Sepsis downregulates myostatin mRNA levels without altering myostatin protein levels in skeletal muscle

Myostatin is a negative regulator of muscle mass and has been reported to be upregulated in several conditions characterized by muscle atrophy. The influence of sepsis on myostatin expression and activity is poorly understood. Here, we tested the hypothesis that sepsis upregulates the expression and downstream signaling of myostatin in skeletal muscle. Because sepsis‐induced muscle wasting is at least in part regulated by glucocorticoids, we also determined the influence of glucocorticoids on myostatin expression. Sepsis was induced in rats by cecal ligation and puncture and control rats were sham‐operated. In other experiments, rats were injected intraperitoneally with dexamethasone (10 mg/kg) or corresponding volume of vehicle. Surprisingly, myostatin mRNA levels were reduced and myostatin protein levels were unchanged in muscles from septic rats. Muscle levels of activin A, follistatin, and total and phosphorylated Smad2 (p‐Smad2) were not influenced by sepsis, suggesting that myostatin downstream signaling was not altered during sepsis. Interestingly, total and p‐Smad3 levels were increased in septic muscle, possibly reflecting altered signaling through pathways other than myostatin. Similar to sepsis, treatment of rats with dexamethasone reduced myostatin mRNA levels and did not alter myostatin protein levels. Fasting, an additional condition characterized by muscle wasting, reduced myostatin mRNA and activin A protein levels, increased myostatin protein, and did not influence follistatin and p‐Smad2 levels. Of note, total and p‐Smad3 levels were reduced in muscle during fasting. The results suggest that sepsis and glucocorticoids do not upregulate the expression and activity of myostatin in skeletal muscle. The role of myostatin may vary between different conditions characterized by muscle wasting. Downstream signaling through Smad2 and 3 is probably regulated not only by myostatin but by other mechanisms as well. J. Cell. Biochem. 111: 1059–1073, 2010. © 2010 Wiley‐Liss, Inc.     

Design and synthesis of potent myostatin inhibitory cyclic peptides

Myostatin is a negative regulator of skeletal muscle growth and myostatin inhibitors are promising lead compounds against muscle atrophic disorders such as muscular dystrophy. Previously, we published the first report of synthetic myostatin inhibitory 23-mer peptide 1, which was identified from a myostatin precursor-derived prodomain protein. Our structure-activity relationship study afforded the potent inhibitory peptide 3. In this paper, we report an investigation of the synthesis of conformationally-constrained cyclic peptide based on the linear peptide 3. To examine the potency of side chain-to-side chain cyclized peptides, a series of disulfide-, lactam- and diester-bridged derivatives were designed and synthesized, and their myostatin inhibitory activities were evaluated. The diester-bridged peptide (11) displayed potent inhibitory activity with an in vitro IC₅₀ value of 0.26?µM, suggesting that it could serve as a new platform for development of cyclic peptide inhibitors.     

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.     

An Antibody Blocking Activin Type II Receptors Induces Strong Skeletal Muscle Hypertrophy and Protects from Atrophy

The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings.     

Structure of myostatin·follistatin-like 3: N-terminal domains of follistatin-type molecules exhibit alternate modes of binding

TGF-β family ligands are involved in a variety of critical physiological processes. For instance, the TGF-β ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-β family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes.     

Myostatin induces cyclin D1 degradation to cause cell cycle arrest through a phosphatidylinositol 3-kinase/AKT/GSK-3 beta pathway and is antagonized by insulin-like growth factor 1

Myostatin is a transforming growth factor beta superfamily member and is known as an inhibitor of skeletal muscle cell proliferation and differentiation. Exposure to myostatin induces G1 phase cell cycle arrest. In this study, we demonstrated that myostatin down-regulates Cdk4 activity via promotion of cyclin D1 degradation. Overexpression of cyclin D1 significantly blocked myostatin-induced proliferation inhibition. We further showed that phosphorylation at threonine 286 by GSK-3beta was required for myostatin-stimulated cyclin D1 nuclear export and degradation. This process is dependent upon the activin receptor IIB and the phosphatidylinositol 3-kinase/Akt pathway but not Smad3. Insulin-like growth factor 1 (IGF-1) treatment or Akt activation attenuated the myostatin-stimulated cyclin D1 degradation as well as the associated cell proliferation repression. In contrast, attenuation of IGF-1 signaling caused C2C12 cells to undergo apoptosis in response to myostatin treatment. The observation that IGF-1 treatment increases myostatin expression through a phosphatidylinositol 3-kinase pathway suggests a possible feedback regulation between IGF-1 and myostatin. These findings uncover a novel role for myostatin in the regulation of cell growth and cell death in concert with IGF-1.     

Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C₂C₁₂ myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C₂C₁₂ myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.     

