Alexandra C. McPherron

Regulation of myostatin activity and muscle growth

Myostatin is a transforming growth factor-β family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may have applications for promoting muscle growth, we investigated the regulation of myostatin signaling. Myostatin protein purified from mammalian cells consisted of a noncovalently held complex of the N-terminal propeptide and a disulfide-linked dimer of C-terminal fragments. The purified C-terminal myostatin dimer was capable of binding the activin type II receptors, Act RIIB and, to a lesser extent, Act RIIA. Binding of myostatin to Act RIIB could be inhibited by the activin-binding protein follistatin and, at higher concentrations, by the myostatin propeptide. To determine the functional significance of these interactions in vivo, we generated transgenic mice expressing high levels of the propeptide, follistatin, or a dominant-negative form of Act RIIB by using a skeletal muscle-specific promoter. Independent transgenic mouse lines for each construct exhibited dramatic increases in muscle mass comparable to those seen in myostatin knockout mice. Our findings suggest that the propeptide, follistatin, or other molecules that block signaling through this pathway may be useful agents for enhancing muscle growth for both human therapeutic and agricultural applications.

Suppression of body fat accumulation in myostatin-deficient mice

Myostatin is a TGF-beta family member that acts as a negative regulator of muscle growth. Mice lacking the myostatin gene (Mstn) have a widespread increase in skeletal muscle mass resulting from a combination of muscle fiber hypertrophy and hyperplasia. Here we show that Mstn-null mice have a significant reduction in fat accumulation with increasing age compared with wild-type littermates, even in the setting of normal food intake (relative to body weight), normal body temperature, and a slightly decreased resting metabolic rate. To investigate whether myostatin might be an effective target for suppressing the development of obesity in settings of abnormal fat accumulation, we analyzed the effect of the Mstn mutation in two genetic models of obesity, agouti lethal yellow (A(y)) and obese (Lep(ob/ob)). In each case, loss of Mstn led to a partial suppression of fat accumulation and of abnormal glucose metabolism. Our findings raise the possibility that pharmacological agents that block myostatin function may be useful not only for enhancing muscle growth, but also for slowing or preventing the development of obesity and type 2 diabetes.

Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases.

Myostatin is a transforming growth factor β family member that acts as a negative regulator of skeletal muscle growth. Myostatin circulates in the blood of adult mice in a noncovalently held complex with other proteins, including its propeptide, which maintain the C-terminal dimer in a latent, inactive state. This latent form of myostatin can be activated in vitro by treatment with acid; however, the mechanisms by which latent myostatin is activated in vivo are unknown. Here, we show that members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteinases can cleave the myostatin propeptide in this complex and can thereby activate latent myostatin. Furthermore, we show that a mutant form of the propeptide resistant to cleavage by BMP-1/TLD proteinases can cause significant increases in muscle mass when injected into adult mice. These findings raise the possibility that members of the BMP-1/TLD family may be involved in activating latent myostatin in vivo and that molecules capable of inhibiting these proteinases may be effective agents for increasing muscle mass for both human therapeutic and agricultural applications.

Loss of myostatin attenuates severity of muscular dystrophy in mdx mice.

Myostatin, a transforming growth factor–β family member, is a negative regulator of skeletal muscle growth. To explore the therapeutic potential of targeting myostatin in settings of muscle degeneration, we crossed myostatin null mutant mice with mdx mice, a model for Duchenne and Becker muscular dystrophy. Mdx mice lacking myostatin were stronger and more muscular than their mdx counterparts. Diaphragm muscle showed less fibrosis and fatty remodeling, suggesting improved muscle regeneration.

Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11.

The bones that comprise the axial skeleton have distinct morphological features characteristic of their positions along the anterior/posterior axis. We previously described a novel TGF-β family member, myostatin (encoded by the gene Mstn, formerly Gdf8), that has an essential role in regulating skeletal muscle mass. We also identified a gene related to Mstn by low-stringency screening. While the work described here was being completed, the cloning of this gene, designated Gdf11 (also called Bmp11), was also reported by other groups. Here we show that Gdf11, a new transforming growth factor β (TGFβ) superfamily member, has an important role in establishing this skeletal pattern. During early mouse embryogenesis, Gdf11 is expressed in the primitive streak and tail bud regions, which are sites where new mesodermal cells are generated. Homozygous mutant mice carrying a targeted deletion of Gdf11 exhibit anteriorly directed homeotic transformations throughout the axial skeleton and posterior displacement of the hindlimbs. The effect of the mutation is dose dependent, as Gdf11+/– mice have a milder phenotype than Gdf11–/– mice. Mutant embryos show alterations in patterns of Hox gene expression, suggesting that Gdf11 acts upstream of the Hox genes. Our findings suggest that Gdf11 is a secreted signal that acts globally to specify positional identity along the anterior/posterior axis.

