Review article

n engl j med 363;17 nejm.org october 21, 2010

1638
franklin h. epstein lecture
Franklin H. Epstein, M.D., served the New England Journal of Medicine for more than 20 years.
A keen clinician, accomplished researcher, and outstanding teacher, Dr. Epstein was Chair and Professor of
Medicine at Beth Israel Deaconess Medical Center, Boston, where the Franklin H. Epstein, M.D., Memorial
Lectureship in Mechanisms of Disease has been established in his memory.
Cardiac Development and Implications
for Heart Disease
Jonathan A. Epstein, M.D.
From the Department of Cell and Devel-
opmental Biology and the Cardiovascular
Institute, University of Pennsylvania School
of Medicine, Philadelphia. Address reprint
requests to Dr. Epstein at the Department
of Cell and Developmental Biology, Univer-
sity of Pennsylvania School of Medicine,
421 Curie Blvd., 1154 BRB II, Philadelphia,
PA 19104, or at epsteinj@mail.med.upenn
.edu.
N Engl J Med 2010;363:1638-47.
Copyright 2010 Massachusetts Medical Society.
D
uring the past decade, our understanding of the development
of the embryonic heart has been improved by a number of discoveries. These
new findings will require changes in standard teachings of how the four-
chambered heart forms, and they have implications for the management of con-
genital and acquired heart disease. In the coming years, additional advances in our
knowledge of cardiac development are likely to further influence the classification
and treatment of congenital heart disease, inform clinicians on the best uses of re-
generative treatment (e.g., stem-cell therapy), and revise our understanding of some
cardiovascular disorders in adults. This review gives examples of recent findings in
the field of cardiac development, with an emphasis on those likely to have the
greatest effect on clinical practice.
Overview
Classic teaching holds that cardiovascular development proceeds from the early spec-
ification of bilateral clusters of progenitor cells that coalesce to form a cardiac
crescent and a midline linear heart tube. This tube, which consists of an inner cell
layer of endothelium surrounded by myocardial precursor cells,
1
undergoes a series
of looping or bending events followed by ballooning or expansion of regions des-
tined to become cardiac chambers. Subsequently, a series of septation events results
in a four-chambered heart with parallel systemic and pulmonary circulations.
Additional cell types that lie outside the heart tube are important in the devel-
opment of the heart and influence its morphogenesis (Fig. 1). For example, neural-
crest cells, which form components of the peripheral nervous system and the cra-
niofacial regions, migrate to the heart, where they are essential for septation of the
cardiac outflow tract.
2
The link between neural-crest cells
3
and septation helps to
explain the association of craniofacial defects with some forms of congenital heart
disease.
Lineage Restriction
The mature heart consists of many different cell types, including myocardial cells,
endothelial cells, smooth-muscle cells, fibroblasts, and specialized conducting cells.
Until recently, the origins and lineages of these various cell types were unclear.
Recently, however, the technique of gene targeting has allowed rigorous fate map-
ping (a method used to determine the cellular derivatives of a cell or population of
cells) and lineage analysis in mammalian embryos and adults.
4-7
The results of
these studies, and of clonal analyses of embryonic stem-cell differentiation in vi-
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Franklin H. Epstein Lecture
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1639
tro,
7-10
convincingly document a progressive lin-
eage restriction of cells engaged in cardiac devel-
opment (Fig. 2). It is now clear that precursor cells
in the embryo have the potential to differentiate
into various types of cardiac cells. As a particular
lineage develops, however, the potential of its
member cells to deviate into alternative lineages
becomes progressively restricted.
Embryonic stem cells are pluripotent that is,
they have the ability to become nearly any kind of
cell. Such stem cells can differentiate into sponta-
neously beating myocardial cells when grown in
tissue culture in the presence of specific growth
factors and under particular conditions.
12-14
Car-
diac precursor cells that arise in vitro from em-
bryonic stem cells express kinase-domainrelated
(KDR) receptor (a receptor for vascular endothe-
lial growth factor) and NKX2-5 (a transcription
factor with a role in cardiac development).
8,10

These early cardiac precursor cells have the po-
tential to become endothelium, smooth muscle, or
myocardium. In vivo genetic studies suggest that
similar precursor cells contribute to multiple lin-
eages in the developing heart.
The phenomenon of progressive lineage re-
striction during cardiac development has impor-
tant implications for the use of stem-cell therapy
in cardiac disease. For treatments intended to
enhance endogenous myocardial repair or to
generate new heart muscle through the delivery
Figure 1. Overview of Cardiac Development.
Traditional descriptions of cardiac development include progression from the cardiac crescent to the linear heart
tube, which loops and becomes septated as it develops into the mature heart. Multiple cell types extrinsic to the ini-
tial cardiac crescent, including neural-crest cells, cells arising from the second heart field, and epicardial progeni-
tors, contribute to cardiac morphogenesis.
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1640
of appropriate cardiac progenitor cells, or to grow
bioprostheses of contractile myocardial patches,
8

researchers and practitioners must consider which
cells can best meet these goals.
