To become an Arnold Schwartzenegger or a Hercules, does one need to pump iron, or be blessed with the appropriate genotype, or both? Four years ago, McPherron et al. (10) showed that mice lacking the gene myostatin (or GDF-8) are heavy muscled without pumping iron (Figure 1). In this review, we discuss the biology of myostatin and the possible role of myostatin in muscle growth and regeneration.
MYOSTATIN STRUCTURE AND FUNCTION
Myostatin, previously known as Growth and Differentiation Factor 8 (GDF-8), is a member of the TGF-β superfamily. The Myostatin gene has been cloned from most of the vertebrate species, and sequence analysis shows that myostatin structure and sequence is highly conserved during evolution. Although expression of only one isoform of myostatin is described in higher vertebrates, recently two myostatin isoforms have been reported from brook trout with 92% identity at the nucleotide level (11).
Structurally, Myostatin appears to share several features with other members of the TGF-β superfamily. Myostatin contains a core of hydrophobic amino acids near the N-terminus that functions as a signal sequence for secretion. It is synthesized as a precursor form that is proteolytically processed to give rise to either 26-kDa or 12-kDa active processed mature myostatin in myoblasts or fibroblasts, respectively (Figure 2) (13). Mature myostatin contains nine cysteine residues that facilitate the active C-terminal mature protein to form a homodimer in a cysteine knot structure (10), and like other TGF-β members, once processed, myostatin appears to be secreted as corroborated by the presence of mature 26-kDa myostatin in circulation. However the proteins that facilitate myostatin secretion and the latency complex of myostatin are not yet characterized.
In addition to structure, myostatin function appears to be very well conserved in the vertebrates. Targeted deletion of the entire C-terminus of myostatin in mice leads to an increase in the size of the animal (10) (Figure 1). The myostatin null mice were significantly larger than wild-type animals, the increase in body weight coming from a two- to three-fold increase in muscle mass. The increase in muscle mass resulted from muscle cell hyperplasia and hypertrophy (10), which suggests that myostatin is a negative regulator of muscle growth. Another mutation in the myostatin coding sequence identified in compact hypermuscular mouse is a deletion in the pro-peptide region preceding the proteolytic processing site of the precursor protein.
In cattle, the muscular hypertrophy (mh) or double-muscle phenotype (Figure 1) is a heritable condition that primarily results from hyperplasia relative to normal cattle. The Belgian Blue and Piedmontese breeds of cattle, which are characterized by an increase in muscle mass, have mutations in the myostatin coding sequence (7). More recently, Grobet et al. (5) have identified seven DNA sequence polymorphisms of which five were predicted to disrupt the function of myostatin in various European double muscled cattle.
Muscle size is considered to be one of the most heritable quantitative traits in humans, with genetic variation accounting for as much as 92%–94% of the total variance in muscle circumference (3). To determine the genetic basis of inter-individual and inter-ethnic differences in muscle mass, Ferrel et al. (3) sequenced the myostatin gene in different populations. Sequence analysis of myostatin cDNA from 40 individuals revealed five missense substitutions -A55T, K153R, E164K, P198A, and I225T, two of these, A55T in exon 1 and K153R in exon 2, are polymorphic in general population. These polymorphisms had different allele frequencies in whites and blacks, and neither of them had a significant effect on muscle mass response to strength training, suggesting that response is not significantly affected by variation at the myostatin locus.
Myostatin, a determinant of prenatal muscle growth is expressed early in gestation. Myostatin expression is first detected prenatally in the dermomyotome compartment of somites during embryonic myogenesis in day 10.5 mouse embryos (10). In cattle, very low levels of myostatin mRNA are detected in day 15 to day 29 embryos, and increased expression is detected from day 31 onwards (7). Similarly, in the whole pig fetus, myostatin mRNA is abundant at day 21 of gestation (6). The increase of myostatin expression in the bovine embryo is suggested to directly relate to the gestational stage when primary myoblasts are starting to fuse and differentiate into myofibers, and the secondary myoblasts are initially proliferating and then fusing. Myostatin then continues to be expressed in adult axial and paraxial muscles, although the expression levels vary among individual muscles (7,10). The pre- and post-natal expression of myostatin may also be associated with muscle fiber type as double-muscled cattle have a greater number of fast glycolytic fibers than normal cattle. Furthermore, higher amounts of myostatin mRNA and protein are observed in fast-twitch muscle compared to slow-twitch muscle in mice, supporting myostatin in fiber type specific function (1).
In addition to skeletal muscle, low levels of myostatin have been detected in other tissues. Myostatin expression has been found in adipose tissue (10) and has been suggested to inhibit preadipocyte differentiation (8). Myostatin is also detected in the tubuloalveolar secretory lobules of the lactating mammary gland (6), and in a report using myostatin specific antibodies, myostatin protein was shown to also be present in cardiomyocytes and purkinje fibers of heart (12). The role myostatin plays in these tissues is, at present, unknown.
