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PHYSIOLOGY 25: 250 259, 2010; doi:10.1152/physiol.00008.2010

A Gene for Speed: The Emerging Role of

-Actinin-3 in Muscle Metabolism
A common polymorphism (R577X) in the ACTN3 gene results in complete deficiency of -actinin-3 protein in 16% of humans worldwide. The presence of

Yemima Berman1,2,3 and

Kathryn N. North1,2
1

Institute for Neuroscience and Muscle Research, The Childrens

Hospital at Westmead, Westmead; 2Faculty of Medicine,
University of Sydney, Sydney; and 3Department of Clinical
Genetics, Royal North Shore Hospital, St. Leonards, Australia
kathryn@chw.edu.au

-actinin-3 protein is associated with improved sprint/power performance in

athletes and the general population. Despite this, there is evidence that the null
genotype XX has been acted on by recent positive selection, likely due to its
emerging role in the regulation of muscle metabolism. -Actinin-3 deficiency
reduces the activity of glycogen phosphorylase and results in a fundamental
shift toward more oxidative pathways of energy utilization.
Deficiency of the fast-fiber skeletal muscle protein
-actinin-3 is common in the general population
due to a polymorphic-null allele in the ACTN3
gene. Numerous independent studies have established that the absence of -actinin-3 is detrimental to sprint and power performance in athletes
and in the general population (1, 25, 55, 63, 66).
The sarcomeric -actinins have long been considered to be primarily structural proteins. However,
recent data suggest that -actinin-3 plays a significant role in the regulation of muscle metabolism.
-Actinin-3 deficiency results in a shift in the characteristics of fast glycolytic muscle fibers toward
those of slow muscle fibers with oxidative metabolism (48, 49, 62). This review examines the emerging role of -actinin-3 in regulation of skeletal
muscle metabolism.

The -Actinin Family of Proteins

The -actinins are a family of actin-binding proteins
that have been identified in a diverse range of organisms, suggesting an ancient origin (3, 8, 33, 50). The
-actinin protein structure is comprised of three domains; an NH2-terminal actin-binding domain, a
central rod domain containing four internal repeated
122-amino acid motifs, and a COOH-terminal region
containing two EF-hand calcium binding motifs. The
four repetitive motifs found in -actinin share homology with spectrin, suggesting a common evolutionary origin of the -actinin proteins and the
spectrin family of actin binding cytoskeletal proteins,
of which dystrophin is a member (13, 75). There is
marked evolutionary conservation of the -actinin
genes across species, particularly within the NH2terminal actin-binding domain (9).
There are four -actinin genes in humans,
ACTN1ACTN4 (9, 85). ACTN1 and ACTN4 contain
functional calcium-sensitive EF hands, whereas
the skeletal muscle or sarcomeric -actinins, encoded by ACTN2 and ACTN3, have EF hands that

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are not calcium sensitive (15). In humans, -actinin-2 is expressed in the heart, in all skeletal muscle fibers, and in the brain, whereas -actinin-3 is
expressed only in fast glycolytic skeletal muscle
fibers, is not present in cardiac muscle, and has
low levels of expression in the brain (50). These two
proteins diverged from one another following a
gene duplication event over 300 million years ago
(mya), but have retained considerable sequence
similarity (43). Human -actinin-2 and -actinin-3
are 79% identical and 91% similar at the amino
acid level (9, 42).
The sarcomeres are repeating units that constitute the contractile apparatus of the muscle
fiber (myofibril) and are comprised of actin-containing thin filaments and thick filaments containing myosin (19). The thin filaments are
anchored to electron-dense bands known as Zlines, in perpendicular orientation to the myofibrils. The ordered alignment of the Z-lines in
adjacent myofibrils enables co-coordinated contractions between myofibrils and allows transmission of contractions to the costameres at
which the Z-line is linked to the muscle membrane. The sarcomeric -actinins are major components of the Z-line and historically have been
thought to have a primarily structural role in
skeletal muscle (10, 11). In addition to actin, they
bind to many of the Z-line-associated proteins
including myotilin, myopalladin, Z-band alternatively spliced PDZ motif protein (ZASP), filamin-,
actinin-, and telethonin-binding protein of the
Z-disc (FATZ), and titin (1, 5, 7, 27, 28, 40, 65).
The -actinins can form antiparallel dimers with
themselves or other -actinins, allowing cross
linking of actin and titin filaments from neighboring sarcomeres and are thought to play a
significant role in maintenance of the structural
integrity of the Z-line of skeletal muscle (9 11,
16, 47, 70).