8,15,16
For example,
the regenerated tissue that results from treatment
with a progenitor cell that has the potential to
produce only cardiac myocytes will be unlike nor-
mal cardiac tissue, which has multiple cellular
components. Progenitors with a restricted differ-
entiation capacity may have to be replaced with
multipotent progenitor cells if the goal is to re-
generate multilineage tissue composed of endo-
thelium, smooth muscle (regenerating vascula-
ture), and contractile myocardium. At this time,
the most appropriate type of progenitor cell to be
used in stem-cell therapy for ischemic cardiomy-
opathy and other forms of heart failure is un-
known, and the markers and gene-expression
signatures that characterize various progenitors
are only now being elucidated.
Progressive lineage restriction is also a feature
of the differentiation of a multipotent hematopoi-
etic stem cell into various blood-cell lineages.
17

The delineation of each stage of lineage restriction
in blood-cell precursors has allowed for identifi-
cation of clinically useful growth factors, such as
granulocyte colony-stimulating factor, granulo-
cytemacrophage colony-stimulating factor, and
erythropoietin, each of which affects a different
progenitor. As is the case with hematopoietic stem
cells, the identification and characterization of
specific cardiac progenitor cells and cardiac
Figure 2. Progressive Lineage Restriction.
In the hematopoietic system, stem cells become progressively restricted during differentiation. Similarly, cardiac progenitors, which may
share a common precursor with hematopoietic stem cells, become progressively restricted in terms of the types of potential mature de-
rivatives that can ultimately be produced. Adapted from Wu et al.
11

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Franklin H. Epstein Lecture
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1641
growth factors may lead to useful treatments for
myocardial infarction or heart failure.
The Second Heart Field
Not all precursors of cardiac muscle reside in the
cardiac crescent and the early linear heart tube.
Many right ventricular myocytes and, to a variable
degree, myocytes in the atria, left ventricle, and
cardiac inflow and outflow tracts enter the devel-
oping heart after its initial looping stages are com-
plete.
18-20
These additional cells arise from a sec-
ond heart field that is medial and ventral to the
primary cardiac crescent. (In the embryo, a field
consists of a group of related cells within a de-
fined boundary.) Cells in the second heart field
migrate first to the pharyngeal regions, where
they can be identified in mouse embryos in early
gestation or midgestation according to the prod-
ucts of specific marker genes, including the tran-
scription factor islet 1.
20
These second-heartfield
cardiac precursors in the pharyngeal regions invade
the developing heart and migrate along its inflow
and outflow tracts. Second-heartfield progeni-
tors that express islet 1 are multipotent cells that
can give rise to smooth-muscle cells at the base
of the aorta and pulmonary arteries, to endothe-
lial cells, or to myocardium.
7
The existence of a second heart field has im-
portant implications for understanding congeni-
tal heart diseases. For example, abnormalities in
the distinct genetic pathways that mediate for-
mation of myocytes in either the right or the left
ventricle could explain congenital defects whose
predominant effect is on the right or left ventri-
cle. The results of induced genetic perturbations in
only second-heartfield cells of embryonic mice
suggest that abnormalities in this population can
cause double-outlet right ventricle, right ventricu-
lar hypoplasia, pulmonic stenosis, and tetralogy of
Fallot.
21,22
Moreover, genetic studies in humans
have shown that haplotypes within the ISL1 lo-
cus are strongly associated with these forms of
congenital heart disease.
23
Perhaps other right-
sided disorders, such as hypoplastic right ventri-
cle, Ebsteins anomaly, and some forms of arrhyth-
mogenic right ventricular dysplasia,
24
are also the
result of abnormalities in second-heartfield cells.
The usual classification of congenital heart
diseases depends on the anatomical characteris-
tics of the abnormality. These traditional classi-
fication systems may have to be changed in light
of emerging evidence that the same developmen-
tal abnormality, or similar developmental abnor-
malities, can underlie anatomically distinct con-
genital heart disorders. An example is the group
of clinically dissimilar congenital heart defects
(e.g., a double-outlet right ventricle and right ven-
tricular hypoplasia) that are related through their
association with abnormalities of second-heart
field progenitor cells.
25
Aberrations of these pre-
cursor cells may also cause anatomical abnor-
malities of the left or right side of the heart (e.g.,
defects of atrial septation, ventricular septation,
conus positioning, and great-vessel alignment)
because the cells contribute to both the inflow
and the outflow tracts of the heart.
26
Moreover,
recent studies of what have come to be known as
second-heartfield cardiac defects suggest that in-
flow and outflow abnormalities frequently coex-
ist.
26
Anatomical classification is undoubtedly
clinically useful, but it is likely that classification
systems based on developmental relationships and
genetic causes will provide additional diagnostic
and prognostic information. Consensus opinions
that explicitly define developmentally based clas-
sifications of congenital heart disorders will also
promote enhanced communication among basic
researchers and their clinical colleagues.
The Epicardium in Cardiac
Development and Repair
The epicardium, a layer of connective tissue lo-
cated between the myocardium and the pericar-
dium,
27
arises from a transient embryonic struc-
ture called the proepicardial organ (Fig. 3). Cells
in this organ derive from the septum transver-
sum, which separates the embryos thorax from
its abdomen, and to the diaphragm and liver. Some
proepicardial cells migrate to the developing heart
and contribute to the formation of the epicardial
layer. Descendants of proepicardial cells invade
the myocardium, where they develop into fibro-
blasts in the heart and smooth-muscle cells of
the coronary arteries.
