). It has also been
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 357

Fig. 15.2 Effects of membrane stretch on LKbg channel activity in c-a and i-o patches. (a). In
the c-a patches, the initially low open probability (Po ) of LKbg channels was markedly increased
by applying 43 mmHg of negative pressure. (b). With 1 mM MgATP on the cytoplasmic side,
a serial negative pressure was applied as indicated above each trace. The membrane stretch by
the negative pipette pressure reversibly increased the open probability (Po ) of LKbg channels in a
pressure-dependent manner at 60 mV

noted that the MKbg in B lymphocytes are not affected by membrane stretch (Nam
et al., 2004).
Apart from the membrane stretch, arachidonic acid (AA) and intracellular acid-
ification activate LKbg (Zheng et al. 2008, 2009). The mechanosensitivity and
chemosensitivity indicate that LKbg might be encoded by TREK-2 (KCNK10),
a member of the tandem two-pore domain K+ channel gene (KCNK) family.
Among the TREK subfamily of KCNK, TREK-2 shows a large but variable unitary
conductance that ranges up to 290 pS (Lotshaw 2007). Despite their common prop-
erties, we were initially reluctant to pinpoint TREK-2 as the molecular identity of
LKbg because of the differential responses to phosphatidylinositol 4,5-bisphosphate
(PIP2 ) between LKbg and cloned TREK-2 (see below).
358 S.J. Kim and J.H. Nam

Fig. 15.3 Mechanism of the inhibitory effects of MgATP and the effects of PLC activation on
LKbg . (a). Direct application of PIP2 (5 M) to the cytoplasmic side of membrane (bath solu-
tion) suppressed the LKbg activity. These results suggest that the cytoplasmic ATP-dependent
inhibition of LKbg is tightly related to PIP2 in the membrane. (b). For the direct stimulation of
membrane-bound PLC, m-3M3FBS (a chemical activator of PLC) was applied to i-o patches after
confirming the inhibition of LKbg channels by MgATP. The application of m-3M3FBS (50 M)
slowly activated LKbg channels in the presence of MgATP (1 mM)

15.2.3 Regulation of LKbg /TREK-2 by PIP2

As mentioned above, the i-o patch recording of LKbg shows remarkable spontaneous
activation that is readily inhibited by the application MgATP to the cytoplasmic
side (IC50 =0.18 mM). The inhibitory effect of ATP is phosphorylation-dependent
because non-hydrolysable adenosine 5-(,-imino)-triphosphate (AMP-PNP) has
no effect. However, the inhibition by ATP is not prevented by protein kinase
inhibitors, but is effectively blocked by PI-kinase inhibitor, wortmannin (Nam et al.,
2004). Such results strongly suggest the inhibitory regulation of LKbg by PIP2
locally produced from the membrane phospholipids and associated enzymes, a
membrane-delimited regulatory mechanism.
PIP2 is well recognized as a key physiological regulator of various types of ion
channels/transporters (Hilgemann et al., 2001; Horowitz et al., 2005; Suh and Hille,
2005). While most PIP2 -sensitive ion channels are positively regulated by the nega-
tively charged phosphoinositol head, LKbg are inhibited by PIP2 (Fig. 15.3a) (Nam
et al., 2007, 2004). Inhibition by intrinsic PIP2 could explain the low activity of
LKbg in intact B cells and the spontaneous activation after the membrane exci-
sion (i.e., inside-out patch formation). In physiological conditions, cytoplasmic ATP
and PI-kinases seem to ensure the repletion of PIP2 . To explain the membrane-
delimited reversible inhibition by ATP in i-o patches, it is strongly suggested that
the PI kinases are tightly associated with plasma membrane and LKbg . Moreover,
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 359