1548-9213/10 2010 Int. Union Physiol. Sci./Am. Physiol. Soc.

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-Actinin-3 Deficiency is Common
in the General Population
In 1999, we described a common single-base transversion (CT) in exon 16 of the ACTN3 gene that
converts an arginine residue (R) to a stop codon (X)
at amino acid position 577 (56). Approximately
16% of the world population is completely deficient in -actinin-3 protein due to homozygosity
for the R577X stop codon (ACTN3 577XX genotype)
(48). There is variation in frequency of the R577X
allele in different ethnic groups, with allele frequencies of 0.55 in Europeans, 0.52 in Asian populations, and 0.09 in Africans (49). -Actinin-3
deficiency does not result in muscle disease, suggesting that it is not essential for baseline muscle
function, and that the closely related isoform,
-actinin-2, can at least partially compensate for
its absence at the Z-line in fast muscle fibers. However, ACTN3 has been highly conserved during
vertebrate evolution, suggesting that the sarcomeric -actinins are not completely functionally
redundant and that ACTN3 has evolved to perform
specific functions in fast fibers (47, 50).
We have studied genetic variation around the
ACTN3 R577X polymorphism across a wide range of
populations using DNA available through the International HapMap project (48). Low levels of genetic
variation and an unusually long region of complete
homozygosity in the region surrounding the 577X
allele suggest a recent and rapid expansion in the
frequency of this allele amongst Eurasian population
due to positive selection. Thus, although ACTN3 appears have been conserved through early evolution(300 mya), there is now recent positive selection
(1533 thousand years ago) favoring the nonfunctional ACTN3 allele. This suggests that both states,
-actinin-3 deficiency and -actinin-3 expression,
may confer benefits to muscle function that have
been acted on through natural selection.

-Actinin-3 Deficiency Improves

Human Sprint Performance
In 2003 in collaboration with the Australian Institute of Sport, we showed that ACTN3 genotype is
strongly associated with elite athlete status (83).
There is a striking and highly significant reduction
in the frequency of -actinin-3-deficient individuals among sprint/power athletes compared with
controls (FIGURE 1). The association between the
ACTN3 genotype and sprint performance has been
replicated in a number of studies in populations of
varied ethnicity, including European, American,
and Israeli athletes (1, 25, 55, 63, 66). A meta-analysis
of existing published data has given a P value of
0.5 1011 of the effect of ACTN3 genotype on
sprint performance (46). Although -actinin-3

deficiency is associated with poorer muscle strength

and sprint performance, loss of -actinin-3 appears to
be beneficial in certain circumstances, with the frequency of the XX-null genotype higher in endurance
athletes than in controls in some studies (26, 83).
Similar ACTN3 genotype associations have also
been demonstrated in nonathletes, with deficiency of
-actinin-3 associated with significantly slower 40-m
sprint times in Greek adolescent males (51), lower
isometric maximal voluntary muscle contractions
(20), and lower knee extensor shortening and lengthening peak torques in untrained adult women and
men (20, 51, 77, 78). In summary, the large number of
human studies that have been performed to date
show that the ACTN3 R577X polymorphism represents an important genetic factor associated with
variations in muscle performance in humans, with
the presence of -actinin-3 associated with improved
sprint and power performance.

