Download Transforming growth factor-β and myostatin signaling in skeletal

J Appl Physiol 104: 579–587, 2008;
doi:10.1152/japplphysiol.01091.2007.
Invited Review
Transforming growth factor-␤ and myostatin signaling in skeletal muscle
Helen D. Kollias1 and John C. McDermott2
1
Department of Neurology, Johns Hopkins Hospital, Baltimore, Maryland; and 2Department of Biology, York University,
Toronto, Ontario, Canada
myogenesis; Smad; MyoD
SKELETAL MUSCLE has a remarkable morphogenic ability to
regenerate and adapt to environmental stimuli. Fundamentally, skeletal muscle regeneration is the recapitulation of
skeletal muscle development in a postnatal context. Thus an
understanding of the regulation of development and regeneration are mutually reinforcing. Both regeneration and
development of skeletal muscle are profoundly sensitive to
the extracellular milieu. In particular, signal transduction
pathways initiated by growth factors such as insulin-like
growth factor (IGF-1), fibroblast growth factor (FGF), and
transforming growth factor-␤ (TGF-␤) exert a potent level
of control over muscle gene expression. The role of the
TGF-␤ superfamily of growth factors is of considerable
interest since many facets of muscle ontogeny and physiology are regulated by some of the ligands belonging to this
cytokine superfamily. In particular, myostatin is of growing
interest since it has been shown to exert profound effects on
muscularity in mice, cattle, and humans (see Fig. 1; Refs.
52, 54, 68), as well as other animals. TGF-␤ superfamily
signaling relies on a relatively parsimonious canonical pathway that interdigitates with particular aspects of the host
cell machinery through cell-specific protein:protein interactions that allow precise cell context-dependent responses to
the ligand. This review will focus on salient aspects of
TGF-␤ superfamily signaling that pertain specifically to
skeletal muscle. Information from a variety of studies offering insights into the molecular events of TGF-␤ signal
transduction, the function of TGF-␤ in cultured muscle
cells, and the effects of TGF-␤ in postnatal skeletal muscle
physiology, will provide the foundation of this review.
Address for reprint requests and other correspondence: J. McDermott, 327
Farquharson, 4700 Keele St., Toronto, Ontario, Canada M3J 1P3 (e-mail:
[email protected]).
http://www. jap.org
AN OVERVIEW OF TGF-␤ SIGNALING
TGF-␤1 ligand is recognized as the prototype of a class of
multifunctional growth factors that regulate key events of
metazoan development, disease, and repair (44, 45). In 1981,
TGF-␤ effects were first reported by Roberts and colleagues
(65), who found that TGF-␤ induced rat kidney fibroblasts to
proliferate. Since then, TGF-␤ has been found to have a
plethora of effects, such as regulation of cell growth, proliferation, differentiation, adhesion, migration, and apoptosis (reviewed in Refs. 44, 46, 48). These effects are highly complex
and sometimes difficult to reconcile; however, one unifying
feature of TGF-␤ signaling is that its effects are profoundly
dependent on the cell context. For example, TGF-␤ causes
increased proliferation in fibroblasts (65) but inhibits proliferation in epithelial cells, causes differentiation of neuronal cells,
but blocks differentiation in mesenchymal cells (2, 18, 19, 66,
73). In skeletal muscle, TGF-␤ superfamily members have
been shown to have potent effects on both muscle development
and postnatal skeletal muscle mass.
The TGF-␤ superfamily consists of over 50 structurally
related ligands, many of which fall into three major subfamilies: TGF-␤, bone morphogenic protein (BMP), and activin
(reviewed in Ref. 17). Exhaustive work on the molecular
characterization of the TGF-␤ signal transduction pathway has
led to the delineation of a canonical signaling pathway for all
TGF-␤ superfamily members consisting of three main components (74): 1) the ligand; 2) the receptors (serine/threonine
kinases); and 3) the intracellular mediators (Smads; see Fig. 2).