Fig. 15.4 Schematic model of the membrane-delimited regulation by PIP2 . Intrinsically generated
PIP2 by PI- and PIP-kinase inhibit LKbg . The mechanical stretch activates membrane-bound PLC
(PLC2) by signalling pathways not yet elucidated. The decrease of PIP2 levels by PLC relieves
the nearby LKbg from their tonic inhibition

the spontaneous activation of LKbg suggests that a lipid phosphatase and/or phos-
pholipase (PLC) that hydrolyzes PIP2 are also tightly associated in the same patch
membrane. Regarding the PLC-dependent regulation, it is also notable that a chem-
ical PLC activator, m-3M3FBS (Fig. 15.3b), and B cell receptor (BCR) ligation
increase the LKbg activity (Nam et al., 2004). Based these data, we suggest a model
for the membrane-delimited regulation of LKbg by PIP2 as shown in Fig. 15.4.
Above we mentioned that the TREK subfamily of KCNK, TREK-2, is the most
likely candidate for the LKbg gene. However, in comparison with the inhibition of
LKbg by PIP2 or by MgATP, TREK-1 was activated by PIP2 in earlier studies of
cloned TREK channels (Chemin et al., 2005). Interestingly, however, a more recent
study by the same group indicated dual effects of PIP2 on TREK-1 depending on the
basal activity of the channel (Chemin et al., 2007), which is partly consistent with
our results. Although TREK-1 is highly homologous with TREK-2, the c-terminal
of TREK-2 is longer than TREK-1, which might have caused the different responses
to PIP2 . To clarify this controversial issue, we compared the properties of rat TREK-
1 and -2 channels permanently expressed in HEK-293 cells with the properties of
LKbg in WEHI-231 cells (Zheng et al., 2009). According to this study, the spon-
taneous activation of both TREK-1 and TREK-2 in ATP-free conditions and their
inhibition by MgATP or by PIP2 were the same as those of LKbg . Furthermore,
TREK-2-specific si-RNA transfection decreased the LKbg current in B cells. In
conclusion, TREK-2 encodes LKbg that are normally suppressed by intrinsic PIP2
formed by PI-kinase and MgATP in intact cells (Zheng et al., 2009).
The question then is why there have been such differences between the studies
of TREKs in terms of their PIP2 -sensitivity. Also, no previous study has reported
the reversible inhibitory effects of ATP on TREK-2 shown in our i-o patch clamp
experiments. Although the question remains unsolved, the inconsistent results seem
to be due to differences in the expression systems for TREKs: our previous studies
were performed in mouse B lymphocytes and HEK-293 cells permanently express-
ing TREKs, whereas other studies were mostly performed in COS-7 cell lines or
Xenopus oocytes for transient overexpression of TREK-1. We recently found that
transiently overexpressed TREK-2 in HEK-293 cells showed irregular responses to
360 S.J. Kim and J.H. Nam

MgATP in the i-o patch recordings (data not shown). We hypothesize that the regu-
lation by PIP2 /PI-kinase/lipid-phosphatase is less effective in transiently transfected
TREKs, while the intrinsically expressed TREKs such as LKbg in B lymphocytes
are more tightly associated with the PIP2 -mediated regulatory complex.
Considering that the plasmalemmal concentration of PIP2 is relatively low
(Hilgemann et al., 2001), the extent of co-localization between PIP2 and TREKs
might vary according to the mode of expression (transient vs. permanent) or cell
types. A locally concentrated, inhomogeneous distribution of PIP2 and the regula-
tion of ion channels by the localized PIP2 could be suggested from the effects of
methyl--cyclodextrin (MCD). MCD is known to scavenge cholesterols, thereby
disrupting cholesterol-rich microdomains (e.g., lipid rafts) of plasma membranes.
The concentration of PIP2 in the lipid rafts is thought to be higher than that in other
regions, and interestingly, the application of MCD facilitates the recovery of LKbg
activity from the inhibition by PIP2 (Fig. 15.5).

15.2.4 Dual Sensitivity of LKbg /TREK-2 to PIP2


One piece of experimental evidence supporting the positive effect of PIP2 on TREK
is the inhibition by poly-L-lysine (poly-L), a PIP2 -scavenging polycationic agent.
Also, in the presence of poly-L, TREK-1 channels are consistently activated by PIP2