Understanding the Role of

-Actinin-3 in Muscle Performance:
The Actn3 KO Mouse
To better understand the mechanisms underlying
the effect of -actinin-3 on skeletal muscle performance and the factors that might contribute toward positive selection for the X allele during
recent human evolution, we generated a knockout
mouse (Actn3KO) completely deficient for -actinin-3 at the protein and mRNA level (49). Similar to
humans, wild-type (WT) mice express -actinin-3
predominantly in fast fibers. Unlike humans, -actinin-2 is usually expressed predominantly in type
1 and type IIa fibers in postnatal mouse muscle. In
the Actn3 KO, -actinin-2 is upregulated and expressed in all fibers, mimicking the pattern of expression seen in ACTN3 577XX humans. Like humans,
-actinin-3 deficiency in the Actn3 KO mouse does
not result in overt muscle disease. The Actn3 KO
mice appear normal and have similar activity levels
to WT mice on open-field testing (49).

-Actinin-3 Deficiency Reduces

Muscle Mass
Actn3 KO mice weigh slightly less than WT mice,
with lower muscle mass seen in all muscles normally expressing -actinin-3 (48). The heart and
slow-twitch soleus muscle (located in the lower
hindlimb underlying the gastrocnemius) do not
normally express -actinin-3 and so provide an
internal negative control. There was no difference
in the size of the heart between WT and Actn3 KO
mice. Unlike the other muscles analyzed, we saw a
trend toward an increase in size in the soleus. This
may reflect hypertrophy of the soleus to compensate for reduced strength in the surrounding muscles. The increase in size of the soleus also argues

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against an overall runt effect among the Actn3
KOs. Rather, it suggests that the reduction in muscle mass in the presence of -actinin-3 deficiency
is specific to the muscles in which it is normally
expressed. As further evidence of a role for -actinin-3 in muscle size, -actinin-3 deficiency has
also been associated with reduced muscle mass in
Japanese and American women (24, 77, 78, 86).
The reduction in muscle mass that we see in the
Actn3KO appears to be due to a reduced diameter
of the type 2B, fast glycolytic fibers that normally
express -actinin-3. We see no change in the number of muscle fibers or significant alteration in fiber
type as defined by myosin heavy chain isoform.
Rather, we see that the type 2B muscle fibers have
a cross-sectional area that is 34% smaller than the
2B fibers found in WT littermates. Similarly, actinin-3
deficiency has been shown to reduce the total muscle
cross-sectional area occupied by fast, glycolytic (type
2X) fibers in ACTN3 577XX humans (77).

-Actinin-3 Deficiency Reduces

Muscle Strength
Grip strength is significantly lower in Actn3 KO
mice (6 7%) compared with WT mice, although

still within the normal range overall (48). This confirms that the Actn3KO mice are modeling normal
variation rather than weakness as a manifestation
of muscle disease (FIGURE 1). Human studies have
also shown reduced muscle strength in XX individuals. In a group of elite male road cyclists (n 46),
individuals with XX-genotypes were found to have
less peak power output and less power to tolerate
high submaximal workloads compared with RR genotypes (35). Reduced peak torque values were
also seen among XX women in a large cohort of
women across a broad span of age range (848
women aged 2290 yr) (78).

-Actinin-3 Deficiency Results in Improved

Endurance Capacity
Intriguingly, we found that Actn3 KO mice have an
increased capacity to run longer distances (FIGURE 1)
(49). Using a modified intrinsic exercise test where mice
are run to exhaustion, Actn3 KO mice were able to run
on average 33% further than WT mice. This data is
consistent with the findings of our original human
association study in which we found a trend toward an increase in the frequency of XX individuals
among endurance athletes, reaching significance

