Mutations in the Myostatin gene leading to hypermuscularity in mammals: indications for a similar mechanism in fish?

The transforming growth factor β (TGF-β) superfamily encodes secreted factors that are important in regulating embryonic development and tissue homeostatis in adults. Myostatin (MSTN, encoded by MSTN) or 'growth and differentiation factor 8', a member of this superfamily, is a negative regulator of skeletal muscle growth and is highly conserved among animal species. In 1997, a mutation associated with the so-called double-muscling phenotype in cattle was found in the MSTN gene. During the years following the discovery of the first MSTN mutation, other mutations were found in cattle and other mammalian species, and MSTN became one of the most thoroughly studied genes in animals. The aim of this review is mainly to describe the functional mutations located in the MSTN genes of several mammalian species, leading to double muscling in these animals. Furthermore, in light of the increasing importance of fish genetics, the possibility of functional mutations in piscine MSTN with a similar effect as in mammals, and a genetic model for MSTN research in fish, will also be discussed.     

Prodomains of transforming growth factor beta (TGFbeta) superfamily members specify different functions: extracellular matrix interactions and growth factor bioavailability

The specific functions of the prodomains of TGFβ superfamily members are largely unknown. Interactions are known between prodomains of TGFβ-1-3 and latent TGFβ-binding proteins and between prodomains of BMP-2, -4, -7, and -10 and GDF-5 and fibrillins, raising the possibility that latent TGFβ-binding proteins and fibrillins may mediate interactions with all other prodomains of this superfamily. This possibility is tested in this study. Results show that the prodomain of BMP-5 interacts with the N-terminal regions of fibrillin-1 and -2 in a site similar to the binding sites for other bone morphogenetic proteins. However, in contrast, the prodomain of GDF-8 (myostatin) interacts with the glycosaminoglycan side chains of perlecan. The binding site for the GDF-8 prodomain is likely the heparan sulfate chain present on perlecan domain V. These results support and extend the emerging concept that TGFβ superfamily prodomains target their growth factor dimers to extracellular matrix macromolecules. In addition, biochemical studies of prodomain·growth factor complexes were performed to identify inactive complexes. For some members of the superfamily, the prodomain is noncovalently associated with its growth factor dimer in an inactive complex; for others, the prodomain·growth factor complex is active, even though the prodomain is noncovalently associated with its growth factor dimer. Results show that the BMP-10 prodomain, in contrast to BMP-4, -5, and -7 prodomains, can inhibit the bioactivity of the BMP-10 growth factor and suggest that the BMP-10 complex is like TGFβ and GDF-8 complexes, which can be activated by cleavage of the associated prodomain.     

Myostatin-deficient medaka exhibit a double-muscling phenotype with hyperplasia and hypertrophy, which occur sequentially during post-hatch development

Myostatin (MSTN) functions as a negative regulator of skeletal muscle mass. In mammals, MSTN-deficient animals result in an increase of skeletal muscle mass with both hyperplasia and hypertrophy. A MSTN gene is highly conserved within the fish species, allowing speculation that MSTN-deficient fish could exhibit a double-muscled phenotype. Some strategies for blocking or knocking down MSTN in adult fish have been already performed; however, these fish show either only hyperplastic or hypertrophic growth in muscle fiber. Therefore, the role of MSTN in fish myogenesis during post-hatch growth remains unclear. To address this question, we have made MSTN-deficient medaka (mstnC315Y) by using the targeting induced local lesions in a genome method. mstnC315Y can reproduce and have the same survival period as WT medaka. Growth rates of WT and mstnC315Y were measured at juvenile (1–2 wk post-hatching), post-juvenile (3–7 wk post-hatching) and adult (8–16 wk post-hatching) stages. In addition, effects of MSTN on skeletal muscle differentiation were investigated at histological and molecular levels at each developmental stage. As a result, mstnC315Y show a significant increase in body weight from the post-juvenile to adult stage. Hyper-morphogenesis of skeletal muscle in mstnC315Y was accomplished due to hyperplastic growth from post-juvenile to early adult stage, followed by hypertrophic growth in the adult stage. Myf-5 and MyoD were up-regulated in mstnC315Y at the hyperplastic growth phase, while myogenin was highly expressed in mstnC315Y at the hypertrophic growth phase. These indicated that MSTN in medaka plays a dual role for muscle fiber development. In conclusion, MSTN in medaka regulates the number and size of muscle fiber in a temporally-controlled manner during posthatch growth.     