Fig. 15.5 Irreversible inhibitory effects by the application of PIP2 (1 M), and the effects of
MCD on recovery from PIP2 -induced inhibition of LKbg channels. (a), The LKbg channel activity
was not recovered by washout of PIP2 up to 10 min by a sustained application of PIP2 (1 M). (b),
The application of methyl--cyclodextrin (MCD, 2 mM), a cholesterol scavenger that disrupts
lipid rafts (Kilsdonk et al., 1995), significantly facilitated the recovery of LKbg channels from the
inhibition of PIP2
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 361

application (Chemin et al., 2005). After the first report about the positive effects of
PIP2 on TREK-1, Chemin et al. also demonstrated dual effects of PIP2 on TREK-1
(Chemin et al., 2007). According to the more recent paper, exogenous PIP2 appli-
cation activated TREK-1 when the basal channel activity was low and, vice versa,
PIP2 application inhibited TREK-1 when the control activity was high.
Interestingly, in our own experiments, the concentration-dependent dual effects
of PIP2 were also demonstrated after poly-L treatment (Fig. 15.6). After confirming
the spontaneous activation of TREKs under i-o conditions, we found that poly-L
(30 g/ml) treatment almost completely inhibited TREKs. The inhibitory effect of
poly-L was inconsistent with our initial hypothesis that TREKs are simply inhibited
by PIP2 . These contradictory responses led us to modify our model to include the
dual effects of PIP2 : the activities of TREKs are positively and negatively regulated
by relatively low and high levels of PIP2 , respectively.
In our experience, however, only an excessive experimental condition such as
poly-L treatment seemed to decrease PIP2 below the level that is minimally required

Fig. 15.6 Dual effects of PIP2 on TREKs and LKbg in the presence of poly-L-lysine (poly-L).
AC, i-o recordings of TREK-1, -2 (overexpressed in HEK-293) and LKbg (WEHI-231) with
symmetrical KCl solution at 60 mV holding voltage. The application of poly-L (30 g/ml) to
the intracellular side (bath solution) completely inhibited the channel activities, and the inhibition
was not reversed by washout of poly-L. After confirming steady-state inhibition, 8 M and 20 M
PIP2 were applied sequentially. Note that 20 M PIP2 initially activated and then inhibited the
channels
362 S.J. Kim and J.H. Nam

for the activity of TREKs. After spontaneous activation of LKbg /TREK-2, only
inhibitory effects on LKbg /TREK-2 are observed in response to ATP and PIP2 . The
removal of ATP and the spontaneous degradation process actually lower the mem-
brane PIP2 level. But the level might still be greater than the putative minimum level
required to activate TREKs. Under such conditions, poly-L might further scavenge
the residual PIP2 that was required for the basal activity of TREKs. To test this
dual-mode hypothesis, we applied different concentrations of PIP2 after confirm-
ing the inhibition of TREKs by poly-L. At relatively low concentration (8 M),
TREK-2 are increased, while at a higher concentration (20 M), the channels are
transiently activated then go into an inhibited state (Fig. 15.6). The dual effects
of PIP2 on TREK-2 are basically consistent with the previous results in TREK-
1-overexpressing COS-7 cells (Chemin et al., 2007). From the responses of various
deletion mutants of TREK-1 to PIP2 and poly-L, Chemin et al. (2007) suggested that
the different c-terminal domains are responsible for the positive and negative effects
of PIP2 . Because the c-terminal domain proximal to the transmembrane domain
is highly conserved between TREK-1 and TREK-2 (Bang et al., 2000), the dual
modes of PIP2 action on TREK-2 might also due to the differential interaction with
the anionic phospholipids.
As a whole, it is evident that TREKs are modulated by PIP2 in a dual manner
at least in vitro. However, the LKbg /TREK-2 channels in B lymphocytes are under
tonic inhibition by physiological levels of intrinsic PIP2 . Then, what is the relation-
ship between the mechanosensitivity of LKbg /TREK-2 and the tonic inhibition by
PIP2 ?