FIGURE 1. -Actinin-3 is associated with altered muscle performance

A: ACTN3 R577X genotype frequencies in controls and elite sprint and endurance athletes. The frequency of the 577XX
(-actinin-3 deficient) genotype is significantly lower in the total power athlete group (6%) than in controls (18%) and
significantly higher in female endurance athletes (29%) than in female controls. B: Actn3 KO mice have improved endurance performance. Actn3 KO mice run farther before exhaustion in an intrinsic exercise capacity test. C: Actn3 KO mice
display reduced grip strength compared with wild-type (WT) mice. D: Actn3 KO mice have reduced muscle mass. Mean
muscle mass of triceps (TRIC), tibialis anterior (TA), gastrocnemius (GST), quadriceps (QUAD), and spinalis thoracis (SPN)
excised from male 8-wk-old mice. Values are means 95% CI. Significant difference: *P 0.05; **P 0.01; ***P
0.001. Figure was adapted from Refs. 48, 49, 83.
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among female athletes. This association was also
significant when road cycling athletes were analyzed separately and in a study of Israeli elite athletes (26, 83). However, other studies have not
replicated the association between ACTN3 genotype and elite endurance performance. This suggests that the association between -actinin-3
deficiency and endurance is not as strong as its
association with reduced performance in sprint
and power activities (2, 45, 53, 55, 59, 67, 84).
It is interesting to speculate why it is that improved endurance is apparent in our Actn3 KO
mice, when some human studies have not demonstrated a significant effect in XX individuals. Not
surprisingly, there are significant differences in
muscle metabolism and contractile properties between mice and humans. Mice have a greater disparity in metabolism between slow and fast muscle
fiber types. In particular, fast fibers in mice display
a greater shift toward glycolytic metabolism than
humans, and mouse muscle has a much higher
proporation of fast, glycolytic fibers (1). Therefore,
in addition to overcoming and environmental and
genetic variability inherent in human studies, the
effect of -actinin-3 on glycolytic muscle metabolism may also be magnified in the mouse model.

-Actinin-3 Deficiency Results in Increased

Activity of Mitochondrial Enzymes
The most striking phenotype in the Actn3 KO
mouse is a metabolic shift in the characteristics of
fast muscle fibers toward those of slow oxidative
fibers (48). At a histochemical level, there is increased intensity for a number of mitochondrialassociated enzyme stains in KO mouse muscle;
NADH-tetrazolium reductase (a reductase that is
present in both mitochondria and the sarcoplasmic reticulum) and succinate dehydrogenase
(SDH), which is associated with the tricarboxylic
acid cycle (49). Immunoblotting also shows increased levels of porin (found in the outer mitochondrial membrane), and cytochrome c oxidase
(CCO), one of the rate-limiting steps of oxidative
phosphorylation in the mitochondria. At an enzyme level, CCO shows activity levels that are 25
39% higher in Actn3 KO mouse muscle compared
with WT (FIGURE 2).
In addition to SDH, another key pacemaking
enzyme in the tricarboxylic acid (TCA) cycle, citrate synthase is also elevated in the Actn3KO48.
The TCA cycle lies at the hub of energy pathways
for utilization of carbohydrates, fats, and proteins.
Once glucose has been converted to pyruvate by
the process of glycolysis, it can pass into the mitochondria where it is converted into acetyl-CoA by
decarboxylation and can enter the TCA cycle. The
TCA cycle does not utilize oxygen directly, so it

depends on subsequent passage through oxidative

phosphorylation.
The activity of two mitochondrial enzymes involved in fatty acid oxidation, (hydroxyacyl-CoA
dehydrogenase and medium chain acyl-CoA dehydrogenase) are elevated in the Actn3 KO mouse
(48). For fat to be metabolized, triglycerides are
hydrolyzed to fatty acids and glycerol. Fatty acids
can be broken down through beta oxidation and
may then be delivered to the TCA cycle as acetyl
CoA or succinyl CoA. At a functional level, the
Actn3KO mouse has an increased capacity to
oxidize fats (palmitate oxidation; Quinlan K, unpublished observations).
Quantification of mitochondrial DNA copy number is unaltered in Actn3 KO mouse muscle (48),
suggesting that the increase in activity of enzymes
located in the mitcohondria is due to increased
mitochondrial activity rather than increased mitochondrial number. Consistent with this, there is no
increase in another mitochondrial enzyme, glutamate dehydrogenase, which is involved in nitrogen
and glutamate metabolism, in the Actn3 KO
mouse. In addition, the levels of PGC1, a known
marker of increased mitochondrial biogenesis, are
similar in WT and Actn3 KO mice.