29-32
Signals from the epi-
cardium are required for proper maturation of the
myocardium and normal development of the cor-
onary arteries.
33-35
Recent studies suggest that epicardial pro-
genitor cells are multipotent, with the ability to
differentiate into smooth muscle, fibroblasts, and
perhaps also cardiac muscle and endothelium
36,37
(although the idea that these cells form coronary
endothelium has been challenged
38
). These stud-
ies used genetic markers expressed by the proepi-
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1642
cardial organ the Wilms tumor 1 gene (Wt1)
37

and the T-box 18 gene (Tbx18)
36
to map the
fate of epicardial precursor cells throughout the
course of their differentiation. The results sug-
gest that some epicardial precursors contribute
to myocardium, an indication of an additional
developmental avenue for the generation of car-
diac muscle. In adult zebrafish, which can re-
generate myocardium after injury,
39
the epicar-
dium becomes activated by surgical resection of
heart muscle
40
; the activated epicardium express-
es fetal genes, including wt1 and tbx18. It remains
unclear whether these activated epicardial cells
contribute directly to the regeneration of myocar-
dium or produce signals that cause cardiac myo-
cytes to enter the cell cycle.
41
Since epicardium
normally gives rise to cardiac fibroblasts, the main
components of scars, it is possible that the abil-
ity of mammals to grow new heart muscle was
lost during evolution in a trade-off for the abil-
ity to rapidly form a scar after injury.
Epicardium-derived progenitor cells (EPDCs)
have been isolated from human, rat, and mouse
hearts and have been grown in tissue culture.
42

In rodents (as in zebrafish), these cells reactivate
fetal genes after myocardial infarction and pro-
liferate.
43,44
Ex vivo, EPDCs can differentiate into
multiple cell types and express contractile proteins
of myocytes. Various growth factors, including
thymosin
4
, have been found to promote the pro-
liferation of EPDCs and to improve recovery and
myocardial function after injury in animals
45-47
;
it remains unclear whether these changes are due
to myocardial regeneration or to factors secreted
Figure 3. Development and Derivatives of the Epicardium.
The epicardium derives from the proepicardial organ (Panel A), a multipotent cluster of cells that arises dorsal to
the looped heart. Epicardial progenitors migrate to and encompass the developing heart and form the mature epi-
cardium (Panel B). Some epicardial progenitors undergo an epithelial-to-mesenchymal transition, invade the myo-
cardium, and differentiate into various mature cardiac cell types, which may include vascular smooth-muscle cells,
fibroblasts, endothelial cells, and cardiac myocytes. Adapted from Schlueter.
28
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Franklin H. Epstein Lecture
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1643
by EPDCs that have paracrine effects influencing
myocyte survival or function. These studies sug-
gest that interventions involving the manipulation
of epicardial activation and EPDCs after injury
whether through the use of systemic therapy, treat-
ments administered within the pericardial space,
or the application of a drug-eluting patch to the
epicardial surface of the damaged heart may
be worthwhile avenues for further investigation.
The Cardiac Conduction System
The specialized cells of the cardiac conduction sys-
tem arise from myocardial precursor cells. The
mature cells have relatively poor contractility and
express specialized ion channels and gap-junction
proteins, including connexins, that mediate elec-
trical coupling with neighboring cells.
48,49
Early
in development, at the time of chamber specifi-
cation, the myocardium between the developing
atria and ventricles has slow conduction charac-
teristics and other properties reminiscent of the
atrioventricular node. Similarly, the myocardium
of the inflow tract acquires autonomous activity
and develops pacemaker function. The sinus node
develops from this tissue. The cells that give rise
to the sinus node express the fetal TBX18 gene,
whereas the cells that give rise to the atrioven-
tricular node and the Purkinje system express the
NKX2-5 transcription factor.
48
The possibility that
precursors of pacemaker cells of the sinus node
are related to the myocardium surrounding the
pulmonary veins could be important (and is cur-
rently under investigation) because atrial fibrilla-
tion commonly arises from an arrhythmia within
the pulmonary veins. The myocardium of the pos-
terior wall of the left atrium extends to and en-
sheathes the proximal pulmonary vein, thereby
providing electrical continuity, and atrial fibrilla-
tion can be successfully treated through electrical
isolation of the pulmonary veins.
50
The develop-
ment of pulmonary-vein myocardium requires
the PITX2 transcription factor,
51
and recent ge-
nomewide association studies have identified
haplotypes at 4q25, near PITX2, that are associated
with atrial fibrillation.
52,53
Thus, the response of
pulmonary-vein myocardial cells to altered PITX2
function may underlie the susceptibility to atrial
fibrillation. It is also possible that melanocyte-
like cells in the heart,
54
which are present in the
atrioventricular ring, the atria, and the pulmo-
nary veins in the developing embryo, play a role
in atrial fibrillation. In animal models, genetic
abnormalities induced in these cells increase the
susceptibility to atrial arrhythmia.