For the TGF-␤, Activin, and BMP subfamilies signal transduction begins with the ligand binding to its type II receptor. The
type II receptor associates with its corresponding type I receptor, forming an activated heterotetrameric receptor complex,
which transphosphorylates the type I receptor activating the
latent kinase activity of the receptor complex. The activated
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Kollias HD, McDermott JC. Transforming growth factor-␤ and myostatin
signaling in skeletal muscle. J Appl Physiol 104: 579 –587, 2008;
doi:10.1152/japplphysiol.01091.2007.—The superfamily of transforming
growth factor-␤ (TGF-␤) cytokines has been shown to have profound effects on
cellular proliferation, differentiation, and growth. Recently, there have been major
advances in our understanding of the signaling pathway(s) conveying TGF-␤
signals to the nucleus to ultimately control gene expression. One tissue that is
potently influenced by TGF-␤ superfamily signaling is skeletal muscle. Skeletal
muscle ontogeny and postnatal physiology have proven to be exquisitely sensitive
to the TGF-␤ superfamily cytokine milieu in various animal systems from mice to
humans. Recently, major strides have been made in understanding the role of
TGF-␤ and its closely related family member, myostatin, in these processes. In this
overview, we will review recent advances in our understanding of the TGF-␤ and
myostatin signaling pathways and, in particular, focus on the implications of this
signaling pathway for skeletal muscle development, physiology, and pathology.
Invited Review
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TGF-␤ AND MYOSTATIN SIGNALING IN SKELETAL MUSCLE
Fig. 1. Phenotype of mysotatin null vertebrates.A: upper forelimb muscles of wild-type mouse; B: upper forelimb of mouse that is myostatin null (52); C: bovine
with myostatin mutation (54); D and E: human with myostatin mutation as a neonate (D) and at 7 mo of age (E; Ref. 68). Copyright permissions: copyright 1997
National Academy of Sciences USA (A and B); reprinted with permission from Macmillan Publishers (C): Nature; copyright 1997 (D) and 2004 (E)
Massachusetts Medical Society. All rights reserved.
SKELETAL MUSCLE DEVELOPMENT AND REGENERATION
Briefly, skeletal muscle ontogeny is comprised of three
developmental stages: 1) determination, 2) differentiation, and
3) maturation (Fig. 3). Determination begins with the aggre-
gation of mesodermal precursors in the paraxial region of the
embryo, which form somites. The somites are composed of
pluripotent cells, of which some will become muscle progenitor cells (33). These myogenic progenitor cells give rise to
proliferating cells that are committed to the myogenic lineage
termed myoblasts and are derived from the myotome compartment of the somite (33). Proliferating myoblasts subsequently
undergo differentiation, a process that includes withdrawal
from the cell cycle, expression of muscle-specific genes, and
fusion resulting in the formation of multinucleated myotubes.
Myotubes eventually give rise to the vast array of muscle fibers
that are used to construct the complex skeletal muscle architecture of the animal. There are two types of myoblasts,
embryonic and fetal. Most embryonic myoblasts differentiate
Fig. 2. Overview of the canonical transforming
growth factor (TGF)-␤ pathway. The TGF superfamily member (ligand) binds to the type II and
type I receptors. The type II receptor then
transphosphorylates and activates the type I receptor. The type I receptor phosphorylates receptorbound Smads (R-Smads), allowing them to interact with Smad4, and this complex translocates
into the nucleus. The ternary Smad complex (RSmads and Smad4) then contributes to higher
order complexes with transcription factors and
cofactors that regulate target gene transcriptions.
Adapted from Ref. 44.
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receptor complex then phosphorylates a receptor-regulated
Smad protein that oligomerizes with a common Smad or
co-Smad termed Smad4. The Smad oligomer translocates into
the nucleus where it interacts with Smad binding partners to
regulate transcription (27). These Smad binding partners regulate, in a cell type-specific manner, subsets of genes that are
target genes of TGF-␤ signaling. Another group of Smads,
inhibitor Smads (Smad6 and Smad7) serve to abrogate TGF-␤
signaling by establishing an autoinhibitory feedback loop.
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Fig. 3. Schematic diagram depicting the
states of skeletal muscle development in the
mouse. Stages of skeletal muscle development: 1) determination; 2) differentiation
(embryonic and fetal); and 3) maturation.
Determination ends with the formation of the
myotome from the dermomyotome. Embryonic myoblasts differentiate and fuse to form
primary muscle fibers. Fetal myoblasts then
undergo differentiation to form secondary
fibers that surround the primary fibers (13,
33). Dpc, days postconception in the mouse.