-Actinin-3 Deficiency Results in

Increased Glycolysis
Hexokinase, the enzyme that catalyses the first step
in glycolysis is significantly upregulated in the Actn3
KO mouse muscle, as is glyceraldehyde-6-phosphate
dehydrogenase (GAPDH). Glycolytic metabolism is
coupled to mitochondrial oxidative metabolism in
the presence of oxygen but can also generate ATP in
its absence. GAPDH is involved in the sixth step of
glycolysis and is typically highly expressed in all
tissues leading to its frequent use as a house-keeping control gene for Western blotting and RT-PCR.
Interestingly, we saw no detectable change in the
activity of phosphofructokinase, a key regulatory
and rate-limiting step in glycolysis.
Despite the striking differences we see in muscle
metabolism properties we see in Actn3KO muscle,
we have not seen changes in muscle fiber-type
proportions, suggesting that the shift in metabolism in the Actn3KO is not due to an increased
proportion of oxidative, type 1, or type 2A fibers.

-Actinin-3 Deficiency Results in Increased

Glycogen Content in Muscle
Using three methods [periodic acid-Schiff (PAS)
staining, glycogen assays, and dual-axis electron
microscopic tomography], we have demonstrated
that muscle glycogen content is significantly increased in Actn3 KO mice (62). By microscopic
tomography, glycogen volume as a percentage of
the cytoplasmic volume was 1.9% in Actn3 KO

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mice compared with 1.0% in WT. In ACTN3 577XX
humans, there is also an increase in glycogen compared with 577RR and 577RX individuals, who express -actinin-3.
Glycogen metabolism is the key pathway for energy production during high-intensity activity, and
depletion of glycogen results in muscle fatigue.
Glycogen metabolism is controlled by complex
feedback mechanisms (38). Glycogen synthesis is
controlled by the delivery of glucose to the cell
(glucose transport) and the enzyme glycogen synthase. Glycogen utilization (glycogenolysis) is catalyzed by glycogen phosphorylase.
Glycogen synthase and glycogen synthase activity levels are increased (by 100% and 50% compared with WT) in Actn3KO mouse muscle (62).
However, when corrected for total glycogen synthase levels, the percentage activity of glycogen
synthase is not increased. Glycogen synthase activity is regulated by a number of factors including
phosphorylation, activation by glucose 6-phosphate, insulin, and exercise. Existing evidence suggests that, when glycogen content is high, strong
feedback decreases glycogen synthase activity, making glycogen synthase the rate-limiting step in glycogenesis (38). Elevation of total glycogen synthase and

FIGURE 2. Actn3 KO mice display a shift toward more oxidative pathways

of metabolism
Enzyme analyses show increased expression of enzymes involved in the glycolysis pathway (PFK, HK, and GAPDH) in the Actn3 KO mice. The anaerobic conversion of pyruvate to lactate by lactate dehydrogenase (LDH) is reduced, whereas the activity of
enzymes involved in mitochondrial oxidative metabolism [citrate synthase (CS), succinate dehydrogenase (SDH), cytochrome c oxidase (CCO)], and fatty acid oxidation
[3-hydroxyacyl-CoA dehydrogenase (BHAD), and medium chain acyl-CoA dehydrogenase (MCAD)] are increased in Actn3 KO mice. Glycogen content is increased in Actn3
KO mice, and glycogen phosphorylase activity is decreased. Hexokinase activity (HK) is
also increased in Actn3Kos, whereas glucose-6-phosphate (G6P) and Phsosphofructokinase (PFK) are not. NADH tetrazoleum reductase (NADH-TR) levels are increased in
Actn3KOs by staining.
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active glycogen synthase in the presence of elevated glycogen in the Actn3 KO mouse suggests
there may be some additional feedback mechanism in the presence of -actinin-3 deficiency that
combats the expected reduction in glycogen synthase activity in this state.