15.3 Mechanosensitivity of TREK-2/LKbg in B Cells

Above we extensively discussed the PIP2 -dependent regulation of TREKs because


this property seems to be critical in the stretch-dependent activation of TREKs as
well as LKbg . According to the model suggested by Chemin et al. (2005), the stretch-
dependent activation of TREK-1 is mediated by an interaction between positively
charged (basic) amino acids in the c-terminus of TREK-1 and PIP2 in the mem-
brane inner leaflet. However, a more recent study from the same group demonstrated
that the stretch-dependent activation of TREK-1 is markedly suppressed by exoge-
nous application of PIP2 (Chemin et al., 2007). Similar to the later report, our study
showed that the stretch-dependent activations of LKbg and TREK-2 are blocked by
PIP2 application (Nam et al., 2007) (Fig. 15.7).
With regard to the tonic inhibition of LKbg /TREK-2 by PIP2 in intact cells, we
previously hypothesized that the hydrolysis and/or dephosphorylation of PIP2 is
an underlying mechanism of the mechanosensitivity. This hypothesis was exten-
sively tested in WEHI-231 and in primary splenic B cells: (1) In the absence of
ATP, the spontaneous activation of LKbg in an i-o patch was significantly acceler-
ated by membrane stretch, (2) pharmacological inhibition of lipid phosphatases did
not affect the stretch-dependent activation of LKbg , (3) hyposmotic swelling also
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 363

Fig. 15.7 Inhibition of the mechanosensitivity of TREK-2 by PIP2 . (a). In cell-attached (c-a)
mode with KCl pipette solution, the application of negative pressure (27 mmHg) through the
patch pipette activated TREK-2 in a reversible manner. (b). The spontaneously activated TREK-
2 current by membrane excision was completely inhibited by 8 M PIP2 . Under this condition,
membrane stretch (27 mmHg) did not recover the channel activity

activated LKbg , and (4) the tyrosine phosphorylation of PLC and PIP2 hydrolysis
was confirmed in WEHI-231 cells under osmotic stress (Nam et al., 2007). More
recently, we confirmed that the stretch-dependent activation of cloned TREK-1 and
TREK-2 was also prevented by PLC inhibitor, U73122, but not by its negative ana-
logue, U73343 (Fig. 15.8). As a whole, we suggest that the stretch-sensitivity of
TREKs might actually be a release from the inhibition by PIP2 through the activa-
tion of PLC and PIP2 hydrolysis, i.e., a mechno-biochemical signalling hypothesis
(Fig. 15.4). The PIP2 hydrolysis by membrane stretch could also be suggested from
irreversible activation (open-locked state) of LKbg after sustained application of
negative pressure (Fig. 15.3c, see also Nam et al., 2007). Under the open-locked
state, LKbg are not affected by ATP alone but can be inhibited only when PI4P is
applied together, indicating that the substrate phosphoinositides for PIP2 generation
are actually depleted by sustained membrane stretch (Nam et al., 2007).
Although the evidence supports the mechano-biochemical coupling hypothesis,
it must also be noted that the stretch effects on TREKs showed different pharma-
cological sensitivity depending on the level of negative pressure. The activation
by relatively weak stretch (e.g. 27 mmHg via patch pipette) was prevented by
PLC inhibitor, U73122. In the presence of U73122, a higher negative pressure
(43 mmHg) could activate TREKs (Fig. 15.8). The PLC-independent activation of
TREKs by the stronger membrane stretch might indicate a more direct link between
the membrane stretch and channel gating, as has been suggested by Chemin et al.
for TREK-1 (Chemin et al., 2005). As a whole, the above concentration-dependent
effects of PIP2 might be associated with dual modes of mechanosensitivity where
both mechanisms are associated with PIP2 , i.e., mechano-biochemical signalling
(PLC-mediated) and direct physical signalling mediated by putative electrostatic
interaction between the c-terminus and the residual PIP2 . Actually, the mechanosen-
sitivity of TREK-1 is inhibited both by scavenging PIP2 with poly-L treatment
(Chemin et al., 2005) and by exogenous application of PIP2 (Chemin et al., 2007;
Nam et al., 2007). In these respects, PIP2 is a double-edged sword in terms of
mediating the mechanosensative regulation of TREKs.
364 S.J. Kim and J.H. Nam