Myostatin Suppression of Akirin1 Mediates Glucocorticoid-Induced Satellite Cell Dysfunction

Glucocorticoids production is increased in many pathological conditions that are associated with muscle loss, but their role in causing muscle wasting is not fully understood. We have demonstrated a new mechanism of glucocorticoid-induced muscle atrophy: Dexamethasone (Dex) suppresses satellite cell function contributing to the development of muscle atrophy. Specifically, we found that Dex decreases satellite cell proliferation and differentiation in vitro and in vivo. The mechanism involved Dex-induced upregulation of myostatin and suppression of Akirin1, a promyogenic gene. When myostatin was inhibited in Dex-treated mice, Akirin1 expression increased as did satellite cell activity, muscle regeneration and muscle growth. In addition, silencing myostatin in myoblasts or satellite cells prevented Dex from suppressing Akirin1 expression and cellular proliferation and differentiation. Finally, overexpression of Akirin1 in myoblasts increased their expression of MyoD and myogenin and improved cellular proliferation and differentiation, theses improvements were no longer suppressed by Dex. We conclude that glucocorticoids stimulate myostatin which inhibits Akirin1 expression and the reparative functions of satellite cells. These responses attribute to muscle atrophy. Thus, inhibition of myostatin or increasing Akirin1 expression could lead to therapeutic strategies for improving satellite cell activation and enhancing muscle growth in diseases associated with increased glucocorticoid production.     

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.     

Skeletal muscle-derived progenitors capable of differentiating into cardiomyocytes proliferate through myostatin-independent TGF-beta family signaling

The existence of skeletal muscle-derived stem cells (MDSCs) has been suggested in mammals; however, the signaling pathways controlling MDSC proliferation remain largely unknown. Here we report the isolation of myosphere-derived progenitor cells (MDPCs) that can give rise to beating cardiomyocytes from adult skeletal muscle. We identified that follistatin, an antagonist of TGF-β family members, was predominantly expressed in MDPCs, whereas myostatin was mainly expressed in myogenic cells and mature skeletal muscle. Although follistatin enhanced the replicative growth of MDPCs through Smad2/3 inactivation and cell cycle progression, disruption of myostatin did not increase the MDPC proliferation. By contrast, inhibition of activin A (ActA) or growth differentiation factor 11 (GDF11) signaling dramatically increased MDPC proliferation via down-regulation of p21 and increases in the levels of cdk2/4 and cyclin D1. Thus, follistatin may be an effective progenitor-enhancing agent neutralizing ActA and GDF11 signaling to regulate the growth of MDPCs in skeletal muscle.     

Haploinsufficiency of myostatin protects against aging‐related declines in muscle function and enhances the longevity of mice

The molecular mechanisms behind aging-related declines in muscle function are not well understood, but the growth factor myostatin (MSTN) appears to play an important role in this process. Additionally, epidemiological studies have identified a positive correlation between skeletal muscle mass and longevity. Given the role of myostatin in regulating muscle size, and the correlation between muscle mass and longevity, we tested the hypotheses that the deficiency of myostatin would protect oldest-old mice (28–30 months old) from an aging-related loss in muscle size and contractility, and would extend the maximum lifespan of mice. We found that MSTN^+/− and MSTN^−/− mice were protected from aging-related declines in muscle mass and contractility. While no differences were detected between MSTN^+/+ and MSTN^−/− mice, MSTN^+/− mice had an approximately 15% increase in maximal lifespan. These results suggest that targeting myostatin may protect against aging-related changes in skeletal muscle and contribute to enhanced longevity.     

Characterization of follistatin-type domains and their contribution to myostatin and activin a antagonism

Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-β family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-β family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists.     

The single nucleotide polymorphisms of the chicken myostatin gene are associated with skeletal muscle and adipose growth

Myostatin, a new member of the TGF-ß superfamily, is predominantly expressed in skeletal muscle cells and functions as a negative regulator of skeletal muscle growth in animals. Recently, we have reported three single nucleotide polymorphisms (SNPs) in the chicken myostatin gene. Herein, we investigate the association of those SNPs with the production traits in a F₂ chicken line derived from Broilers crossing to Silky with the least square analysis. The results show that the BB and AA genotypes are strongly associated with abdominal fat weight (AFW), abdominal fat percentage (AFP), and birth weight (BW) (P < 0.05). Breast muscle percentage (BMP) of the AA type is higher than that of the AB type. The breast muscle weight and breast muscle percentages of F₂ individuals have significant difference between CC and DD genotypes (P < 0.05). Breast muscle weight (BMW) of EF birds is higher than that of EE birds (P < 0.05). In this report, we present the first genetic evidence to show that chicken myostatin not only plays an important role in controlling skeletal muscle growth and differentiation, but also may be involved in regulation of adipose growth in chicken.     

Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Normal and Dystrophic Mice

Background

Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies.

Methodology/Principal Findings

Here we describe a unique method of myostatin inhibition utilizing recombinant adeno-associated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophin-deficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose–dependent manner.

Conclusions/Significance

Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition on striated muscle.