Myostatin Propeptide Research Articles

Myostatin Directly Regulates Skeletal Muscle Fibrosis

Skeletal muscle fibrosis is a major pathological hallmark of chronic myopathies in which myofibers are replaced by progressive deposition of collagen and other extracellular matrix proteins produced by muscle fibroblasts. Recent studies have shown that in the absence of the endogenous muscle growth regulator myostatin, regeneration of muscle is enhanced, and muscle fibrosis is correspondingly reduced. We now demonstrate that myostatin not only regulates the growth of myocytes but also directly regulates muscle fibroblasts. Our results show that myostatin stimulates the proliferation of muscle fibroblasts and the production of extracellular matrix proteins both in vitro and in vivo. Further, muscle fibroblasts express myostatin and its putative receptor activin receptor IIB. Proliferation of muscle fibroblasts, induced by myostatin, involves the activation of Smad, p38 MAPK and Akt pathways. These results expand our understanding of the function of myostatin in muscle tissue and provide a potential target for anti-fibrotic therapies.

Myostatin Propeptide Gene Delivery by Adeno-Associated Virus Serotype 8 Vectors Enhances Muscle Growth and Ameliorates Dystrophic Phenotypes inmdxMice

Myostatin has been extensively documented as a negative regulator of muscle growth. Myostatin inhibition is therefore considered an attractive strategy for the treatment of muscle-wasting diseases such as muscular dystrophies. To investigate whether systemic gene delivery of myostatin propeptide (MRPO), a natural inhibitor of myostatin, could enhance body-wide skeletal muscle growth, we used adeno-associated virus serotype 8 (AAV8) vectors to deliver the MRPO gene into either normal mice or mdx mice, a murine model of Duchenne muscular dystrophy (DMD). In normal mice, a significant increase in skeletal muscle mass was observed after either an intraperitoneal injection of AAV-MPRO into neonates, or an intravenous injection of AAV-MPRO76AFc (a modified MPRO fused with IgG Fc) into adults. Enhanced muscle growth occurred because of myofiber hypertrophy, not hyperplasia. In mdx mice, a significant increase in skeletal muscle mass was also observed after AAV-MPRO76AFc injection. The treated mdx mice showed larger and more uniform myofibers, fewer infiltrating mononuclear cells, less fibrosis, and lower serum creatine kinase levels. In addition, a grip force test and an in vitro tetanic contractile force test showed improved muscle strength. A treadmill test, however, showed reduced endurance of the treated mdx mice compared with their untreated counterparts. Importantly, no cardiac hypertrophy was observed in either normal or mdx mice after myostatin inhibition by gene delivery. These results clearly demonstrate the efficacy of AAV8-mediated myostatin propeptide gene delivery in a rodent model of DMD, and warrant further investigation in large animal models and eventually in human patients.

Enhanced muscle by myostatin propeptide increases adipose tissue adiponectin, PPAR-α, and PPAR-γ expressions

Muscle tissue utilizes a large portion of metabolic energy for its growth and maintenance. Previously, we demonstrated that transgenic over-expression of myostatin propeptide in mice fed a high-fat diet enhanced muscle mass and circulating adiponectin while the wild-type mice developed obesity and insulin resistance. To understand the effects of enhanced muscle growth on adipose tissue metabolism, we analyzed adiponectin, PPAR-α, and PPAR-γ mRNA expressions in several fat tissues. Results indicated muscled transgenic mice fed a high-fat diet displayed increased epididymal adiponectin mRNA expression by 12 times over wild-type littermates. These transgenic mice fed either a high or normal fat diet also displayed significantly high levels of PPAR-α and PPAR-γ expressions above their wild-type littermates in epididymal fat while their expressions in mesenteric fats were not significantly different between transgenic mice and their littermates. This study demonstrates that enhanced muscle growth has positive effects on fat metabolisms through increasing adiponectin expression and its regulations.

Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass and ameliorates dystrophic pathology inmdxmice

Myostatin is a potent negative regulator of skeletal muscle growth. Therefore, myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. The known myostatin inhibitors include myostatin propeptide, follistatin, follistatin-related proteins, and myostatin antibodies. Although follistatin shows potent myostatin-inhibiting activities, it also acts as an efficient inhibitor of activins. Because activins are involved in multiple functions in various organs, their blockade by follistatin would affect multiple tissues other than skeletal muscles. In the present study, we report the characterization of a myostatin inhibitor derived from follistatin, which does not affect activin signaling. The dissociation constants (K(d)) of follistatin to activin and myostatin are 1.72 nM and 12.3 nM, respectively. By contrast, the dissociation constants (K(d)) of a follistatin-derived myostatin inhibitor, designated FS I-I, to activin and myostatin are 64.3 microM and 46.8 nM, respectively. Transgenic mice expressing FS I-I, under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. Hyperplasia and hypertrophy were both observed. We crossed FS I-I transgenic mice with mdx mice, a model for Duchenne muscular dystrophy. Notably, the skeletal muscles in the mdx/FS I-I mice showed enlargement and reduced cell infiltration. Muscle strength is also recovered in the mdx/FS I-I mice. These results indicate that myostatin blockade by FS I-I has a therapeutic potential for muscular dystrophy.

Effects of Enhanced Muscle Growth by Myostatin Propeptide Transgene on Adiponectin and Adiponectin Receptors Gene Expression

Effects of Enhanced Muscle Growth by Myostatin Propeptide Transgene on Adiponectin and Adiponectin Receptors Gene Expression

Production of a Polyclonal Anti-Myostatin Antibody and the Effects of In Ovo Administration of the Antibody on Posthatch Broiler Growth and Muscle Mass

In this study, we produced a polyclonal antibody against unprocessed chicken myostatin and examined the effect of in ovo administration of the antibody on posthatch chicken growth and muscle mass. A PCR-amplified unprocessed chicken myostatin cDNA was cloned into an Escherichia coli expression vector, and myostatin proteins were expressed. Recombinant myostatin purified by electro-elution of the SDS-PAGE fractionated myostatin band was used as an immunogen to produce rabbit polyclonal antimyostatin antibody (pAb-AVM46). In Western blot analysis, the pAb-AVM46 showed high affinity to the myostatin propeptide, but little affinity to the mature myostatin. Two experiments examined the effect of in ovo administration of the pAb-AVM46 on posthatch chicken growth and skeletal muscle mass. In experiment 1, broilers from eggs injected once with 35 μg of the antibody into the yolk on d 3 of incubation had significantly lower combined thigh and leg weight at 4 wk posthatch than the controls that received no injection, or the broilers from eggs received the same dose of antibody into the albumen. In experiment 2, 2 different doses of the antibody (9 or 70 μg) were injected into the yolk, and the effects on body and muscle weight were examined at 5 wk posthatch. Birds from eggs injected with 70 μg of the antibody had significantly lighter (11.6%) combined thigh and leg weight than the control birds. The percentage of the combined thigh and leg weight to BW of the 70-μg group was also significantly lower than that of the control group (20.95 vs. 23.08%). The results of this study indicate that unprocessed full-length myostatin as an immunogen produced antibody populations having affinity mostly to the propeptide with little to the mature form. The decreased muscle weight observed in broilers injected with the antibody in the yolk indicates that myostatin activity was probably elevated by the binding of the antibody to the propeptide, and provides evidence that myostatin propeptide inhibits the biological activity of myostatin in broilers.

AAV-mediated delivery of a mutated myostatin propeptide ameliorates calpain 3 but not α-sarcoglycan deficiency

Myostatin is a negative regulator of muscle mass whose inhibition has been proposed as a therapeutic strategy for muscle-wasting conditions. Indeed, blocking myostatin action through different strategies has proved beneficial for the pathophysiology of the dystrophin-deficient mdx mouse. In this report, we tested the inhibition of myostatin by AAV-mediated expression of a mutated propeptide in animal models of two limb-girdle muscular dystrophies: LGMD2A caused by mutations in the calpain 3 (CAPN3) gene and LGMD2D caused by mutations in the alpha-sarcoglycan gene (SGCA). In the highly regenerative Sgca-null mice, survival of the alpha-sarcoglycan-deficient muscle fibers did not improve after transfer of the myostatin propeptide. In calpain 3-deficient mice, a boost in muscle mass and an increase in absolute force were obtained, suggesting that myostatin inhibition could constitute a therapeutic strategy in this predominantly atrophic disorder.