Fig. 15.8 Effects of PLC inhibitor on the mechanosensitive activation of TREK-1 and -2. After
confirming the basal activities of TREK-1 and -2 in c-a configuration, relatively mild (27 mmHg)
or strong (43 mmHg) suction was applied as indicated above each trace. (a): Both TREK-1 and -2
were slowly activated by 27 mmHg of suction (upper panels), and these responses were signif-
icantly suppressed by pretreatment with U73122 (2 M, lower panels). (b): Summaries of the
changes in channel activity (NPo ) by relatively low membrane stretch (27 mmHg) and the effects
of pretreatment with U73122 (2 M) or with U73343 (10 M). In each patch, NPo during the
initial 15 s and the later 15 s of membrane stretch were normalized to the pre-stretch activity.
The averaged values are shown in the bar graphs (n=6). (c): Representative traces of TREK-1
and TREK-2 responses to the higher level of membrane stretch (43 mmHg). Pretreatment with
U73122 did not block the activation of TREKs by the stronger negative pressure. Representative
cases from five similar experiments are shown. (d): Summaries of the NPo changes by relatively
high membrane stretch (43 mmHg) and the effects of pretreatment with U73122 (2 M, n = 5).
This figure was adopted and modified from the figure 5 in Zheng et al. (2009) with permission
from the American Physical Society
15 Mechanosensitive K+ Channels in Mouse B Lymphocytes 365

15.4 PLC-Dependent Mechanosensitivity of Cells


Mechanosensitive- or stretch-dependent PLC activation has been suggested in other
cell types such as hepatocyte and cardiomyocytes (Moore et al., 2002; Ruwhof
et al., 2001). However, considering that PLCs are not transmembrane proteins,
it is still unclear how mechanical stimuli actually induce PLC activation. Recent
studies suggest various types of indirect mechanisms: (1) mechanosensitive activa-
tion of Gq/11 -protein coupled receptors (GPCRs) and (2) mechanosensitive release
of arachidonic acid metabolites (e.g., epoxyeicosanoids) that subsequently acti-
vate the PLC-coupled receptors (Miah et al., 2004; Zhu et al., 2005). The former
mechanism was suggested from studies in arterial smooth muscle where the
increased intravascular pressure and wall tension activate multiple intracellular sig-
nals resulting in reactive constriction called myogenic tone or myogenic response.
Mederos-y-Schniztler et al. (2008) recently suggested that Gq -coupled receptors are
stimulated by membrane stretch, which subsequently activates PLC/IP3 /PKC path-
ways. According to this model, the activation of diacylglycerol (DAG)-sensitive
TRPC6 and Ca2+ -sensitive TRPM4 channels might explain the stretch-activated
nonselective cation (SAC) channels in arterial myocytes (Sharif-Naeini et al., 2008).
However, the activation mechanism of arterial SAC is still controversial, and the
stretch-dependent release of AA metabolite (20-HETE) has also been suggested in
arterial myocytes (Inoue et al., 2009).
Literature search reveals that PLA2 is more widely reported as the mechano-
biochemical signalling mechanism than the PLC pathway. Because LKbg /TREK-2
are sensitive to AA and polyunsaturated fatty acids (Zheng et al., 2008), the AA-
dependent activation might also play a role in the mechanosensation of LKbg /TREK-
2 in vivo (see below).

15.5 Role of Mechanosensitive TREK-2 in B Cells

Variable modes of mechanical stimuli could be encountered by lymphocytes


throughout cell motility and adhesion during their migration through capillary walls.
In inflamed tissue, lymphocytes are also exposed to osmotic stresses in addition to
chemical signals. The strong Ca2+ signalling of B cells under osmotic swelling has
been described (Liu et al., 2005) where the osmosensitive activation of nonselective
cation channels such as TRPV4 and the subsequent Ca2+ influx are suggested as key
mechanisms. In addition, it was suggested that the osmotic activation of TRPV4-like
channels in B cells is mediated by AA-metabolites; osmosensitive activation of PLC
releases DAG, and the DAG is further metabolized into AA by DAG lipase (Zhu
et al., 2005). In this respect, the AA-sensitive activation of LKbg /TREK-2 indicates
an additional role of facilitating the Ca2+ signals in B cells under inflammatory con-
ditions (King and Freedman 2009; Ma and Finkel 2009). Because AA inhibits other
types of K+ channels in lymphocytes such as Kv1.3 and SK4/IKCa1 (Zheng et al.,
2008), the activation of LKbg /TREK-2 by AA and membrane stretch has greater
366 S.J. Kim and J.H. Nam

