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419
G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness,
DOI 10.1007/978-90-481-9713-2_18, © Springer Science+Business Media B.V. 2011
Abstract Myostatin is a secreted growth and differentiating factor that belongs
to TGF-b super-family. Myostatin is expressed in skeletal muscle predominantly.
Low levels of myostatin expression are seen in heart, adipose tissue and mammary
gland. Naturally occurring mutations in bovine, ovine, canine and human myostatin
gene or inactivation of the murine myostatin gene lead to an increase in muscle
mass due to hyperplasia. Molecularly, myostatin has been shown to regulate muscle
growth not only by controlling myoblast proliferation and differentiation during
fetal myogenesis, but also by regulating satellite cell activation and self-renewal
postnatally. Consistent with the molecular genetic studies, injection of several
myostatin blockers including Follistatin, myostatin antibodies and the Prodomain
of myostatin have all been independently shown to increase muscle regeneration
and growth in muscular dystrophy mouse models of muscle wasting. Furthermore,

prolonged absence of myostatin in mice has also been shown to reduce sarcopenic
muscle loss, due to efficient satellite cell activation and regeneration of skeletal
muscle in aged mice. Similarly, treatment of aged mice with Mstn-ant 1 also
increased satellite cell activation and enhanced the efficiency of muscles to regen-
erate. Given that antagonism of myostatin leads to significant increase in postnatal
muscle growth, we propose that myostatin antagonists have tremendous therapeutic
value in alleviating sarcopenic muscle loss.
Keywords Myostatin • GDF-8 • Skeletal muscle • Smad • Wnt • Proliferation
• Differentiation • Satellite cells • Muscle wasting • Atrophy • Cachexia • Sarcopenia
R. Kambadur (*)
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore
e-mail:
C. McFarlane and R. Kambadur
Singapore Institute for Clinical Sciences, Singapore
M. Sharma
Department of Biochemistry, National University of Singapore, Singapore
Role of Myostatin in Skeletal Muscle Growth
and Development: Implications for Sarcopenia
Craig McFarlane, Mridula Sharma, and Ravi Kambadur
420 C. McFarlane et al.
1 Myostatin
1.1 The Myostatin Gene, Structure and Processing
Myostatin, or growth and differentiation factor-8 (GDF-8), is a TGF-b superfamily
member that was initially characterised in 1997 as a specific regulator of skeletal
muscle mass in mice (McPherron et al. 1997). Targeted disruption of the myostatin
gene in mice (Fig. 1) resulted in a generalised increase in skeletal muscle mass
(double-muscling); in particular a two to threefold increase in muscle weight was
observed with no corresponding increase in adipose tissue (Fig. 1). The enhanced
muscle phenotype in the myostatin-null mice was determined to result from a com-
bination of both muscle hyperplasia and hypertrophy (McPherron et al. 1997).

Myostatin has a number of characteristics common to the TGF-b superfamily
(Fig. 2). In particular, the precursor myostatin molecule contains an N-terminal
(NH2) core of hydrophobic amino acids that functions as a signal sequence
for secretion (McPherron et al. 1997). In addition, the C-terminal (COOH)
region of myostatin contains nine conserved cysteine residues which are critical
for homodimerisation and for the formation of the “cysteine knot” structure, a
characteristic feature of the TGF-b superfamily (McPherron and Lee 1996;
McPherron et al. 1997). Furthermore, myostatin is synthesised in myoblasts as a
376 amino acid precursor protein which, like other members of the TGF-b super-
family, is proteolytically cleaved at the RSRR site (Fig. 2), a process which occurs
within the Golgi apparatus under the control of the serine protease furin or other
members of the proprotein convertase family (Lee and McPherron 2001; McPherron
et al. 1997; Sharma et al. 1999). Proteolytic processing of the myostatin 52 kDa
precursor protein by furin results in the formation of a 36/40 kDa Latency-
Associated Peptide (LAP) and a 12.5/26 kDa mature portion, which is suggested
to correspond to a C-terminal monomer or dimer respectively (Lee and McPherron
2001; McFarlane et al. 2005; Thomas et al. 2000). The processed mature form of
Fig. 1 Double-muscling in myostatin-null mice. (a) Photograph showing the difference between
the forelimbs of wild-type and myostatin-null mice. A dramatic increase in skeletal muscle mass
is observed in the myostatin-null mice compared to wild-type mice (Adapted from McPherron
et al. 1997). (b) Photograph showing the size difference between wild-type and myostatin-null
mice at the same age. Myostatin-null mice were generated by McPherron et al. (1997)
421Role of Myostatin in Skeletal Muscle Growth and Development
myostatin, together with LAP, is subsequently secreted from myoblasts and it is
the C-terminal mature region that is able to bind to the receptor and elicit biologi-
cal function. The importance of proteolytic processing is clear, as generation of a
dominant-negative form of myostatin, through mutation of the RSRR site to the
amino acids GLDG, results in widespread skeletal muscle hypertrophy (Zhu et al.
2000). Previously it has been demonstrated that processing of myostatin is devel-
opmentally regulated, whereby reduced myostatin processing is observed during