The region of slow-conducting myocardium
that separates atria from ventricles in the embryo
and provides atrioventricular delay initially oc-
cupies the entire atrioventricular ring.
48,49,55
As
cardiac development proceeds, fibroblasts derived
from the epicardium invade the atrioventricular
sulcus and form the annulus fibrosis,
56,57
which
isolates the atria from the ventricles electrically.
The atrioventricular canal myocardium regresses,
and the property of slow conduction becomes
restricted to the specialized cells of the atrioven-
tricular node. In animal models, deficient devel-
opment of the annulus fibrosis causes abnormal
electrical connectivity between the atria and ven-
tricles, pre-excitation, and characteristics of the
WolffParkinsonWhite syndrome.
56,58
Ectopic
myocardium that bridges the atrioventricular re-
gion is, however, more common than breakdown
of the annulus fibrosis in humans with this syn-
drome. Consequently, some forms of the syn-
drome may result when the normal regression of
the atrioventricular canal myocardium fails to oc-
cur during development.
Proper formation of the atrioventricular node
depends on transcription factors that play reiter-
ated roles during cardiac development. These fac-
tors include NKX2-5, TBX5, and GATA4.
48
In mice,
Tbx5 and Gata4 regulate the expression of con-
nexin 30.2, which is required for slow conduction
in the atrioventricular node.
59
Haploinsufficiency
of Gata4 in mice causes a short PR interval.
59
Mu-
tation of NKX2-5, TBX5, or GATA4 has been associ-
ated with an atrial septal defect in humans as well
as in animal models.
60-63
Hence, structural inter-
ference of conducting fibers by the septal defect
may not entirely explain the association between
atrial septal defects and conduction abnormalities.
Rather, a single underlying genetic defect may
affect septal closure and specialized conduction
cells independently.
64
Indeed, patients with
NKX2-5 mutations can have isolated conduction
defects. These factors, and the cellular processes
that these factors regulate, may continue to play
important roles in conduction tissues through-
out adult life. For example, progressive degenera-
tion of the atrioventricular node and heart block
develop when the Nkx2-5 gene has been inacti-
vated in adult mice.
65
It seems likely that an as-
sociation between certain risk alleles of this gene
or related genes will be found in and may
predict heart block in elderly patients.
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1644
Cardiovascular Maturation
At the time of birth, the cardiovascular system un-
dergoes a series of abrupt and critical changes.
Blood must be diverted to the lungs for oxygen-
ation, and the portal circulation must perfuse the
liver when enteric feeding begins. Increased oxy-
gen tension associated with a babys first breaths,
coupled with withdrawal from exposure to mater-
nal prostaglandins, stimulates closure of the duc-
tus arteriosus, sending blood from the right ven-
tricle to the lungs and establishing the parallel
pulmonary and systemic circulations. The foramen
ovale of the atrial septum closes. The ductus veno-
sus constricts, sending portal blood to the liver.
Less obvious but equally important changes
occur in myocardial cells during the neonatal pe-
riod. There is a shift in gene-expression profiles
within the heart; many fetal isoforms of genes
become down-regulated or replaced by their adult
counterparts. Examples include genes encoding
contractile components of the sarcomere, calcium-
handling machinery, energy-utilization enzymes,
and natriuretic factors.
66-68
Re-expression of fe-
tal genes occurs in nearly every form of heart
failure in adults, and this phenomenon is thought
to contribute to the progression of heart failure.
Elucidation of the mechanisms that regulate the
re-expression of fetal genes during disease states
may reveal new targets for the treatment of heart
failure. In addition, an improved understanding
of the genetic programs governing myocyte mat-
uration should inform the development of regen-
erative treatments; the use of such treatments
for cardiac disease has been hampered by a lim-
ited ability to engineer fully mature adult cardiac
myocytes from progenitor cells.
Our knowledge of the mechanisms that regu-
late gene expression has increased considerably
in recent years. Gene expression requires spe-
cific transcription factors proteins that acti-
vate or inhibit transcription from genomic DNA
to messenger RNA (mRNA) by binding to pro-
moter or enhancer regions of genes. Changes in
DNA packaging that are effected by chromatin
and the enzymatic modification of histones, the
principal protein component of chromatin, also
influence gene expression through a mechanism
termed epigenetic modification. This mechanism
regulates gene expression by affecting the enzy-
matic acetylation of histones (and causing other
chemical modifications) and triggering the un-
winding of chromatin (which exposes actively
transcribed loci); it also represses gene transcrip-
tion by enzymatically deacetylating histones and
condensing chromatin. Epigenetic control of chro-
matin structure, a mechanism that mediates glob-
al changes in gene-expression programs, is critical
to cellular reprogramming (the directed altera-
tion in which one cell type is changed to another
e.g., a fibroblast becomes a pluripotent stem
cell) and to the determination of cell fate.
There are indications that the activities of spe-
cific histone deacetylase enzymes are necessary
for regulation of the expression of the fetal gene
program in the heart during development and
that these enzymes are involved in heart failure
in adults. Genetic inactivation of histone deacety-
lase 2 in mice, for example, decreases the expres-
sion of fetal cardiac genes and inhibits reactiva-
tion of the fetal gene program in situations
involving cardiac stress in adults.