ROLE OF TGF-␤ AND MYOSTATIN IN SKELETAL
MUSCLE DEVELOPMENT
Since it would be untenable to consider all TGF-␤ ligands,
we will focus on the protypical TGF-␤1 and myostatin ligands
because of their potent role in skeletal muscle (35, 37, 38, 47).
TGF-␤1 and myostatin signaling in skeletal muscle involve two
strikingly similar pathways, which mediate some overlapping
and also mutually exclusive effects. TGF-␤1 plays an important role in skeletal muscle development but also influences
different tissues producing a plethora of outcomes. TGF-␤1
activity is very specific to the cellular context, that is, its
signaling outcome is determined by the intracellular milieu of
the cell. This duplicitous nature of TGF-␤1 proves to be
necessary for its in vivo function since it regulates many tissues
and processes. Conversely, myostatin plays a much more
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restricted role in skeletal muscle development, muscle mass
regulation, and adipose tissue. The elucidation of the role of
TGF-␤1 in skeletal muscle began in experiments using cultured
muscle cells in vitro, while the understanding of the role of
myostatin began in vivo through gene targeting experiments in
mice. These two different starting points led to different
insights into the role of these interrelated pathways. TGF-␤1 is
the archetypal cytokine of this superfamily and much of the
understanding of the components of the TGF-␤ signaling
pathway have been delineated by the study of TGF-␤1. Conversely, because of the pervasive hypermuscular phenotype of
myostatin null mice, myostatin research has focused more on
clinical applications in skeletal muscle and less on the detailed
biochemical dissection of its cognate signaling pathway.
In the late 1980s, several groups made three key observations that enabled the identification of an important in vivo role
for TGF-␤ in skeletal muscle development. First, TGF-␤ is
present in large amounts in the limb bud, is produced in the
ectoderm, and affects the adjacent mesenchymal cells. Second,
TGF-␤ inhibits differentiation of fetal, but not embryonic,
myoblasts (80). Last, the treatment of limb bud organ cultures
with TGF-␤ neutralizing antibodies results in the premature
appearance of large myotubes (secondary; Ref. 14). These
observations suggest that TGF-␤ prevents premature differentiation in migrating myoblasts, allowing for proper muscle
formation in the developing embryonic limb.
Myostatin was discovered during a screen for novel mammalian members of the TGF-␤ superfamily (54). Expression of
myostatin mRNA is first seen at embryonic day 9.5 post
conception in the myotome of developing somites and continues throughout muscle development. The myostatin gene is
expressed in adult skeletal muscle, heart, and adipose tissue
(69). In addition to its restricted pattern of expression, mice
homozygous null for the myostatin gene are two to three times
larger in mass than the wild type due to an enhanced mass of
the musculature (Fig. 1, A and B). The increase in skeletal
muscle mass is attributed to both hyperplasia (increase in
muscle fiber number) and hypertrophy (increase in muscle fiber
size). Myostatin homozygous null mice also have reduced
stores of adipose tissue (53, 54). Furthermore, heterozygous
(⫹/⫺) myostatin mice have a 25% increase in body weight,
which is a lesser effect than that seen in the homozygous
(⫺/⫺) mice, suggesting a dose-dependent effect of myostatin
protein levels on skeletal muscle mass. Naturally occurring
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and fuse to form primary muscle fibers, but some continue to
proliferate and become fetal myoblasts. These fetal myoblasts
form secondary fibers, smaller than the primary fibers, which
surround the primary fibers (13). Fetal myogenesis is more
similar to postnatal muscle regeneration than embryonic myogenesis. Postnatally, myotubes mature into muscle fibers. In
the adult, skeletal muscle regeneration begins with quiescent
satellite cells, located between the basal lamina and the sarcolemma of the muscle fiber, that become activated by trauma, be
it mechanical or biological (e.g., disease). Recent work has
classified satellite cells as a heterogeneous population of cells
comprised of stem cells and committed progenitors based on
the combined Pax7 and Myf5 status of the cells (32). Once
activated, satellite cells proliferate, undergo differentiation,
and then fuse to an existing muscle fiber or alternately create
fibers de novo (see review, Refs. 9, 26).