The two isoforms of myostatin that have been identified in brook trout are expressed in different tissues. One isoform is predominately expressed in muscle and brain, whereas the other is expressed in ovarian tissue (11). Within the brain, myostatin mRNA was localized to the optic lobes, hindbrain, and hypothalamus. This suggests that the biological role of myostatin in the fish may not be limited to myogenesis.
Myostatin gene expression appears to be transcriptionally regulated during myogenesis, in postnatal muscle, and in response to some physiological conditions in adults. An analysis of approximately 1.6-kb of promoter sequence immediately upstream of myostatin gene from cattle, pig, mouse, and human (Patent Database) reveals that several DNA sequence motifs for muscle-specific gene transcription are present and that some of these elements are conserved in these species.
The most notable of the muscle-specific transcription factor binding motifs present are the E boxes to which MRFs such as MyoD, Myf5, Myogenin, and MRF4 bind. Of the MRFs, both Myf5 and MyoD have been shown to be expressed very early in embryonic myogenesis (E8 and E10 stages in mice) and are implicated in the determination and proliferation of myoblasts. Results from our laboratory show that during bovine myogenesis, the peak expression of MyoD is at day 70–120 and that of myostatin is between days 90–120, which coincides with onset of secondary fiber formation. Because timing of myostatin expression during development coincides with that of MyoD, and both MyoD and myostatin are predominately expressed in fast-twitch muscle (1), MyoD may be one of the key regulators of myostatin expression during the proliferation and differentiation of myoblasts during myogenesis.
MYOSTATIN AND THE MECHANISM OF MYOSTATIN FUNCTION
During myogenesis, myoblasts proliferate and withdraw from the cell cycle and commit to a differentiation pathway to form myotubes. The size of muscle, therefore, is in part dependent on the extent of myoblast proliferation that occurs prior to differentiation. Recent studies (13) show that myostatin inhibits the proliferation of exponentially growing myoblasts in culture without causing overt differentiation or increasing apoptotic cell death.
Fundamental to myoblast proliferation is the cell cycle. Regulation of the cell cycle predominantly occurs at the gap phases, G1 and G2, where cyclin-dependent kinases (Cdks) and their cognate partners, cyclins, phosphorylate substrates, which serve to promote gap-phase transitions. Regulating these Cdks are the p16 and p21 families of Cdk inhibitors (CKIs). When myostatin-treated myoblasts were analyzed by FACS, the distributions revealed cell-cycle arrest in the G1 and G2 phases. Based on the analysis of the cell-cycle control proteins, we have proposed a mechanism for myostatin function in muscle growth (13) (Figure 3) whereby in response to myostatin signaling, p21 is upregulated, inhibiting cyclin-E·Cdk2 activity, which causes the hypophosphorylation of Rb protein and G1 arrest. This model also, in part, explains the muscular hyperplasia seen in animals lacking functional myostatin in which myoblast proliferation becomes partially deregulated.
It is interesting to note that Rb is not the only substrate of Cdk2. Cdk2 and its cognate partner cyclin-E are expressed in late G1 and have many substrates including Rb, E2Fs, SWI/SNF, spliceosome-associated proteins, NPAT, p27, CDC25A, and unknown substrates involved in chromosome duplication and DNA replication initiation (2). The majority of these are fundamental for entry into, and progression through, S-phase. At present, it is unknown how myostatin signaling affects these substrates and the degree that they contribute to the G1-phase cell-cycle arrest.
MYOSTATIN IN MUSCLE MISUSE
Myostatin is a secreted negative regulator of skeletal muscle growth, hence its role in muscle wasting conditions is being investigated in various laboratories. Serum concentrations of myostatin protein were found to increase in HIV-infected men with weight loss, which suggests that increased myostatin levels may contribute to the pathophysiology of muscle wasting during HIV infection (4). A transient increase in myostatin mRNA during unloading-induced atrophy of fast-twitch muscle was observed in mice (1). In another study by Wehling et al. (14), atrophy of rat hindlimb muscles induced by 10 d of unloading resulted in a 16% decrease in plantaris mass and a 110% increase in myostatin mRNA. However, this increase in myostatin mRNA levels was not sufficient to cause significant loss of muscle mass. In these experiments, animals subjected to long periods of muscle unloading with intermittent muscle loading experienced no significant loss of muscle mass but showed higher levels of myostatin. One of the explanations for the elevated levels of myostatin in hindlimb unloading is that myostatin may function as an inhibitor of satellite cell proliferation (1). This is supported by the fact that myostatin-null mutant mice have both muscle hyperplasia and hypertrophy, which can be related to increased satellite cell activity. Furthermore, hindlimb unloading inhibits satellite cell proliferation. Unpublished results from our laboratory indicate that myostatin indeed inhibits the satellite cell proliferation. The rats exposed to the microgravity environment of the space shuttle flight also show loss of skeletal muscle mass is associated with increased myostatin mRNA and protein levels in the skeletal muscle.