69
Chemical in-
hibitors of this enzyme, now being studied in
phase 3 clinical trials for the treatment of cer-
tain cancers, can prevent reactivation of the fetal
gene program, cardiac hypertrophy, and heart
failure in animal models (e.g., ClinicalTrials.gov
numbers NCT00773747 and NCT01023308).
70-75
These results suggest that epigenetic mechanisms
regulate the transition of fetal cardiac gene pro-
grams to adult programs and that these mecha-
nisms could be targets for new treatments of
heart failure.
MicroRNA (miRNA) molecules are short, single
strands of RNA that modulate gene expression
by binding to complementary regions in mRNA
transcripts. The miRNA genes in chromosomal
DNA can be expressed as independent genes un-
der the control of their own promoters or can be
coexpressed with other genes in which they are
embedded. In mice, the fetal heart expresses beta-
myosin heavy chain (-MHC), whereas the adult
heart expresses alpha-myosin heavy chain (-MHC).
In adult mice, cardiac stressors such as pressure
overload or chronic adrenergic stimulation in-
duce re-expression of -MHC and suppression of
-MHC.
66
The basis of this reciprocal regulation
is the embedding of a regulatory miRNA gene in
the intron of the genes for each of the two MHC
isoforms.
76
The expression of -MHC results in
coexpression of miR-208 (a cardiac-specific miRNA
gene), which, in the presence of stress-induced
signals, indirectly activates transcription of -MHC
(Fig. 4). In the absence of miR-208, stress signals
fail to activate -MHC and other fetal genes, and
both the hypertrophic response and associated
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Franklin H. Epstein Lecture
n engl j med 363;17 nejm.org october 21, 2010
1645
fibrosis are blunted. The possibility of targeting
miRNA genes with therapeutic agents is an
emerging area of investigation.
77-79
Summary
Recent studies have revealed a surprising num-
ber of previously unappreciated aspects of car-
diac morphogenesis that are relevant to both
congenital and adult cardiovascular disease. It
is now clear that cell populations extrinsic to
the primary heart field and the linear heart tube
of the embryo contribute to the development of
the mature heart and modulate cardiac mor-
phogenesis. These cell populations include neu-
ral-crest cells, the cells arising from a second
heart field, and epicardial cells. The full devel-
opmental potential and unique defining charac-
teristics of various cardiac progenitor cells are
only partially known, and specific stages of the
progressive lineage restriction of these cardiac
progenitors require further characterization. The
ability to expand cardiac progenitor populations,
either in situ or ex vivo, and to direct cell fate
will have important implications for regenera-
tive cardiovascular therapies. The cell biology of
myocyte maturation and the effects of the tran-
sition from the fetal heart to the adult heart re-
main areas of investigation that are likely to
inform our understanding of heart failure. Stud-
ies of the regulation of gene-expression programs
in the heart are likely to suggest new therapeu-
tic targets for cardiovascular disease.
Supported by a grant from the National Institutes of Health
(U01 HL100405), a DeHaan Cardiac Myogenesis Award from the
American Heart Association, and the W.W. Smith endowed chair
for cardiovascular research.
Disclosure forms provided by the author are available with the
full text of this article at NEJM.org.
Figure 4. MicroRNA-Mediated Regulation of Myosin Heavy-Chain Gene Isoform Expression.
MicroRNA-208 (miRNA-208), which is embedded within an intron of the gene encoding alpha-myosin heavy chain
(-MHC), indirectly regulates the expression of beta-myosin heavy chain (-MHC) in response to stress-induced
signals. AAA represents the polyadenylated tail of messenger RNA; LA denotes left atrium, LV left ventricle, RA right
atrium, and RV right ventricle. Adapted from a drawing provided courtesy of Dr. E. Olson.
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1646
References
1. Harvey RP, Rosenthal N, eds. Heart
development. San Diego, CA: Academic
Press, 1999.
2. Kirby ML, Gale TF, Stewart DE. Neu-
ral crest cells contribute to normal aorti-
copulmonary septation. Science 1983;220:
1059-61.
3. Stoller JZ, Epstein JA. Cardiac neural
crest. Semin Cell Dev Biol 2005;16:704-15.
4. Cui C, Cheuvront TJ, Lansford RD,
Moreno-Rodriguez RA, Schultheiss TM,
Rongish BJ. Dynamic positional fate map
of the primary heart-forming region. Dev
Biol 2009;332:212-22.
5. Hsieh PC, Segers VF, Davis ME, et al.
Evidence from a genetic fate-mapping study
that stem cells refresh adult mammalian
cardiomyocytes after injury. Nat Med
2007;13:970-4.
6. Ma Q, Zhou B, Pu WT. Reassessment
of Isl1 and Nkx2-5 cardiac fate maps us-
ing a Gata4-based reporter of Cre activity.
Dev Biol 2008;323:98-104.
7. Moretti A, Caron L, Nakano A, et al.
Multipotent embryonic isl1+ progenitor
cells lead to cardiac, smooth muscle, and
endothelial cell diversification. Cell 2006;
127:1151-65.