implications. In addition to the amplification of Ca2+ signalling, the mechanosen-


sitive and AA-dependent activation of K+ channels in B cells might also prevent
excessive cell swelling.
In summary, mouse B lymphocytes express stretch-activated two-pore domain
K+ channels, TREK-2 (LKbg ). The activity of TREK-2 is under tonic inhibition
by PIP2 , and the relatively mild membrane stretch relieves this inhibition via acti-
vation of PLC hydrolysing PIP2 . Apart from the mechano-biochemical signalling
mechanism, more direct regulation by stronger membrane stretch is also suggested.
Acknowledgement This work was supported by the Korea Science and Engineering Foundation
(KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-11203-0 and R11-
2007-040-01003-0).

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Index

A D
Actin-crosslinking proteins, 27, 30, 4652, 156 Dystrophin, 35, 143, 147, 149150
Actin cytoskeleton, 2558, 89, 9294, 98, 152,
156, 225, 244, 264, 288, 301 E
Actin filament associated protein (AFAP), ECM, see Extracellular matrix (ECM)
262265 Endothelial cells, 7, 26, 194203, 224232,
Actin microfilaments, 27, 6970, 7778, 86, 240, 242, 245, 248, 265268, 279280,
8890, 9296 282283, 307, 333334
Acute lung injury, 240, 256, 267 Epoxyeicosatrienoic acids, 240241
Acute respiratory distress syndrome (ARDS), Extracellular matrix (ECM), 48, 1013,
256257, 267268 15, 52, 6768, 7778, 83, 85, 9193,
AFAP, see Actin filament associated protein 98, 135, 141149, 151152, 154156,
(AFAP) 158, 194, 210, 222, 225, 228229, 256,
ARDS, see Acute respiratory distress 260261, 278279, 298302, 304305,
syndrome (ARDS) 307, 332
Articular cartilage, 7799, 297298, 301, 305
F
B FAK, see Focal adhesion kinase (FAK)
Background K+ channel, 355356 Fluid flow, 27, 85, 94, 202, 228230,
B lymphocyte, 353366 282283, 285286, 300, 304, 325,
Bone, 2627, 7074, 8384, 195, 202, 329330, 332
277288, 297308, 321, 332333 Fluid shear stress, 69, 71, 73, 230, 279281,
283284, 288, 303, 325, 328330,
C 333334
Cadherin, 146147, 152154, 156, 267 Focal adhesion, 67, 11, 27, 46, 5253, 55,
Calcium (Ca), 4, 8, 11, 111, 116, 119, 85, 94, 98, 144147, 156157, 201, 220,
157, 240, 243, 250, 260, 262, 268, 224, 229, 232, 258, 261262, 278280,
281282, 285286, 301, 303, 328330, 282284, 287288, 301302, 307
333335 Focal adhesion kinase (FAK), 6, 11, 52, 55,
Calcium-activated channels, 11 85, 145147, 201, 224, 229, 258, 279,
Calcium spark, 911 282284, 301302
Cardiac myocyte, 210
Cartilage, 7799, 297302, 304308, 332333 G
Chondrocyte, 12, 80, 8399, 298302, 304, GsMTx-4 MscS, 168, 170, 177, 180, 184185
306307, 326, 328, 332
Cytoskeleton, 315, 2558, 6774, 7799, H
135, 142, 152, 156, 194, 205, 208209, HaTx, 168, 171172, 174178, 180, 182184
220, 225, 244, 256, 260261, 263265, Heart, 6, 109135, 141158, 168169, 173,
278, 288, 300301, 303, 326 193, 256257