Two families of transcriptional regulatory proteins are critical in regulating developmental myogenesis: the muscle regulatory factors (MRFs) and the myocyte enhancer factor 2
(MEF2) proteins. MRF members are considered the prototypic
master regulatory genes in skeletal muscle based on their
capacity to convert some nonmyogenic fibroblast cells into a
myogenic cell phenotype (61). MEF2 family members function
as necessary coactivators for the MRFs in an evolutionarily
conserved ancient code to specify skeletal muscle ontogeny
(7). Both MRFs and MEF2 members have functional ciselements within many skeletal muscle-specific promoters and
enhancers (7).
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TGF-␤ SIGNALING IN SKELETAL MUSCLE
TGF-␤ represses skeletal muscle specific gene expression
and has also been reported to modulate proliferation in satellite
cells (3, 12, 19, 24, 47, 59). Taken together, these studies
indicate that TGF-␤ reprograms gene expression in muscle
cells resulting in an alteration of proliferative control and a
potent inhibition of the program of gene expression underlying
myogenic differentiation. As key orchestrators of muscle gene
expression, the MRFs were shown to be targeted by TGF-␤
signaling. Therefore, logical targets of TGF-␤1 are the myogenic transcription factors, the MRFs and the MEF2s, as the
quintessential regulators of myogenesis. The MRFs are comprised of MyoD, Myf5, myogenin, and MRF4, whereas the
MEF2s include MEF2A-D. To that end, Brennan and colJ Appl Physiol • VOL
leagues (8) showed that TGF-␤ inhibited the transcriptional
activity of myogenin without affecting its DNA binding affinity. Furthermore, it has been shown that TGF-␤1 targets the
basic helix-loop-helix (bHLH) region of all MRFs, decreasing
their DNA transcriptional activity without affecting their binding properties (43). Not only does TGF-␤1 inhibit the transcriptional activity of the MyoD protein it also inhibits the
transcription of the MyoD gene, thus reducing both levels and
activity (76).
How does TGF-␤1 inhibit myogenesis? What role do Smad2
and Smad3 have in myogenesis? In recent years several studies
have begun to elucidate the role of Smad2 and Smad3 during
TGF-␤1-mediated myogenic inhibition and in “normal” differentiation (39, 62). It has been shown that Smad3 is the key
mediator of TGF-␤ inhibition of myogenesis. Liu and colleagues (39) showed that Smad3 alone, and not Smad2, via
MyoD inhibition is critical in TGF-␤ signaling in myoblasts.
Smad3 was found to physically interact with MyoD, inhibiting
the transactivation properties of MyoD. Furthermore, Smad3
was also shown to physically interact with MEF2C and decrease its transcriptional activity (40). However, the relevance
of this biochemical interaction in vivo is controversial because
the MEF2C gene is not expressed in myoblasts endogenously
(50), although MEF2C does play a role in the late stages of
differentiation and conditional deletion in skeletal muscle
affects sarcomere integrity (60).
The role of Smad2 in TGF-␤ inhibition of myogenesis is less
clear. Using myoblasts De Angelis et al. (15) reported that MEF2,
normally found in the nucleus, translocates to the cytoplasm upon
TGF-␤ treatment. Forced expression of MEF2C rescued TGF-␤
inhibition by maintaining a pool of MEF2C in the nucleus.
Furthermore, endogenous Smad2 and MEF2 are complexed
together in the nuclei of differentiating myotubes, but not in
myoblasts, where Smad2 is cytoplasmic and where MEF2A
and 2C are not highly expressed (62). Thus the link between
MEF2 and Smad2 protein is complex and may depend on the
differentiation status of the cells. Together, these data suggest
that Smad2 is an important component of TGF-␤-mediated
repression of differentiation in myoblasts, although the precise
mechanism mediating this effect still requires further characterization (60).