8. Kattman SJ, Huber TL, Keller GM.
Multipotent flk-1+ cardiovascular progen-
itor cells give rise to the cardiomyocyte,
endothelial, and vascular smooth muscle
lineages. Dev Cell 2006;11:723-32.
9. Wu SM, Fujiwara Y, Cibulsky SM, et
al. Developmental origin of a bipotential
myocardial and smooth muscle cell precur-
sor in the mammalian heart. Cell 2006;
127:1137-50.
10. Yang L, Soonpaa MH, Adler ED, et al.
Human cardiovascular progenitor cells
develop from a KDR+ embryonic-stem-
cell-derived population. Nature 2008;453:
524-8.
11. Wu SM, Fujiwara Y, Cibulsky SM, et
al. Developmental origin of a bipotential
myocardial and smooth muscle cell pre-
cursor in the mammalian heart. Cell
2006;127:1137-50.
12. Chen VC, Stull R, Joo D, Cheng X,
Keller G. Notch signaling respecifies the
hemangioblast to a cardiac fate. Nat Bio-
technol 2008;26:1169-78.
13. Laflamme MA, Chen KY, Naumova
AV, et al. Cardiomyocytes derived from
human embryonic stem cells in pro-sur-
vival factors enhance function of infarcted
rat hearts. Nat Biotechnol 2007;25:1015-
24.
14. Murry CE, Keller G. Differentiation of
embryonic stem cells to clinically relevant
populations: lessons from embryonic de-
velopment. Cell 2008;132:661-80.
15. Sui R, Liao X, Zhou X, Tan Q. The cur-
rent status of engineering myocardial tis-
sue. Stem Cell Rev 2010 March 3 (Epub
ahead of print).
16. Chien KR, Domian IJ, Parker KK. Car-
diogenesis and the complex biology of
regenerative cardiovascular medicine. Sci-
ence 2008;322:1494-7.
17. Morrison SJ, Uchida N, Weissman IL.
The biology of hematopoietic stem cells.
Annu Rev Cell Dev Biol 1995;11:35-71.
18. Kelly RG, Brown NA, Buckingham
ME. The arterial pole of the mouse heart
forms from Fgf10-expressing cells in pha-
ryngeal mesoderm. Dev Cell 2001;1:435-
40.
19. Waldo KL, Kumiski DH, Wallis KT, et
al. Conotruncal myocardium arises from
a secondary heart field. Development
2001;128:3179-88.
20. Cai CL, Liang X, Shi Y, et al. Isl1 iden-
tifies a cardiac progenitor population that
proliferates prior to differentiation and
contributes a majority of cells to the
heart. Dev Cell 2003;5:877-89.
21. High FA, Jain R, Stoller JZ, et al. Mu-
rine Jagged1/Notch signaling in the sec-
ond heart field orchestrates Fgf8 expres-
sion and tissue-tissue interactions during
outflow tract development. J Clin Invest
2009;119:1986-96.
22. Ward C, Stadt H, Hutson M, Kirby
ML. Ablation of the secondary heart field
leads to tetralogy of Fallot and pulmonary
atresia. Dev Biol 2005;284:72-83.
23. Stevens KN, Hakonarson H, Kim CE,
et al. Common variation in ISL1 confers
genetic susceptibility for human congeni-
tal heart disease. PLoS One 2010;5(5)
e10855.
24. Lombardi R, Dong J, Rodriguez G, et
al. Genetic fate mapping identifies second
heart field progenitor cells as a source of
adipocytes in arrhythmogenic right ven-
tricular cardiomyopathy. Circ Res 2009;
104:1076-84.
25. Dyer LA, Kirby ML. The role of sec-
ondary heart field in cardiac develop-
ment. Dev Biol 2009;336:137-44.
26. Bajolle F, Zaffran S, Losay J, Ou P,
Buckingham M, Bonnet D. Conotruncal
defects associated with anomalous pulmo-
nary venous connections. Arch Cardio-
vasc Dis 2009;102:105-10.
27. Mnner J, Prez-Pomares JM, Macas
D, Muoz-Chpuli R. The origin, forma-
tion and developmental significance of the
epicardium: a review. Cells Tissues Organs
2001;169:89-103.
28. Schlueter J. The origin and therapeu-
tic potential of the coronary blood vessel
system. Presented at the European Society
of Cardiology, Barcelona, August 29Sep-
tember 2, 2009. abstract.
29. Guadix JA, Carmona R, Muoz-Ch-
puli R, Prez-Pomares JM. In vivo and in
vitro analysis of the vasculogenic poten-
tial of avian proepicardial and epicardial
cells. Dev Dyn 2006;235:1014-26.
30. Vrancken Peeters MP, Gittenberger-
de Groot AC, Mentink MM, Poelmann RE.
Smooth muscle cells and fibroblasts of
the coronary arteries derive from epitheli-
al-mesenchymal transformation of the epi-
cardium. Anat Embryol (Berl) 1999;199:
367-78.
31. Mikawa T, Gourdie RG. Pericardial
mesoderm generates a population of cor-
onary smooth muscle cells migrating into
the heart along with ingrowth of the epi-
cardial organ. Dev Biol 1996;174:221-32.