fetal muscle development when comparted to post-natal stages of growth
(McFarlane et al. 2005). Furthermore it was demonstrated that there is reduced
proteolytic processing of myostatin during myogenic differentiation and more
importantly myostatin has the ability to negatively regulate the expression of the
serine protease furin. Myostatin inhibition of furin expression was proposed to be
a mechanism through which myostatin negatively auto-regulates its processing
during the critical periods of fetal growth, thereby facilitating the differentiation
of myoblasts (McFarlane et al. 2005)
1.2 Expression of Myostatin
Myostatin is first detected in mice embryos at day 9.5 post-coitum, where it is
specifically located within the most rostral somites (McPherron et al. 1997). By
day 10.5 post-coitum, myostatin is expressed in the majority of the somites, spe-
cifically located in the myotome layer of developing somites (McPherron et al.
1997). In cattle, low levels of myostatin mRNA are detected in day 15 to day 29
embryos with increasing expression detected from day 31 onwards (Kambadur
et al. 1997; Bass et al. 1999; Oldham et al. 2001). Furthermore, in the pig foetus
myostatin mRNA expression is abundant at days 21 and 35 of gestation, with an
increase in expression by day 49 (Ji et al. 1998). In the chicken myostatin expres-
sion is first detected as early as embryonic day 0 (the blastoderm stage) with rela-
tively low levels detected through to embryonic day 6. From day 7, myostatin
mRNA levels rapidly increase and level off through to day 16 (Kocamis et al.
1999). Post-natal skeletal muscle continues to express myostatin, although variation
in myostatin expression is observed between individual muscles (Kambadur
et al. 1997; McPherron et al. 1997). The expression of myostatin is primarily
RSRR
LAP matureNH2 SP
aa
1
aa
264-267

aa
376
COOH
Fig. 2 The structure of myostatin. Schematic representation of the structure of myostatin.
Myostatin shares characteristics common to the TGF-b superfamily, including a signal peptide
(SP) for secretion and a RSRR proteolytic processing site. Proteolytic processing of myostatin
gives rise to LAP and mature myostatin regions (Adapted from Joulia-Ekaza and Cabello
[2006])
422 C. McFarlane et al.
restricted to skeletal muscle (Kambadur et al. 1997; McPherron et al. 1997;
Ji et al. 1998; Bass et al. 1999; Carlson et al. 1999; Kocamis et al. 1999; Sazanov
et al. 1999; Jeanplong et al. 2001; Oldham et al. 2001), however, low levels of
myostatin expression have been detected in various other tissues; in particular in
the secretory lobules of lactating mammary glands (Ji et al. 1998), in adipose tis-
sue (McPherron et al. 1997), and in cardiomyocytes and Purkinje fibres of the
heart (Sharma et al. 1999). More recently it has been shown that both myostatin
mRNA and protein are expressed in human placental tissue. The presence of
myostatin in the placenta is suggested to be involved with uptake of glucose
(Mitchell et al. 2006).
Myostatin expression may also be associated with specific fibre types in skeletal
muscle. Carlson et al. have shown that higher amounts of myostatin mRNA and
protein are detected in fast-twitch muscle (type-II fibres) as compared to slow-
twitch muscle (type-I fibres) (Carlson et al. 1999). Furthermore, it has been shown
that in myostatin-null mice there is an increase in fast fibres (type-II) in the typi-
cally slow fibre-dominated M. soleus muscle, and a switch from oxidative (type-
IIA) to glycolytic fibres (type-IIB) in the predominantly fast-twitch EDL muscle
(Girgenrath et al. 2005). Therefore, suggesting a fibre type-specific role for myo-
statin in regulation of muscle physiology.
1.3 Regulation of Myostatin
Myostatin is synthesised as a precursor protein, proteolytically processed and