TGF-␤ in skeletal muscle: a role in vivo. Recently, an in
vivo role for TGF-␤1 in skeletal muscle regeneration was
confirmed. Mice deficient in fibrillin-1 have increased TGF-␤
signaling activity that causes a failure of skeletal muscle to
regenerate (11). These mice are used as a model for the Marfan
syndrome (MFS) in humans, which is caused by a mutation of
the FBN1gene that encodes fibrillin-1 (an extracellular matrix
protein). MFS symptoms include bone overgrowth, ocular lens
dislocation, emphysema, cardiac complications, and an inability to increase skeletal muscle mass in response to exercise and
other physiological signals (16). Antagonizing TGF-␤ signaling with a TGF-␤ neutralizing antibody or by losartan treatment rescues skeletal muscle regeneration in the fibrillin-1deficient mice. Increased TGF-␤ signaling was also found in
the skeletal muscle of dystrophin-deficient mice, a model of
Duchenne muscular dystrophy, and antagonizing TGF-␤ signaling restored the regeneration program in these mice (11).
Furthermore, the fibrillin-1-deficient mice exhibited increased
satellite cell numbers when TGF-␤ was antagonized. Thus
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myostatin mutations in cattle (Belgian Blue, Piedmontese, and
Marchigiana) and in humans have subsequently been shown
also to lead to pronounced hypermuscularity (28, 42, 54, 68;
Fig. 1, C-E). The consistency of the hypermuscular phenotype
corresponding to myostatin null mutation across species is
consistent with the evolutionary conservation of the myostatin
gene and protein product between humans, mice, rats, pigs,
cows, chickens, and turkeys.
Since both TGF-␤1 and myostatin have been shown to
regulate myogenesis and postnatal muscle physiology, a comparative analysis of the two may provide useful insights into
the common and divergent aspects of the intracellular signaling
of these pathways. TGF-␤1 signal transduction begins with the
active ligand binding to the TGF-␤ type II receptor (T␤RII)
and either ALK-1 (activin like kinase-1) or T␤RI/ALK-5
(TGF-␤ type I receptor) receptors. Myostatin signaling begins
with the active myostatin ligand binding to either activin
receptor type IIA (ActR-IIA) or ActR-IIB and either T␤RI/
ALK-5 or ALK-4, type I receptors (63). The myostatin ligand
has a higher affinity for ActR-IIB in vitro (36, 63). Both
pathways converge in the activation of Smad2 and Smad3
followed by oligomerization with Smad4. Next, the Smad
complex translocates into the nucleus, where it regulates transcription of genes such as MyoD (39, 64). Additional nonSmad-dependent signaling has also been implicated in TGF-␤1
effects, although this is so far less well characterized (51). To
counteract TGF-␤1/myostatin signaling, repression of the signal can be achieved by the I-Smads, Smad6 and Smad7, or a
ubiquitin-mediated proteasomal degradation pathway mediated
by the Smad-ubiquitin regulatory factors (SMURFS; Ref. 5).
Smad7 has been previously shown to inhibit both TGF-␤1 and
myostatin signaling (31, 82). Furthermore, Smad7 may have
additional pro-myogenic functions. A recent study from our
group (31) has shown a dramatic enhancement of skeletal
muscle differentiation and growth by the myostatin signaling
inhibitor Smad7, which interacts with and potentiates MyoD
transcriptional activity. However, although Smad7 is able to
inhibit TGF-␤1 signaling, as assessed by a TGF-␤ responsive
promoter (3TP-lux), it was not able to rescue TGF-␤-mediated
inhibition of myogenesis. Therefore, consideration of how the
TGF-␤1/myostatin downstream effectors integrate with
skeletal muscle-specific transcriptional regulators to control
gene expression will be paramount in understanding how
these growth factors modulate the muscle phenotype during
development and regeneration.
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TGF-␤ AND MYOSTATIN SIGNALING IN SKELETAL MUSCLE
MYOSTATIN SIGNALING IN SKELETAL MUSCLE
While the relevance of TGF-␤1 in vivo has just begun to be
understood, the in vivo importance of myostatin was obvious
from the early phase of its discovery. However, characterization of the intracellular signaling pathway for myostatin is still
in its infancy. Much of what is known has been inferred by
knowledge of the TGF-␤ signaling pathway but there may still
be some surprises to unfold since studies are now identifying
differences in TGF-␤1 and myostatin signaling (31).
Myostatin: a role in vivo. Myostatin null mice (Mstn⫺/⫺)
have proved to be an excellent model for understanding the
genetic role of myostatin during both muscle development and
postnatal muscle physiology (52). In addition to alterations in
muscle mass, changes in fiber contractile properties, fiber type,
and fiber recovery have now been reported in this genetic
model (4, 55, 79).