Myostatin as a therapeutic target in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis is a devastating neurological disease that is inevitably fatal after 3–5 years duration. Treatment options are minimal and as such new therapeutic modalities are required. In this review, we discuss the role of the myostatin pathway as a modulator of skeletal muscle mass and therapeutic approaches using biological based therapies. Both monoclonal antibodies to myostatin and a soluble receptor decoy to its high affinity receptor have been used in clinical trials of neuromuscular diseases and while there have been efficacy signals with the latter approach there have also been safety issues. Our approach is to target the high affinity receptor-binding site on myostatin and to develop a next generation set of therapeutic reagents built on a novel protein scaffold. This is the natural single domain VNAR found in sharks which is extremely versatile and has the ability to develop products with superior properties compared to existing therapeutics.     

Production of bioactive extracellular domain of pig and chicken activin type IIB receptors in Pichia pastoris

Myostatin (MSTN) is a potent negative regulator for skeletal muscle growth, and binds to activin type IIB receptor (ActRIIB) for its cellular signal transduction. Administration of the extracellular domain of ActRIIB (ActRIIB-ECD) improved skeletal muscle growth in laboratory animals, suggesting that ActRIIB-ECD can be a useful pharmacological agent to improve skeletal muscle growth of meat-producing animals. In the current study, pig and chicken ActRIIB-ECDs were produced in the Pichia pastoris GS115, and the recombinant proteins were purified from induced culture media by Ni-NTA affinity chromatography. The digestion of pig and chicken ActRIIB-ECDs with PNGase F and glycoprotein staining demonstrated an N-linked glycosylation of these recombinant proteins. Glycoprotein staining also indicated an additional presence of glycosylation in chicken ActRIIB-ECD. Both the pig and chicken ActRIIB-ECDs were shown to inhibit MSTN activity in a reporter gene assay system in vitro. When MSTN-inhibitory potencies were compared by analyzing EC₅₀ values, no difference in MSTN-inhibitory potency was observed between the glycosylated and N-deglycosylated forms of pig or chicken ActRIIB-ECD, suggesting that glycosylation does not affect the bioactivity of ActRIIB-ECD. MSTN-inhibitory potency of chicken ActRIIB-ECD was greater (P < 0.01) than that of pig ActRIIB-ECD. Results of this study demonstrate that bioactive pig and chicken ActRIIB-ECDs can be produced from P. pastoris. In addition, the study indicates that the N-glycosylation status of ActRIIB-ECD does not affect its bioactivity in vitro.     

Enhancement of C2C12 myoblast proliferation and differentiation by GASP-2, a myostatin inhibitor

BackgroundGASP-2 is a secreted multi-domain glycoprotein known as a specific inhibitor of myostatin and GDF-11. Here we investigate the role of GASP-2 on myogenesis and the effect of its glycosylation on its activity.MethodsGASP-2 overexpression or knockdown by shRNAs were carried out on C2C12 myoblasts cells. In silico analysis of GASP-2 protein was performed to identify its glycosylation sites. We produced a mouse recombinant GASP-2 protein in a prokaryotic system to obtain a fully deglycosylated protein allowing us to study the importance of this post-translational modification on GASP-2 activity.ResultsBoth mature and deglycosylated GASP-2 proteins increase C2C12 proliferation and differentiation by inhibiting the myostatin pathway. In silico and western-blot analyses revealed that GASP-2 presents one consensus sequence for N-glycosylation and six potential sites of mucin-type O-glycosylation.ConclusionsGASP-2 promotes myogenesis and thus independently of its glycosylation.General significanceThis is the first report demonstrating that GASP-2 promotes proliferation and differentiation of myoblasts by inhibiting the canonical pathway of myostatin.     