secreted to elicit its biological function. Studies have highlighted the impor-
tance of several proteins that interact with myostatin to regulate its action.
Myostatin has been shown to interact with the sarcomeric protein Titin-cap
(Nicholas et al. 2002); specifically, titin-cap interacts with the C-terminal
mature portion of myostatin (Nicholas et al. 2002). Over-expression of titin-cap
had no effect on myostatin synthesis and processing, however, increased titin-
cap expression results in enhanced cell proliferation and accumulation of pro-
cessed myostatin within myoblasts. Thus, titin-cap appears to function by
regulating the secretion of mature myostatin (Nicholas et al. 2002). In addition,
human small glutamine-rich tetratricopeptide repeat-containing protein (hSGT)
has been shown to associate with intracellular myostatin (Wang et al. 2003).
The C-terminal region of hSGT and the N-terminal signal peptide region of
myostatin were shown to be critical for this interaction. It is suggested that
hSGT likely plays a role in mediating myostatin secretion and activation (Wang
et al. 2003). Latent TGF-b binding proteins (LTBPs) are extracellular matrix
proteins which have been previously identified to interact with the TGF-b
superfamily (Saharinen et al. 1999). LTBPs associate with TGF-b superfamily
members to allow for secretion; once secreted, removal of LTBPs from the
latent complex is essential for TGF-b activation (Saharinen et al. 1999).
Although LTBPs play an essential role in the secretion and activation of TGF-b
423Role of Myostatin in Skeletal Muscle Growth and Development
superfamily members, published results from this thesis suggest that LTBPs do
not play a role in the regulation of myostatin (McFarlane et al. 2005). Following
secretion, the majority of myostatin (>70%), like TGF-b, has been shown to
exist in an inactive latent complex both in vitro and in vivo, whereby the mature
processed portion of myostatin is bound non-covalently to the propeptide
(LAP) region of myostatin (Lee and McPherron 2001; Thies et al. 2001; Yang
et al. 2001). Recently it has been demonstrated that members of the bone mor-
phogenetic protein-1/tolloid (BMP-1/TLD) family can cleave the myostatin
LAP region from the latent myostatin complex, thus resulting in activation of

mature myostatin (Wolfman et al. 2003). Furthermore, Wolfman et al. demon-
strated that a mutation of LAP to confer resistance to cleavage by BMP/TLD
resulted in enhanced muscle mass in vivo. Previous studies have demonstrated
that follistatin is capable of binding and inhibiting various members of the
TGF-b superfamily (Fainsod et al. 1997; Hemmati-Brivanlou et al. 1994;
Michel et al. 1993). Follistatin has been shown to bind directly to the mature
portion of myostatin blocking the ability of myostatin to bind with the ActRIIB
receptor (Lee and McPherron 2001). Furthermore, interaction with follistatin
interferes with the intrinsic ability of myostatin to inhibit muscle differentiation
(Amthor et al. 2004). In support, mice over-expressing follistatin show a drastic
increase in muscle mass, significantly greater than that of myostatin-null ani-
mals (Lee and McPherron 2001). Additionally, follistatin-null mice demonstrate
reduced muscle mass at birth (Matzuk et al. 1995), consistent with increased
myostatin activity. Follistatin-related gene (FLRG), like follistatin, is able to
bind and inhibit members of the TGF-b superfamily (Tsuchida et al. 2000,
2001; Schneyer et al. 2001). In addition, FLRG has been shown to interact
directly with the mature portion of myostatin, resulting in a dose-dependent
reduction in the activity of myostatin, as assessed through reporter gene assay
analysis (Hill et al. 2002). Growth and differentiation factor-associated serum
protein-1 (GASP-1) has been shown to associate with myostatin in circulation;
specifically associating with both mature and LAP regions of myostatin.
Functionally GASP-1 has been shown to interfere with the activity of myostatin
as determined by reporter gene analysis (Hill et al. 2003). More recently, deco-
rin, a leucine-rich repeat extracellular proteoglycan, has been shown to interact
with the mature region of myostatin, in a Zn
2+
-dependent manner (Miura et al.
2006). This interaction was demonstrated to relieve the inhibitory effect of
myostatin on myoblast proliferation in vitro. One of the intrinsic features of
myostatin is its ability to negatively auto-regulate its expression. In particular,