In addition to the increased muscle mass and decreased fat in
the Mstn⫺/⫺ mice there are also corresponding increases in
strength and changes in fiber type distribution with a shift
toward the type IIb fibers. Furthermore, the contractile properties of Mstn⫺/⫺ mice differed from heterozygous (Mstn⫹/⫺)
and wild-type mice (55). In physiological studies of skeletal
muscle derived from the mice, the extensor digitorum longus
(EDL) muscle and the soleus muscle are often used as indicators of fast twitch and slow twitch muscle fibers, respectively.
In vitro contractile analysis of EDL and soleus muscles from
wild-type, Mstn⫹/⫺, and Mstn⫺/⫺ mice revealed that the maximum tetanic force (Po) in the EDL muscle and soleus muscle
was greater in the Mstn⫺/⫺ than in the wild-type animals (55).
Additionally, the specific maximum tetanic force (sPo; corrected for cross sectional area, N/mm2) was reduced in the
Mstn⫺/⫺compared with the wild-type EDL muscle. However,
in the soleus, no difference was found in the sPo of the
Mstn⫺/⫺ compared with mice with the wild-type genotype. In
the case of the EDL muscle, the phenomenon of decreased sPo
with increased Po occurs because as the whole muscle cross
sectional area increases the angle of pennation by muscle fibers
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increases, thus decreasing the transmission of force, a phenomenon previously described by Maxwell et al. (49). While the
lack of any difference in the sPo of the soleus muscle is more
difficult to explain, one possibility is that Po in the soleus
muscle of the Mstn⫺/⫺ compared with the wild type was not
large enough to affect the sPo. Interestingly, following two
lengthening contractions, the EDL muscle from the Mstn⫺/⫺
mice had a greater force deficiency than the wild type. However, no difference in force production was found in the soleus
muscle. Molecular analysis revealed that the levels of ActRIIB
in the EDL muscle were twice as high as those in the soleus
muscle suggesting that the higher levels of ActRIIB in the EDL
muscle could prime it for greater sensitivity to the ligand
compared with the soleus muscle.
While Mendias et al. (55) have reported an increase in
strength of the EDL of Mstn⫺/⫺ null mice, there is some
conflict in this area since another study reported that Mstn⫺/⫺
null mice do not have increased Po generation in their EDL
muscle compared with wild-type mice despite their larger
muscle mass (4). However, consistent with other studies, the
sPo in the myostatin-null state was reduced compared with that
of their wild-type littermates. As well, the Po was slightly
higher in the EDL muscle of the Mstn⫺/⫺ mice compared with
the wild-type mice. Further comparison of the contractile
properties of the EDL muscle from Mstn⫺/⫺ and wild-type
mice exhibited differences in single twitch characteristics.
EDL muscle from Mstn⫺/⫺ mice contracted and relaxed faster
during a single twitch (4).
A greater proportion of type IIb fibers in the Mstn⫺/⫺ mice
can explain the shorter contraction and relaxation times reported. Assessed by myosin heavy chain (MHC) isoform
expression, Mstn⫺/⫺ mice had less IIa and IIx fibers in EDL,
but had more IIb fibers than the wild-type mice (4, 21). In the
soleus muscle, Mstn⫺/⫺ mice had less IIa mRNA, but more IIx
and IIb mRNA than the wild type, although this change at the
mRNA level needs to be confirmed at the protein level.
However, functional assessment using ATPase staining and
succinate dehydrogenase (SDH) activity did confirm a shift
toward more fast fibers and glycolytic fibers, respectively (21).
The analysis of oxidative enzymes in the EDL muscle reported
a decrease in mitochondrial enzymes, SDH, cytochrome oxidase (COX), and NADH reductase, in the Mstn⫺/⫺ mice
consistent with the shift to fast glycolytic IIb fibers. Furthermore, the number of mitochondria per unit area was reduced.
Taken together these data suggest that modification of
myostatin levels does alter adult muscle contractile properties.