-Actinin-3 Deficiency Results in Reduced

Glycogen Phosphorylase Activity in Muscle
The key enzyme in glycogenolysis, glycogen phosphorylase (GPh), is significantly reduced in Actn3
KO mouse muscle (62). Enzyme quantitation
shows activity in the Actn3 KO mouse muscle is
26% compared with 53% in WT mice. An interaction between glycogen phosphorylase and sarcomeric -actinins has previously been reported (18).
By confocal microscopy, we have also shown that
-actinin-3 and glycogen phosphorylase colocalize
at the Z-line.
A reduced capacity to break down glycogen for
energy would likely be disadvantageous to sprint
athletes, who rely on endogenous fuels such as
muscle glycogen to rapidly produce energy for
contraction. Reduced availability of glucose may,
in turn, result in a compensatory shift toward aerobic metabolism, as observed in Actn3 KO mice.
Such changes could be advantageous to endurance
athletes, allowing them to preferentially use other
fuels such as fatty acids for energy generation.
At the electron microscopic (EM) level, Actn3 KO
muscle type 2B fibers contain concentric ring-like
structures surrounded by and filled by glycogen
particles (FIGURE 3C). These structures also stain
with antibodies to glycogen phosphorylase and Zline proteins desmin and myotilin (62) (FIGURE 4).
Glycogen is typically stored near the contractile
apparatus of muscle. However, when glycogen
stores located at the contractile apparatus are filled
up, further glycogen synthesis can occur in other
regions of the cell (54). The glycogen accumulations that we see in Actn3 KO mouse muscle may
reflect increased glycogen storage and loosening of
the association between glycogen and the myofibril. Alternately, -actinin-3 deficiency may destabilize complexes usually reliant on -actinin-3
homodimers or heterodimers for structural integrity, leading to displacement of glycogen from its
usual location within the muscle fiber (43).
Utilizing global proteomic analysis, we found
differential expression of phosphorylated forms of
GPh in Actn3 KO muscle compared with WT (62).
Since phosphorylation is one of the methods by
which GPh activity is regulated, we hypothesise
that -actinin-3 plays a role in regulation of GPh
activity by altering its posttranslational phosphorylation and that -actinin-3 deficiency results in
decreased activity of GPh due to altered phosphorylation. The reduction in GPh activity may explain

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the observed increase in muscle glycogen content
and decrease the capacity of muscle to use glycogen as a fuel. This, in turn, could explain the switch
in preferred fuel source, from anaerobic metabolism toward more oxidative metabolism as seen in
Actn3 KO mice.

-Actinin-3 Deficiency:
A Pretrained State for
Improved Endurance and Poorer
Sprint Performance?
The improved endurance performance in Actn3
KO mice and in ACTN3 577XX humans, and the
shift in muscle metabolism toward a slow oxidative
phenotype with increased glycogen content in
Actn3 KO mice are all consistent with -actinin-3deficient muscle being pretrained for endurance
performance. It has long been argued that there is
an evolutionary trade-off between sprint and endurance performance, as well as a functional
trade-off between the effects of endurance and
resistance training (34). Sprint and resistance
training utilize exercise of short duration and high
intensity, resulting in muscle hypertrophy, with
increased fiber cross-sectional area, protein content, and an increased ability to generate force (1,
14, 21, 73). Endurance training (in which the length

of exercise is increased and intensity is reduced)