A. Kamkin, I. Kiseleva (eds.), Mechanosensitivity and Mechanotransduction, 369


Mechanosensitivity in Cells and Tissues 4, DOI 10.1007/978-90-481-9881-8,

C Springer Science+Business Media B.V. 2011
370 Index

Heart rhythm, 110 O


Heparan sulfate, 220224, 227232 Osteoarthritis, 91, 96, 297
Osteoblast, 2627, 6774, 278288,
I 302306, 332
ILK, see Integrin-linked kinase (ILK)
Inhibitory cysteine knot peptides, 169 P
Integrin, 315, 2558, 6774, 7799, P130Cas, 260263, 268, 279, 282283,
143150, 157158, 197198, 200201, 287288
203, 205, 208, 220, 224, 232, 258, 260, Phospholipase C, 170, 223, 262, 280, 302, 354
266267, 278280, 282, 287, 299303, PI3K, 145152, 154155, 158, 198, 205, 249,
305, 307, 326 259, 266267
Integrin-linked kinase (ILK), 6, 10, 15, 55, PIP2, 49, 5455, 148, 152, 157, 353366
141, 145, 147150, 155, 157 Primary cilia/primary cilium, 90, 281282,
Isolated ventricular myocytes, 114132 317336
Proteoglycans, 83, 8990, 96, 208, 219232,
L 298299, 307
L-NAME, 109110, 130, 132 PTEN, 148, 150152
LNMMA, 109110, 130, 132 PTIO, 118123, 131132
Pulmonary edema, 241, 246
M Pulmonary hypertension, 1314, 256
Mechanically gated channels, 109135
R
Mechanical stimuli, 3, 12, 2627, 37,
Remodelling, 89, 9394, 142, 146, 149, 152,
5354, 73, 82, 9293, 98, 112, 114,
155158, 298, 306
145, 148, 157, 194, 220, 278282, 288,
298308, 365 S
Mechanical stretch, 6, 53, 114, 116, 193210, Shear stress, 69, 71, 73, 90, 193203, 205206,
220, 231232, 241, 258, 260264, 268, 359 210, 219220, 224225, 227230, 240,
Mechanosensing, 4, 7, 2627, 4652, 54, 258, 260, 266268, 279281, 283284,
57, 197, 199200, 202, 207, 209, 220, 286288, 298301, 303, 319, 325,
277288, 297308, 321, 328 328330, 333335
Mechanosensitive channels, 5, 11, 168, 170, Signal transduction, 26, 57, 6870, 80, 92, 133,
177, 184186 205206, 259260, 262, 278, 282, 318
Mechanosensitivity, 315, 2558, 6774, Smooth muscle cell, 315, 194, 220, 224225,
7799, 117, 121, 126, 150, 154155, 228232, 249, 333
193210, 219232, 268, 297308, 353366 Spider venom, 168, 171
Mechanosome, 278280, 284, 286, 288 Src, 6, 89, 11, 15, 53, 55, 145146, 158,
Mechanotransduction, 315, 2558, 6774, 223, 244, 249, 251, 256, 259, 261269,
7799, 109135, 141158, 168169, 282283
193210, 219232, 239251, 255269, Stretch, 47, 11, 26, 42, 46, 5354,
277288, 297308, 317336, 353366 57, 70, 8586, 92, 94, 98, 109,
Molecular dynamics simulation, 36, 167 113132, 135, 143146, 154155,
MscK, 168, 170, 177, 180 157, 168170, 193210, 220,
Myocardium contraction, 112, 134 228229, 231232, 240242, 246,
Myogenic response, 45, 713, 15, 256258, 260265, 268, 301302, 307,
228229, 365 354359, 362365
Myosin II, 2730, 3435, 37, 4351, 5354 Stretch-activated channels, 54, 85, 98,
169170, 198
N Syndecans, 208, 221224, 232
Nitric oxide (NO), 27, 74, 109135, 198199,
202203, 206, 208, 225, 227229, 239, T
242244, 249, 280, 286287, 300, 302, Transient receptor potential vanilloid, 198, 240
306, 334335 TREK-2, 353366
NO donors, 112113, 122130, 132, 244 Tubulin microtubules, 78, 8183, 86, 88,
NOS/mice, 110, 113, 131 9091, 9596
Index 371

V Ventilator induced lung injury, 245, 249,


Vascular remodeling, 4, 67, 1215, 203, 255269
208210, 230, 232 Vimentin intermediate filaments, 80, 86,
Vascular smooth muscle cells, 315, 194, 220, 8890, 95
224225, 228232

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