The role of myostatin and bone morphogenetic proteins in muscular disorders

Skeletal muscle is the largest organ in the human body, and plays an important role in body movement and metabolism. Skeletal muscle mass is lost in genetic disorders such as muscular dystrophy, muscle wasting and ageing. Chemicals and proteins that restore muscle mass and function are potential drugs that can improve human health and could be used in the clinic. Myostatin is a muscle-specific member of the transforming growth factor (TGF)-β superfamily that plays an essential role in the negative regulation of muscle growth. Inhibition of myostatin activity is a promising therapeutic method for restoring muscle mass and strength. Potential inhibitors of myostatin include follistatin domain-containing proteins, myostatin propeptide, myostatin antibodies and chemical compounds. These inhibitors could be beneficial for the development of clinical drugs for the treatment of muscular disorders. Bone morphogenetic protein (BMP) plays a significant role in the development of neuromuscular architecture and its proper functions. Modulation of BMP activity could be beneficial for muscle function in muscular disorders. This review will describe the current progress in therapy for muscular disorders, emphasising the importance of myostatin as a drug target.

Postnatal expression of myostatin propeptide cDNA maintained high muscle growth and normal adipose tissue mass in transgenic mice fed a high‐fat diet

Myostatin plays a robust, negative role in controlling muscle mass. A disruption of myostatin function by transgenic expression of its propeptide (the 5'region, 866 nucleotides) results in significant muscle growth (Yang et al., 2001. Mol Rep Dev 60:351-361). Studies from myostatin and the propeptide transgene mRNA indicated that myostatin mRNA was detected at day 10.5 postcoitum in fetal mice. Its level remained low, but increased by 180% during the postnatal fast-growth period (day 0-10). An early, high-level postnatal expression of the transgene was identified as being responsible for a highly muscled phenotype. High-fat diet induces adiposity in rodents. To study the effects of dietary fat on muscle growth and adipose tissue fat deposition in the transgenic mice, we challenged the mice with a high-fat diet (45% kcal fat) for 21 weeks. Transgenic mice showed 24%-50% further enhancement of growth on the high-fat diet compared to the normal-fat diet (P = 0.004) from 17 to 25 weeks of age. The total mass of the main muscles of transgenic mice showed a 27% increase on the high-fat diet compared to the normal-fat diet (P = 0.004), while the white adipose tissue mass of the transgenic mice was not significantly different from that of wild-type mice fed a normal-fat diet (P = 0.434). The high-fat diet induced wild-type mice developed 190% greater mass of white adipose tissues compared to the normal-fat diet (P = 0.008), which primarily resulted from enlarged adipocytes. These results demonstrate that disruption of myostatin function by its propeptide shifted dietary fat utilization toward muscle tissues with minimal effects on adiposity. These results suggest that enhancing muscle growth by myostatin propeptide or other means during the early developmental stage may serve as an effective means for obesity prevention.

Transgenic expression of myostatin propeptide prevents diet-induced obesity and insulin resistance

Obesity and insulin resistance cause serious consequences to human health. To study effects of skeletal muscle growth on obesity prevention, we focused on a key gene of skeletal muscle named myostatin, which plays an inhibitory role in muscle growth and development. We generated transgenic mice through muscle-specific expression of the cDNA sequence (5′-region 886 nucleotides) encoding for the propeptide of myostatin. The transgene effectively depressed myostatin function. Transgenic mice showed dramatic growth and muscle mass by 9 weeks of age. Here we reported that individual major muscles of transgenic mice were 45–115% heavier than those of wild-type mice, maintained normal blood glucose, insulin sensitivity, and fat mass after a 2-month regimen with a high-fat diet (45% kcal fat). In contrast, high-fat diet induced wild-type mice with 170–214% more fat mass than transgenic mice and developed impaired glucose tolerance and insulin resistance. Insulin signaling, measured by Akt phosphorylation, was significantly elevated by 144% in transgenic mice over wild-type mice fed a high-fat diet. Interestingly, high-fat diet significantly increased adiponectin secretion while blood insulin, resistin, and leptin levels remained normal in the transgenic mice. The results suggest that disruption of myostatin function by its propeptide favours dietary fat utilization for muscle growth and maintenance. An increased secretion of adiponectin may promote energy partition toward skeletal muscles, suggesting that a beneficial interaction between muscle and adipose tissue play a role in preventing obesity and insulin resistance.

