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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: Kravi@ntu.edu.sg 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 . markers and loss of muscle mass (sarcopenia) and strength. The American Journal of Medicine, 119(526), e9–e17. Schwander, J. C., Hauri, C., Zapf, J., Froesch, E. R. (1983). Synthesis and secretion. interventions for age-related muscle wast- ing: importance of innervation and exercise for preventing sarcopenia. In S. Rattan (Ed.), Modulating aging and longevity. The Netherlands: Kluwer. Shavlakadze,. interleukin-6 and tumor necrosis factor- alpha with muscle mass and muscle strength in elderly men and women: The Health ABC Study. The Journals of Gerontology. Series A: Biological Sciences and Medical