Reduced myostatin levels have been shown to be potentially
beneficial in aging muscle. Comparing older Mstn⫺/⫺ mice (24
mo) to older wild-type mice it was shown that Mstn⫺/⫺ mice
had less IIb fibers and size losses compared with wild-type
mice (70). Genetic removal of myostatin from conception
exhibits profound effects on muscle mass and fiber type, but
another extant question concerns whether postnatal myostatin
repression can also have the same effects. Reducing myostatin
in mice postweaning through the use of myostatin neutralizing
antibodies, or by postnatal conditional genetic targeting, also
leads to increased muscle mass (77–79). Depending on the
length of treatment and the age of the mice there is a 10 –30%
increase in muscle mass with neutralizing antibodies (77).
Whittemore and colleagues (79) reported that mice treated with
neutralizing antibodies had increased skeletal muscle mass and
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inhibition of TGF-␤ signaling results in an improved skeletal
muscle profile in several genetically invoked myopathies.
In vivo studies have also documented the presence of TGF-␤
during muscle injury. Muscle injury caused by eccentric contractions (6), cardiotoxin injection (22), and muscle strain
injury (72) resulted in increased TGF-␤ transcript and protein
levels within 48 h of injury. However, increases in TGF-␤1 and
TGF-␤2 precursors did not correspondingly lead to an increase
in TGF-␤ activity following muscle strain, suggesting a delay
in TGF-␤ activation after injury (72). Furthermore, TGF-␤
type I receptor expression is regulated by myotube excitability.
T␤RI was downregulated when primary rat myotubes were
electrically stimulated and upregulated in adult rat muscle 72 h
after denervation (75). Thus careful dissection of the role of
TGF-␤ signaling in these in vivo contexts is warranted to
further understand the role played by the endogenous cytokine.
Nearly three decades have passed since the potent effects of
TGF-␤ on myogenesis were first reported and only recently has
an in vivo role for TGF-␤ begun to be elucidated. Future
studies using tissue-specific gene targeting and the discovery of
new TGF-␤ pathway mediators may further elucidate the role
of TGF-␤ in skeletal muscle.
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myostatin as a negative regulator of fiber size. However, the
decrease in myostatin mRNA during running is more difficult
to reconcile as one would expect myostatin levels to increase
with this perturbation.
A possible mechanism for exercise modulation of myostatin
mRNA may be through calcium-dependent signaling via calcineurin. Calcineurin is a calcium-dependent phosphatase that
has been shown to play a key role in the control of muscle gene
expression by its regulation of the nuclear factor of activated
transcription (NFAT) transcriptional regulator (10). Myostatin
mRNA was reduced in mouse plantaris muscle that was overloaded. However, when calcineurin is inhibited through the use
of cyclosporin A, myostatin mRNA was maintained at normal
levels, suggesting that calcineurin may play a potentially important role in the regulation of myostatin levels during
muscle remodeling (56, 57). Furthermore, expression of
constitutively activated calcineurin in transgenic mice results in higher levels of myostatin mRNA levels (56). In
addition, a number of experimental manipulations that result
in reduced levels of calcineurin activity, such as calmodulin-binding protein (CaMBP)-overexpressing mice, parvalbumin-overexpressing mice, and NFATc2 null mice also exhibited reduced myostatin mRNA levels (56). Collectively, these
data clearly indicate a connection between calcineurin activity
and myostatin mRNA levels.
The next and, probably, most important question to address
is whether the alteration in myostatin levels is fundamental to
the adaptation of the muscle in these contexts. The question as
to whether myostatin levels are functionally linked to muscle
performance is indeed intriguing. A recent study suggests that
it may be, based on the observation that in a particular breed of
racing dogs (whippets), performance characteristics were correlated with a mutation in the myostatin gene in that the dogs
with the mutation are faster than those with the wild-type allele
(58). This is the first report of myostatin gene mutation leading
to enhanced athletic performance. Although increased muscle
mass and strength in myostatin null animals is firmly established, there has been little evidence to date that it necessarily
translates to increased athletic performance. With regard to this
issue, whippets heterozygous for a mutant copy of myostatin
(mh/⫹) are more muscular than normal whippets, ⬃20% more
mass/height compared with normals, and are among the fastest,
as determined by their racing grade. Interestingly, whippets
homozygous for the mutant copy of myostatin (mh/mh) are
considerably more muscular (60% more mass/height) compared with normal whippets, but were not as fast as the
heterozygous dogs. Thus it is important to consider that the
balance between muscle mass and muscular performance may
be more optimal in the heterozygous rather than the homozygous null state.