Myostatin deficiency but not anti‐myostatin blockade induces marked proteomic changes in mouse skeletal muscle

Pharmacologic blockade of the myostatin (Mstn)/activin receptor pathway is being pursued as a potential therapy for several muscle wasting disorders. The functional benefits of blocking this pathway are under investigation, in particular given the findings that greater muscle hypertrophy results from Mstn deficiency arising from genetic ablation compared to post‐developmental Mstn blockade. Using high‐resolution MS coupled with SILAC mouse technology, we quantitated the relative proteomic changes in gastrocnemius muscle from Mstn knockout (Mstn^?/?) and mice treated for 2‐weeks with REGN1033, an anti‐Mstn antibody. Relative to wild‐type animals, Mstn^?/? mice had a two‐fold greater muscle mass and a >1.5‐fold change in expression of 12.0% of 1137 quantified muscle proteins. In contrast, mice treated with REGN1033 had minimal changes in muscle proteome (0.7% of 1510 proteins >1.5‐fold change, similar to biological difference 0.5% of 1310) even though the treatment induced significant 20% muscle mass increase. Functional annotation of the altered proteins in Mstn^?/? mice corroborates the mutiple physiological changes including slow‐to‐fast fiber type switch. Thus, the proteome‐wide protein expression differs between Mstn^?/? mice and mice subjected to specific Mstn blockade post‐developmentally, providing molecular‐level insights to inform mechanistic hypotheses to explain the observed functional differences.     

Quercetin glycosides prevent dexamethasone-induced muscle atrophy in mice

Although quercetin has numerous biological benefits, including preventing muscle atrophy due to disuse, no reports have been published to date about the preventive effects and molecular mechanisms underlying drug-induced muscle atrophy. Highly soluble and bioavailable quercetin glycosides (QGs) were used to examine the inhibition of dexamethasone (DEX)-induced muscle atrophy in vivo. Male BALB/cCrSlc mice were treated with or without QGs for 7 days ad libitum, followed by addition of DEX to their drinking water for a further 7 days. The weight of gastrocnemius (GM) adjusted by body weight was significantly decreased on day 7 after DEX treatment. DEX-induced decrease of GM weight was improved by QG co-administration on day 7. The mRNA levels of muscle atrophy-related genes in the gastrocnemius were significantly lowered by QGs on day 1. In particular, the expression of myostatin, a master regulator of muscle mass homeostasis, was suppressed to that of the control level. In murine C2C12 myotubes, quercetin elevated the phosphorylation of Akt, which are downstream of the myostatin pathway, as well as expression of atrogenes. We demonstrated the protective effect of QGs in DEX-induced muscle atrophy, which might depend on the suppression of myostatin signaling.     

The depletion of skeletal muscle satellite cells with age is concomitant with reduced capacity of single progenitors to produce reserve progeny

Satellite cells are myogenic progenitors that reside on the myofiber surface and support skeletal muscle repair. We used mice in which satellite cells were detected by GFP expression driven by nestin gene regulatory elements to define age-related changes in both numbers of satellite cells that occupy hindlimb myofibers and their individual performance. We demonstrate a reduction in satellite cells per myofiber with age that is more prominent in females compared to males. Satellite cell loss also persists with age in myostatin-null mice regardless of increased muscle mass. Immunofluorescent analysis of isolated myofibers from nestin-GFP/Myf5^nLacZ/+ mice reveals a decline with age in the number of satellite cells that express detectable levels of βgal. Nestin-GFP expression typically diminishes in primary cultures of satellite cells as myogenic progeny proliferate and differentiate, but GFP subsequently reappears in the Pax7⁺ reserve population. Clonal analysis of sorted GFP⁺ satellite cells from hindlimb muscles shows heterogeneity in the extent of cell density and myotube formation among colonies. Reserve cells emerge primarily within high-density colonies, and the number of clones that produce reserve cells is reduced with age. Thus, satellite cell depletion with age could be attributed to a reduced capacity to generate a reserve population.