32. Mikawa T, Fischman DA. Retroviral
analysis of cardiac morphogenesis: discon-
tinuous formation of coronary vessels.
Proc Natl Acad Sci U S A 1992;89:9504-8.
33. Stuckmann I, Evans S, Lassar AB.
Erythropoietin and retinoic acid, secreted
from the epicardium, are required for car-
diac myocyte proliferation. Dev Biol 2003;
255:334-49.
34. Chen TH, Chang TC, Kang JO, et al.
Epicardial induction of fetal cardiomyocyte
proliferation via a retinoic acid-inducible
trophic factor. Dev Biol 2002;250:198-207.
35. Prez-Pomares JM, Phelps A, Sedmero-
va M, et al. Experimental studies on the
spatiotemporal expression of WT1 and
RALDH2 in the embryonic avian heart:
a model for the regulation of myocardial
and valvuloseptal development by epicar-
dially derived cells (EPDCs). Dev Biol
2002;247:307-26.
36. Cai CL, Martin JC, Sun Y, et al. A myo-
cardial lineage derives from Tbx18 epicar-
dial cells. Nature 2008;454:104-8.
37. Zhou B, Ma Q, Rajagopal S, et al. Epi-
cardial progenitors contribute to the car-
diomyocyte lineage in the developing heart.
Nature 2008;454:109-13.
38. Red-Horse K, Ueno H, Weissman IL,
Krasnow MA. Coronary arteries form by
developmental reprogramming of venous
cells. Nature 2010;464:549-53.
39. Poss KD, Wilson LG, Keating MT.
Heart regeneration in zebrafish. Science
2002;298:2188-90.
40. Lepilina A, Coon AN, Kikuchi K, et al.
A dynamic epicardial injury response sup-
ports progenitor cell activity during ze-
brafish heart regeneration. Cell 2006;127:
607-19.
41. Jopling C, Sleep E, Raya M, Mart M,
Raya A, Belmonte JC. Zebrafish heart re-
generation occurs by cardiomyocyte de-
differentiation and proliferation. Nature
2010;464:606-9.
42. Smart N, Riley PR. Derivation of epi-
cardium-derived progenitor cells (EPDCs)
from adult epicardium. In: Current proto-
cols in stem cell biology. New York: John
Wiley, 2009:Unit2C.2.
43. Limana F, Bertolami C, Mangoni A, et
al. Myocardial infarction induces embry-
onic reprogramming of epicardial c-kit(+)
cells: role of the pericardial fluid. J Mol
Cell Cardiol 2010;48:609-18.
44. Limana F, Zacheo A, Mocini D, et al.
Identification of myocardial and vascular
precursor cells in human and mouse epi-
cardium. Circ Res 2007;101:1255-65.
45. Smart N, Risebro CA, Melville AA, et
al. Thymosin beta-4 is essential for coro-
The New England Journal of Medicine
Downloaded from nejm.org on July 14, 2014. For personal use only. No other uses without permission.
Copyright 2010 Massachusetts Medical Society. All rights reserved.
Franklin H. Epstein Lecture
n engl j med 363;17 nejm.org october 21, 2010
1647
nary vessel development and promotes
neovascularization via adult epicardium.
Ann N Y Acad Sci 2007;1112:171-88.
46. Bock-Marquette I, Shrivastava S, Pipes
GC, et al. Thymosin beta4 mediated PKC
activation is essential to initiate the em-
bryonic coronary developmental program
and epicardial progenitor cell activation
in adult mice in vivo. J Mol Cell Cardiol
2009;46:728-38.
47. Smart N, Risebro CA, Melville AA, et
al. Thymosin beta4 induces adult epicar-
dial progenitor mobilization and neovas-
cularization. Nature 2007;445:177-82.
48. Christoffels VM, Smits GJ, Kispert A,
Moorman AF. Development of the pace-
maker tissues of the heart. Circ Res
2010;106:240-54.
49. Moorman AF, Christoffels VM. Devel-
opment of the cardiac conduction system:
a matter of chamber development. Novar-
tis Found Symp 2003;250:25-34.
50. Peters NS, Schilling RJ, Kanagarat-
nam P, Markides V. Atrial fibrillation:
strategies to control, combat, and cure.
Lancet 2002;359:593-603.
51. Mommersteeg MT, Brown NA, Prall
OW, et al. Pitx2c and Nkx2-5 are required
for the formation and identity of the pul-
monary myocardium. Circ Res 2007;101:
902-9.
52. Damani SB, Topol EJ. Molecular ge-
netics of atrial fibrillation. Genome Med
2009;1:54.
53. Gudbjartsson DF, Arnar DO, Hel-
gadottir A, et al. Variants conferring risk
of atrial fibrillation on chromosome
4q25. Nature 2007;448:353-7.
54. Levin MD, Lu MM, Petrenko NB, et al.
Melanocyte-like cells in the heart and
pulmonary veins contribute to atrial ar-
rhythmia triggers. J Clin Invest 2009;
119:3420-36.
55. Rutenberg JB, Fischer A, Jia H, Gessler
M, Zhong TP, Mercola M. Developmental
patterning of the cardiac atrioventricular
canal by Notch and Hairy-related tran-
scription factors. Development 2006;133:
4381-90.