FUTURE PERSPECTIVES
Repressing TGF-␤ and myostatin signaling in muscle could
have potential therapeutic applications since both TGF-␤ and
myostatin activity are elevated in a variety of clinical conditions associated with muscle loss. Increased TGF-␤ signaling
in mice deficient in fibrillin-1 have impaired skeletal muscle
regeneration (11). A correlation between enhanced myostatin
levels and muscle atrophy has also been observed in AIDS
(23), bed rest (81), limb unloading, and exposure to micro-
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increased grip strength. Critically, this was the first study in
postnatal animals to show that repressing myostatin signaling
not only increases muscle mass but also enhances functional
muscle strength. Recently, postnatal conditional genetic targeting of myostatin also showed increase muscle mass (78). Older
mice that have ceased normal muscle growth also respond to
myostatin repression with increased muscle mass but to a lesser
degree. Additionally, short-term inhibition of myostatin in
aged mice (14 –16 mo) enhanced muscle regeneration and
activated satellite cell activation (71). Since older mice retain
the capability to respond to myostatin repression with increased muscle mass it is tantalizing to consider the possibility
of treating sarcopenia, age-related loss of muscle mass and
strength, by inhibiting myostatin. These findings indicate that
repression of myostatin could proffer a treatment for sarcopenia. Furthermore, inhibiting the myostatin pathway using the
iSmads could be another potentially therapeutic strategy for
maintaining muscle mass in ageing animals.
In concert with the functional consequence of loss of myostatin signaling, when myostatin signaling is increased, mice
undergo a predicted severe muscle loss. In experiments in
which mice were systemically administered myostatin, pronounced skeletal muscle loss was observed (83). These mice
exhibited symptoms similar to those of cachexia, which occurs
in many disease states such as cancer, heart failure, AIDS, and
sepsis. Thus it is now becoming clear that myostatin has the
capacity to act as a specific and potent negative regulator of
skeletal muscle mass during development, which is also paralleled in postnatal skeletal muscle.
Recently, several studies have implicated modulation of
myostatin mRNA levels in response to altered functional demand imposed by exercise training. It is clear that resistance
training represses myostatin mRNA. Long-term resistance
training reduces myostatin levels in rodents and humans (20,
67). An acute bout of resistance exercise also inhibits myostatin mRNA expression in rodents and humans (25, 29). Furthermore, resistance training mitigates atrophy and increases in
myostatin caused by skeletal muscle unloading in rodents (1).
Interestingly, hypertrophic capability of an individual had no
impact on the level of myostatin reduction caused by resistance
exercise (30). Thus both long-term and acute resistance training inhibit myostatin mRNA expression. However, individuals
with greater hypertrophy in response to training did not
concomitantly have the lowest levels of myostatin.
The effect of submaximal intensity running exercise on
myostatin mRNA levels has also been reported (41). Acute
bouts of both resistance exercise (3 sets of 10 repetitions at
70% 1RM) and submaximal running (75% V̇O2 max for 30 min)
reduced myostatin mRNA levels in young untrained participants. Postexercise time points comparing the two modes of
acute exercise showed that resistance exercise has a more
pronounced and longer suppression of myostatin. Resistance
exercise reduced myostatin over sixfold for 23 h (1–24 h
postexercise) while submaximal running decreased myostatin
over threefold for 3 h (8 –12 h postexercise). Myostatin expression is suppressed by both resistance exercise and running, but
with the former being a much more robust suppressor. Thus
these data implicate an inverse correlation between exercise
training and myostatin mRNA levels in a variety of exercise
regimens. Decreases in myostatin mRNA levels in response to
resistance exercise are consistent with the known role of
Invited Review
TGF-␤ AND MYOSTATIN SIGNALING IN SKELETAL MUSCLE
12.
13.
14.
15.
16.
17.
18.
19.
GRANTS
This work is supported by grants to J. C. McDermott from the Canadian
Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council (NSERC) of Canada.
20.
21.
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