Taken together, these results suggest that there may be fundamental differences in myostatin expression in various muscle-wasting conditions. That is, depending on the nature of the stimulus, whether chronic or acute, the expression of myostatin would differ. When animals are subjected to unloading, the stimulus is drastic and occurs rapidly, which results in the quick and transient change in myostatin mRNA expression (1). The signaling events that regulate myostatin expression in response to such stimuli are currently under investigation.
MYOSTATIN IN MUSCLE REGENERATION
Myostatin has also been hypothesized to play a role in the muscle regeneration process. Following skeletal muscle injury, the degeneration process, such as inflammation, neutrophil infiltration, and macrophage phagocytosis of necrotic cells, occurs within the damaged region. This process is then followed by the activation of satellite cells, which exist in skeletal muscle in a quiescent state. Activated satellite cells proliferate and migrate to the site of injury where they fuse to form myotubes and mature into skeletal muscle fibers.
Unfortunately, studies to date examining myostatin in skeletal muscle regeneration are correlative and do not show a direct causative role for myostatin in muscle regeneration after injury. However, in saying this, many of the studies strongly implicate and lend good support to such a role. Kirk et al.(9), show high levels of myostatin protein in necrotic fibers and connective tissue of muscle damaged by injection of the myotoxin, notexin. The simplest interpretation of these findings is that myostatin may be inhibiting satellite cell activation, proliferation, and differentiation in the necrotic tissue while the degeneration process takes place. Myostatin may also be acting as a chemoattractant for phagocytes and inflammatory cells, a role that has been ascribed to the related TGF-β1.
In contrast to the high myostatin expression seen during the necrotic fiber stage, no myostatin was found in the mononucleated cells located in regenerating areas where activated satellite cells are most abundant. Similarly, no myostatin expression was found in nascent myotubes, and low levels of myostatin were detected later in the maturing myotubes. These findings are consistent with myostatin acting as an inhibitor of myoblast proliferation whereby the absence of myostatin permits myoblast cell cycle progression via cyclin·Cdk2 inactivation of Rb.
Other evidence that myostatin may play a role in muscle regeneration comes from the examination of myostatin expression in muscle with degenerative disorders such as muscular dystrophy (15). In mdx mice, which lack dystrophin and are a model for Duchenne muscular dystrophy, and gsg-/- mice, which lack γ-sarcoglycan and are a model for limb girdle muscular dystrophy, a significant degree of regeneration takes place after repetitive cycles of degeneration. Consistent with this, myostatin expression is significantly lower in mdx and gsg-/- mice compared with wild-type mice. Perhaps the best evidence for myostatin having a role in regeneration, however, will come from regeneration studies in myostatin-null mice.
PERSPECTIVES AND CONCLUSION
Close to half a decade of research in myostatin has established that it restricts the growth of embryonic skeletal muscle. Although the function of myostatin in early myogenesis is well established, its role in the adult muscle is poorly understood. Detection of myostatin in post-natal skeletal muscle, combined with studies from induced atrophy, muscle wasting, and regeneration, provide strong circumstantial evidence for the role of myostatin in regulating muscle growth after birth. Future studies using myostatin over expression and conditional– or temporal–knock-out strategies will be useful in elucidating myostatin function in postnatal skeletal muscle. In addition to the skeletal muscle, various other tissues such as heart, mammary gland, and adipocytes express myostatin and possibly contribute to the circulatory levels of myostatin. Future investigations into local and systemic levels in regulating myostatin function will be very crucial in understanding the significance of myostatin in tissue homeostasis.
Understanding myostatin as a regulator of skeletal muscle growth will be of great benefit to the fields of biomedicine and agriculture. The biology of myostatin with respect to its proteolytic processing, binding to the specific receptor, and the subsequent signaling events is extremely important to design antagonists or mimetics of myostatin. Myostatin antagonists will be of tremendous use in medical intervention of old age and other muscle wasting conditions because high systemic levels of myostatin are associated with muscle wasting in humans. Such antagonists may also be sought by the farming industry to improve the muscle mass of farm animals.
We thank members of Functional Muscle Genomics group for their continued support. We apologize sincerely to those whose work we have overlooked or omitted because of space limitations. We are indebted to Foundation for Research, Science and Technology (New Zealand) for financial support.
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