induces a shift in skeletal muscle metabolism toward a more oxidative form of metabolism.
Oxidative metabolism produces a longer lasting
and more stable supply of ATP, making oxidative
fibers more fatigue resistant. Endurance training
results in reduced fast-fiber cross-sectional area,
increased mitochondrial mass, increased oxidative
enzymes, and reduced glycolytic enzymes (37, 60,
71, 74). Training for both strength and endurance
appears to limit the amount of strength gains an
individual can make, suggesting that endurance
training may somehow limit skeletal muscle
growth (36).
It is possible, therefore, that improved endurance capacity in the presence of -actinin-3 deficiency may result in a limitation in explosive/
power abilities. Unfortunately, there are not
adequate or reliable methods by which to test
sprint capacity in mice. However, the presence of
reduced grip strength in Actn3 KO mice compared
with WT does suggest a reduction in explosive
power.
Exercise training improves utilization of fat as an
energy source and reduces the rate at which glycogen is utilized, thereby delaying glycogen depletion. Similar to our Actn3 KO mouse, exercise
training results in higher glycogen stores. Also

FIGURE 3. Actn3 KO mice have increased glycogen content and reduced glycogen phosphorylase activity in skeletal muscle
A: representative PAS staining images of male 8-wk-old mouse quadriceps muscle cross sections demonstrate glycogen levels are higher in Actn3
KO muscle compared with WT. B: glycogen assays on lower hind leg muscles tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL),
and gastrocnemius (GST), quadriceps (QUAD), and spinalis thoracis (SPN) from female 8-wk-old mice. C: glycogen synthase and glycogen synthase
activity are increased in KO muscle, but percentage activity of glycogen synthase is not increased. Glycogen phosphorylase activity and percentage
activity are reduced in the Actn3KO. Values are means 95% CI. Significant difference: *P 0.05; **P 0.01; ***P 0.001. Figure adapted from
Ref. 62.

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similar to our Actn3 KO mice, endurance training
in humans has been shown to result in increased
hexokinase activity and reduced lactate dehydrogenase in muscle (6, 41, 52, 69). A reduction in LDH
can result in poorer sprint performance since LDH
is needed to convert pyruvate, the final product of
glycolysis, to lactate when oxygen is absent or in
short supply.

How Does -Actinin-3 Alter

Skeletal Muscle Metabolism?
Historically, the sarcomeric -actinins have been
considered primarily structural proteins, but we
have mounting evidence that the principle role of
-actinin-3 is an effect on muscle metabolism and
that it has evolved specialized expression in fast

FIGURE 4. Actn3KOs have intramuscular accumulations of

glycogen that stain for GPh and MYOT
Antibodies to myotilin (MYOT; top) and GPh (second panel) used on a single muscle section demonstrate that these proteins co-localise in cytoplasmic inclusions in the KO muscle. Electron microscopy (EM) shows
concentric ring-like structures in Actn3 KO highlighted with black asterisks.
An asterisk has been used on each of the WT and KO images to allow individual fibers to be readily compared between images. Scale bars are 50
m wide. Images are from Ref. 62 and used with the permission of Hum
Mol Genet.
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muscle fibers because of an important role in the