exogenous addition of recombinant myostatin protein results in both a decrease
in myostatin mRNA and repression of myostatin promoter activity (Forbes et al.
2006). Furthermore, myostatin appears to signal through Smad7 to regulate its
own activity (Forbes et al. 2006; Zhu et al. 2004). In support, addition of myo-
statin resulted in enhanced Smad7 expression, while over-expression of Smad7
resulted in repression of myostatin promoter activity and mRNA, an effect
abolished through incubation with siRNA specific for Smad7 (Forbes et al.
2006; Zhu et al. 2004).
424 C. McFarlane et al.
1.4 Mutations in Myostatin
In addition to the targeted disruption of myostatin in mice, several naturally occurring
mutations have been identified in various double-muscled cattle breeds including
Belgian Blue (Fig. 3a) and Piedmontese (Kambadur et al. 1997; McPherron and
Lee 1997; Grobet et al. 1998). Specifically two separate mutations in the coding
region of the myostatin gene have been reported to result in a non-functional myo-
statin product. The phenotype seen in Belgian Blue cattle (Fig. 3a) is caused by an
Fig. 3 Natural mutations in myostatin. (a) Photograph showing the heavy muscling observed in
the Belgian Blue cattle breed (Reproduced from Haliba ‘96 Catalogue). (b) Photograph of a Texel
sheep demonstrating the heavy muscle phenotype oberved in response to a G to A transition muta-
tion in the 3¢ UTR of the myostatin gene, which results in the formation of mir1 and mir206
miRNA sites (Reproduced from Skipper [2006]). (c) Photographs of a heavy muscled Whippet
dog (left) and a Whippet dog demonstrating more typcial muscle mass (right) (Reproduced form
Shelton and Engvall [2007]). (d) Photograph of a human child at 7 months of age possessing a
G to A transition mutation in the myostatin gene, resulting in a non functional myostatin protein
product. Arrows highlight protruding muscles from the boy’s calf and thigh regions (Modified
from Schuelke et al. [2004]).
425Role of Myostatin in Skeletal Muscle Growth and Development
11-nucleotide deletion, which ultimately results in expression of a non-functional
truncated protein product (Kambadur et al. 1997). Conversely, the Piedmontese
cattle express a non-functional myostatin protein through a missense mutation in the

gene sequence, resulting in a G to A transition and substitution of cysteine for
tyrosine (Kambadur et al. 1997; Berry et al. 2002). Furthermore, a mutation in the
myostatin gene has been reported to result in the hyper-muscularity observed in
compact (Cmpt) mice (Szabo et al. 1998). More recently, the heavy muscled
phenotype of the Texel sheep breed has been traced to a mutation in the myostatin
gene resulting in a G to A transition in the 3¢ untranslated region (UTR) (Fig. 3b)
(Clop et al. 2006). This mutation creates a target site for two microRNAs abundant
in skeletal muscle, namely mir1 and mir206 (Clop et al. 2006). MicroRNAs are short
non-coding RNAs which diminish gene activity post-transcriptionally by binding to
target genes, resulting in destabilisation of mRNA and/or inhibition of protein trans-
lation (Tsuchiya et al. 2006). In addition to the Texel breed, a mutation in the myo-
statin gene has been demonstrated to result in the increased muscle mass phenotype
observed in the Norwegian Spælsau sheep breed. Specifically a one base pair inser-
tion mutation at nucleotide 120 from the translation start site (c.120insA) results in
the formation of a premature stop codon at amino acid 49 resulting in the formation
of a non-functional protein product (Boman and Vage 2009).
Recently a mutation in the myostatin gene has been shown to result in dramatic
muscle hypertrophy in the Whippet racing dog breed (Fig. 3c) (Mosher et al. 2007).
The pheotype results form a two base pair deletion in the third exon of the myosta-
tin gene and leads to the formation of a premature stop codon at amino acid 313
resulting in a non-functional protein product. Interestingly, Whippet dogs heterozy-
gote for the mutation are not only more muscular than wildtype but are significantly
faster as well which, for the first time, demonstrates the utility of mutations in
myostatin and enhanced atheletic performance (Mosher et al. 2007).
A mutation in the myostatin gene has also been shown to result in dramatic
hypertrophy in a human child (Schuelke et al. 2004) (Fig. 3d). Cross-sectional
measurements determined that the M. quadriceps muscle was more than twofold
larger than age- and sex-matched controls, while the thickness of the sub-cutaneous
fat pad was significantly lower than controls. The mutation was shown to result
from a G to A transition within intron 1 of the myostatin gene. This transition

resulted in mis-splicing of the precursor mRNA and insertion of the first 108 base
pairs of intron 1 (Schuelke et al. 2004).
2 Physiological Actions of Myostatin
2.1 Myostatin Signaling
Members of the TGF-b superfamily elicit biological functions by binding to spe-
cific type-I and type-II serine/threonine kinase receptors. Studies have shown that