MYOSTATIN AND THE CONTROL OF SKELETAL MUSCLE MASS

The mechanisms by which tissue size is controlled are poorly understood. Over 30 years ago, Bullough proposed the existence of chalones, which act as tissue-specific negative growth regulators. The recent discovery of myostatin suggests that negative regulation of tissue growth may be an important mechanism for controlling skeletal muscle mass and raises the possibility that growth inhibitors may also be involved in regulating the size of other tissues.

Myostatin inhibition in muscle, but not adipose tissue, decreases fat mass and improves insulin sensitivity.

Myostatin (Mstn) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Mstn−/− mice have a dramatic increase in muscle mass, reduction in fat mass, and resistance to diet-induced and genetic obesity. To determine how Mstn deletion causes reduced adiposity and resistance to obesity, we analyzed substrate utilization and insulin sensitivity in Mstn−/− mice fed a standard chow. Despite reduced lipid oxidation in skeletal muscle, Mstn−/− mice had no change in the rate of whole body lipid oxidation. In contrast, Mstn−/− mice had increased glucose utilization and insulin sensitivity as measured by indirect calorimetry, glucose and insulin tolerance tests, and hyperinsulinemic-euglycemic clamp. To determine whether these metabolic effects were due primarily to the loss of myostatin signaling in muscle or adipose tissue, we compared two transgenic mouse lines carrying a dominant negative activin IIB receptor expressed specifically in adipocytes or skeletal muscle. We found that inhibition of myostatin signaling in adipose tissue had no effect on body composition, weight gain, or glucose and insulin tolerance in mice fed a standard diet or a high-fat diet. In contrast, inhibition of myostatin signaling in skeletal muscle, like Mstn deletion, resulted in increased lean mass, decreased fat mass, improved glucose metabolism on standard and high-fat diets, and resistance to diet-induced obesity. Our results demonstrate that Mstn−/− mice have an increase in insulin sensitivity and glucose uptake, and that the reduction in adipose tissue mass in Mstn−/− mice is an indirect result of metabolic changes in skeletal muscle. These data suggest that increasing muscle mass by administration of myostatin antagonists may be a promising therapeutic target for treating patients with obesity or diabetes.

Myostatin promotes the terminal differentiation of embryonic muscle progenitors

Myostatin, a TGF-beta family member, is an important regulator of adult muscle size. While extensively studied in vitro, the mechanisms by which this molecule mediates its effect in vivo are poorly understood. We addressed this question using chick and mouse embryos. We show that while myostatin overexpression in chick leads to an exhaustion of the muscle progenitor population that ultimately results in muscle hypotrophy, myostatin loss of function in chick and mouse provokes an expansion of this population. Our data demonstrate that myostatin acts in vivo to regulate the balance between proliferation and differentiation of embryonic muscle progenitors by promoting their terminal differentiation through the activation of p21 and MyoD. Previous studies have suggested that myostatin imposes quiescence on muscle progenitors. Our data suggest that myostatins effect on muscle progenitors is more complex than previously realized and is likely to be context-dependent. We propose a novel model for myostatin mode of action in vivo, in which myostatin affects the balance between proliferation and differentiation of embryonic muscle progenitors by enhancing their differentiation.

Redundancy of myostatin and growth/differentiation factor 11 function.

BackgroundMyostatin (Mstn) and growth/differentiation factor 11 (Gdf11) are highly related transforming growth factor β (TGFβ) family members that play important roles in regulating embryonic development and adult tissue homeostasis. Despite their high degree of sequence identity, targeted mutations in these genes result in non-overlapping phenotypes affecting distinct biological processes. Loss of Mstn in mice causes a doubling of skeletal muscle mass while loss of Gdf11 in mice causes dramatic anterior homeotic transformations of the axial skeleton, kidney agenesis, and an increase in progenitor cell number in several tissues. In order to investigate the possible functional redundancy of myostatin and Gdf11, we analyzed the effect of eliminating the functions of both of these signaling molecules.ResultsWe show that Mstn-/-Gdf11-/- mice have more extensive homeotic transformations of the axial skeleton than Gdf11-/- mice in addition to skeletal defects not seen in single mutants such as extra forelimbs. We also show that deletion of Gdf11 specifically in skeletal muscle in either Mstn+/+ or Mstn-/- mice does not affect muscle size, fiber number, or fiber type.ConclusionThese results provide evidence that myostatin and Gdf11 have redundant functions in regulating skeletal patterning in mice but most likely not in regulating muscle size.

The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding.

Myostatin is a member of the transforming growth factor‐β (TGF‐β) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF‐β class members and that this region alone can be swapped into activin A to confer signalling through the non‐canonical type I receptor Alk5. Furthermore, the N‐terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle‐wasting disorders.