56. Kolditz DP, Wijffels MC, Blom NA, et
al. Epicardium-derived cells in develop-
ment of annulus fibrosis and persistence
of accessory pathways. Circulation 2008;
117:1508-17.
57. Zhou B, von Gise A, Ma Q, Hu YW, Pu
WT. Genetic fate mapping demonstrates
contribution of epicardium-derived cells
to the annulus fibrosis of the mammalian
heart. Dev Biol 2010;338:251-61.
58. Arad M, Moskowitz IP, Patel VV, et al.
Transgenic mice overexpressing mutant
PRKAG2 define the cause of Wolff-Par-
kinson-White syndrome in glycogen stor-
age cardiomyopathy. Circulation 2003;107:
2850-6.
59. Munshi NV, McAnally J, Bezprozvan-
naya S, et al. Cx30.2 enhancer analysis
identifies Gata4 as a novel regulator of
atrioventricular delay. Development 2009;
136:2665-74.
60. Maslen CL. Molecular genetics of
atrioventricular septal defects. Curr Opin
Cardiol 2004;19:205-10.
61. Basson CT, Cowley GS, Solomon SD,
et al. The clinical and genetic spectrum of
the HoltOram syndrome (hearthand syn-
drome). N Engl J Med 1994;330:885-91.
[Erratum, N Engl J Med 1994;330:1627.]
62. Schott JJ, Benson DW, Basson CT, et al.
Congenital heart disease caused by muta-
tions in the transcription factor NKX2-5.
Science 1998;281:108-11.
63. Garg V, Kathiriya IS, Barnes R, et al.
GATA4 mutations cause human congeni-
tal heart defects and reveal an interaction
with TBX5. Nature 2003;424:443-7.
64. Benson DW, Silberbach GM, Kava-
naugh-McHugh A, et al. Mutations in the
cardiac transcription factor NKX2.5 af-
fect diverse cardiac developmental path-
ways. J Clin Invest 1999;104:1567-73.
65. Pashmforoush M, Lu JT, Chen H, et al.
Nkx2-5 pathways and congenital heart
disease: loss of ventricular myocyte lineage
specification leads to progressive cardio-
myopathy and complete heart block. Cell
2004;117:373-86.
66. Feldman AM, Weinberg EO, Ray PE,
Lorell BH. Selective changes in cardiac
gene expression during compensated hy-
pertrophy and the transition to cardiac
decompensation in rats with chronic aor-
tic banding. Circ Res 1993;73:184-92.
67. Parker TG, Packer SE, Schneider MD.
Peptide growth factors can provoke fe-
tal contractile protein gene expression in
rat cardiac myocytes. J Clin Invest 1990;
85:507-14.
68. Taegtmeyer H, Sen S, Vela D. Return
to the fetal gene program: a suggested
metabolic link to gene expression in the
heart. Ann N Y Acad Sci 2010;1188:191-8.
69. Trivedi CM, Luo Y, Yin Z, et al. Hdac2
regulates the cardiac hypertrophic re-
sponse by modulating Gsk3 beta activity.
Nat Med 2007;13:324-31.
70. Antos CL, McKinsey TA, Dreitz M, et
al. Dose-dependent blockade to cardio-
myocyte hypertrophy by histone deacety-
lase inhibitors. J Biol Chem 2003;278:
28930-7.
71. Bush EW, McKinsey TA. Targeting
histone deacetylases for heart failure. Ex-
pert Opin Ther Targets 2009;13:767-84.
72. Gallo P, Latronico MV, Grimaldi S, et
al. Inhibition of class I histone deacety-
lase with an apicidin derivative prevents
cardiac hypertrophy and failure. Cardio-
vasc Res 2008;80:416-24.
73. Kong Y, Tannous P, Lu G, et al. Sup-
pression of class I and II histone deacety-
lases blunts pressure-overload cardiac hy-
pertrophy. Circulation 2006;113:2579-88.
74. Kee HJ, Sohn IS, Nam KI, et al. Inhibi-
tion of histone deacetylation blocks car-
diac hypertrophy induced by angiotensin II
infusion and aortic banding. Circulation
2006;113:51-9.
75. Kook H, Lepore JJ, Gitler AD, et al.
Cardiac hypertrophy and histone deacety-
lase-dependent transcriptional repression
mediated by the atypical homeodomain
protein Hop. J Clin Invest 2003;112:863-71.
76. van Rooij E, Sutherland LB, Qi X,
Richardson JA, Hill J, Olson EN. Control
of stress-dependent cardiac growth and
gene expression by a microRNA. Science
2007;316:575-9.
77. Brown BD, Naldini L. Exploiting and
antagonizing microRNA regulation for
therapeutic and experimental applications.
Nat Rev Genet 2009;10:578-85.
78. Latronico MV, Condorelli G. MicroR-
NAs and cardiac pathology. Nat Rev Car-
diol 2009;6:419-29.
79. van Rooij E, Marshall WS, Olson EN.
Toward microRNA-based therapeutics for
heart disease: the sense in antisense. Circ
Res 2008;103:919-28.
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