1125. AAV-Mediated Myostatin Propeptide Gene Delivery Results in Growth and Hypertrophy of Skeletal but Not Cardiac Muscles

Muscular dystrophies are inherited myogenic disorders characterized by progressive muscle wasting and weakness. Because of the lack of effective treatment with traditional pharmaceutical agents, gene therapy and stem-cell therapy have been vigorously explored. Besides gene replacement therapy, a different form of gene therapy, i.e., expression of |[ldquo]|booster genes|[rdquo]|, has been investigated, aiming at alleviating the secondary deficiencies in muscular dystrophies rather than the primary ones. Blockade of myostatin is one of such strategies. Myostatin is a member of the TGF-beta family and a negative regulator of skeletal muscle growth. Studies using transgenic mice or antagonist antibody against myostatin showed promotion of muscle growth and amelioration of muscular dystrophy. However, gene delivery of myostatin inhibitors has not been reported.

Myostatin propeptide‐mediated amelioration of dystrophic pathophysiology

Mutations in myostatin (GDF8) cause marked increases in muscle mass, suggesting that this transforming growth factor-beta (TGF-beta) superfamily member negatively regulates muscle growth. Myostatin blockade therefore offers a strategy for reversing muscle wasting in Duchenne's muscular dystrophy (DMD) without resorting to genetic manipulation. Here, we demonstrate that pharmacological blockade using a myostatin propeptide stabilized by fusion to IgG-Fc improved pathophysiology of the mdx mouse model of DMD. Functional benefits evidenced by specific force improvement, exceeded those reported previously using myostatin antibody-mediated blockade. More importantly, use of a propeptide blockade strategy obviates possibilities of anti-idiotypic responses that could potentially limit the effectiveness of antibody-mediated myostatin blockade strategies over time. This study provides a novel pharmacological strategy for treatment of diseases associated with muscle wasting such as DMD and since it uses an endogenous inhibitor of myostatin should help circumvent technical hurdles and toxicity associated with conventional gene or cell based therapies.

Factors Affecting Morphology of Skeletal Muscles

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

Myostatin is a transforming growth factor beta 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.

Regulation of Myostatin in Vivo by Growth and Differentiation Factor-Associated Serum Protein-1: A Novel Protein with Protease Inhibitor and Follistatin Domains

Myostatin, a member of the TGFbeta superfamily, is a potent and specific negative regulator of skeletal muscle mass. In serum, myostatin circulates as part of a latent complex containing myostatin propeptide and/or follistatin-related gene (FLRG). Here, we report the identification of an additional protein associated with endogenous myostatin in normal mouse and human serum, discovered by affinity purification and mass spectrometry. This protein, which we have named growth and differentiation factor-associated serum protein-1 (GASP-1), contains multiple domains associated with protease-inhibitory proteins, including a whey acidic protein domain, a Kazal domain, two Kunitz domains, and a netrin domain. GASP-1 also contains a domain homologous to the 10-cysteine repeat found in follistatin, a protein that binds and inhibits activin, another member of the TGFbeta superfamily. We have cloned mouse GASP-1 and shown that it inhibits the biological activity of mature myostatin, but not activin, in a luciferase reporter gene assay. Surprisingly, recombinant GASP-1 binds directly not only to mature myostatin, but also to the myostatin propeptide. Thus, GASP-1 represents a novel class of inhibitory TGFbeta binding proteins.

The Myostatin Propeptide and the Follistatin-related Gene Are Inhibitory Binding Proteins of Myostatin in Normal Serum

Myostatin, also known as growth and differentiation factor 8, is a member of the transforming growth factor beta superfamily that negatively regulates skeletal muscle mass (1). Recent experiments have shown that myostatin activity is detected in serum by a reporter gene assay only after activation by acid, suggesting that native myostatin circulates as a latent complex (2). We have used a monoclonal myostatin antibody, JA16, to isolate the native myostatin complex from normal mouse and human serum. Analysis by mass spectrometry and Western blot shows that circulating myostatin is bound to at least two major proteins, the myostatin propeptide and the follistatin-related gene (FLRG). The myostatin propeptide is known to bind and inhibit myostatin in vitro (3). Here we show that this interaction is relevant in vivo, with a majority (>70%) of myostatin in serum bound to its propeptide. Studies with recombinant V5-His-tagged FLRG protein confirm a direct interaction between mature myostatin and FLRG. Functional studies show that FLRG inhibits myostatin activity in a reporter gene assay. These experiments suggest that the myostatin propeptide and FLRG are major negative regulators of myostatin in vivo.

Regulation of myostatin activity and muscle growth.

Myostatin is a transforming growth factor-beta 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.