regulation of energy metabolism.
We have shown that -actinin-3 colocalizes with
and increases glycogen phosphorylase activity, but
the precise molecular mechanisms involved are yet
to be determined. There is strong evidence to suggest that glycogen levels play a role in regulating
how fuel is utilized in muscle. Increased muscle
glycogen has been shown to increase carbohydrate
oxidation during exercise (4, 12, 22, 79). When glycogen is depleted, skeletal muscle may also oxidize
free fatty acids to produce ATP and preserve muscle glycogen (61). If muscle glycogen is low before
exercise, there is a shift toward decreased carbohydrate oxidation and increased lipid oxidation.
Patients with McArdles disease lack functional
glycogen phosphorylase in muscle and cannot
break down glycogen stores. These patients suffer
from exercise intolerance and a shift toward lipid
utilization for fuel (44). Interestingly, the ACTN3
577XX genotype is associated with improved muscle performance in these patients (45). The mechanism by which -actinin-3 deficiency improves
exercise tolerance in these patients is unknown.
Given that most patients with McArdles disease
have no functional muscle glycogen phosphorylase
and are therefore unable to utilize glycogen stores,
it is unlikely that increased muscle glycogen would
improve exercise capacity in these patients. It is
possible that the increased fatty acid oxidation capacity seen in Actn3 KO mice could improve fuel
utilization in the absence of glycogenolysis, however, this functional link is yet to be tested.
Interestingly, sprint training also increases the
ability for rapid glycogen breakdown (glycogenolysis) during shirt bursts of maximal or submaximal
activity. It is interesting to speculate whether reduced glycogen phosphorylase activity associated
with -actinin-3 deficiency might reduce the ability to utilize glycogen during sprint activity.
We are in the process of trying to unravel the
pathway of events that lead to the metabolic phenotype in Actn3 KO mice. We have examined the
time course of appearance of the structural and
metabolic phenotypes in Actn3 KO muscle. The
reduction of glycogen phosphorylase activity,
higher muscle glycogen content, and increased
glycolytic and mitochondrial enzymes occur concurrently at 4 wk postnatally. These metabolic
changes are preceded by upregulation of -actinin-2 and interacting proteins at the Z-line, suggesting that structural alterations may lie upstream
of the metabolic changes. Since -actinin-2 and -3
and glycogen phosphorylase are colocated at the
Z-line, loss of -actinin-3 may alter the three-dimensional conformation of the Z-line, which in
turn could alter the availability of glycogen phosphorylase for phosphorylation and activation. Al-

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ternately, the structural and metabolic effects of
-actinin-3 deficiency may be due to independent
and unrelated actions of the -actinin-3 protein.
A review of the known interaction partners of the
sarcomeric -actinins provide tantalizing hints at
alternate possible mechanisms underlying the effects of -actinin-3 on metabolism. In addition to
their structural cross-linking functions at the Zline, the -sarcomeric -actinins interact with a
number of signaling molecules. -Actinin-2 and -3
interact with the calsarcin family of proteins,
which, through their interaction with calcineurin,
are involved in muscle fiber-type determination
and regulation of expression of fiber type-specific
genes (17, 27, 30, 31, 72). -Actinin-2 has been
shown to interact with membrane-bound signaling
proteins such as the NMDA glutamate receptor,
Kv1.4 and Kv1.5 potassium channels, and cardiac
L-type calcium channels (23, 64, 80, 81).
The -actinins also bind to the soluble signaling
factors phosphoinositol 3-kinase (PI3K) and phosphoinositol-4,5-bisphosphate (PIP2), and G-protein-coupled receptor kinase (29, 32, 68). PIP2 acts
as a substrate for enzymes as well as promoting the
recruitment of proteins to the plasma membrane and
subsequent activation of signaling cascades. In the
presence of -actinin-3 deficiency, any alteration of
total -actinin levels or differential binding between
-actinin-2 and -actinin-3 could affect regulation of
one or many of these important signaling pathways.

Conclusion
Over one billion people worldwide are deficient in
-actinin-3, and there is increasing evidence to
suggest that ACTN3 genotype is an important genetic variant that influences the metabolic function of human muscle. -Actinin-3 deficiency
results in a fundamental shift in metabolism away
from the anaerobic pathway toward the oxidative
pathways of muscle metabolism, which provides an
explanation for the association between -actinin-3 deficiency, poorer sprint and power performance, and
enhanced endurance performance. The increase in
metabolic efficiency of -actinin-3-deficient muscle
could also provide an explanation for the adaptive benefit of the 577X allele during recent human evolution.
The next challenge will be to dissect the molecular
mechanisms underlying this metabolic phenotype and
explore whether ACTN3 genotype influences glucose
homeostasis and adaptive responses to diet in the
modern world.

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