Myostatin Propeptide Research Articles

Myostatin inhibitory peptides in sports drug testing.

Across species, skeletal muscle mass is negatively regulated by the TGF-β cytokine myostatin (MSTN). Inhibitors of this growth factor and its signaling pathways are therefore not only promising therapeutics for muscular diseases but also potential performance-enhancing agents in sports. Within this study, protein precipitation and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) were employed to develop a detection method for six novel MSTN inhibitory peptides derived from the regulatory MSTN propeptide and the natural MSTN inhibitor follistatin (FST) from doping control serum samples. The approach was comprehensively characterized and found to allow for a specific detection down to concentrations of 3-9ng/mL. Moreover, several potential metabolites of the drug candidates referred to as DF-3, DF-25, and Peptide 7 were identified as valuable complementary analytical targets for doping control analytical assays. Overall, the acquired data pave the way for an implementation of MSTN inhibitory peptides into routine sports drug testing. Even though no drug candidate has obtained clinical approval yet, a proactive development of detection assays is of utmost importance to deter athletes from misusing such compounds, which are readily available for research purposes and on the black market.

Electrophoretic detection of black market myostatin propeptide.

Myostatin propeptide is prohibited according to chapter S4 of the "WADA 2022 List of Prohibited Substances and Methods." So far, no approved myostatin-propeptide pharmaceuticals are available. Nevertheless, myostatin-propeptides can be bought on the black market for "research purposes." A study on black market myostatin propeptide products is presented as well as electrophoretic detection methods for serum and urine. Out of the 12 tested products, only nine actually contained the protein. Separation by SDS-PAGE revealed that the nine products were relatively impure and that the main compound had a much higher mass (approximately 54-55 kDa) than expected (approximately 33 kDa). Further analyses by mass spectrometry showed that the elevated molecular mass was due to the presence of a full length GST-tag on the propeptide. The developed detection method for serum is based on immunoprecipitation (IP) followed by SDS-PAGE and Western blotting. In total, three anti-myostatin propeptide antibodies were tested. All of them were well suited for either IP or immunoblotting. The final protocol applies a biotinylated polyclonal antibody, streptavidin-coated magnetic beads, and a monoclonal detection antibody. For a sample volume of 500 μL serum, the detection limit of the method is approximately 2.5 ng/mL. The urine method applies a commercial ELISA for IP and performs with a limit of detection (LOD) of approximately 0.4 ng/mL. Furthermore, practically all currently available myostatin propeptide standards were also investigated. Due to the significant molecular mass difference of the black market products, an unambiguous differentiation from endogenous myostatin propeptide is possible.

Local myostatin inhibition improves skeletal muscle glucose uptake in insulin-resistant high-fat diet-fed mice.

Myostatin inhibition is thought to improve whole body insulin sensitivity and mitigate the development of insulin resistance in models of obesity. However, although myostatin is known to be a major regulator of skeletal muscle mass, the direct effects of myostatin inhibition in muscle on glucose uptake and the mechanisms that may underlie this are still unclear. We investigated the effect of local myostatin inhibition by adeno-associated virus-mediated overexpression of the myostatin propeptide on insulin-stimulated skeletal muscle glucose disposal in chow-fed or high fat diet-fed mice and evaluated the molecular pathways that might mediate this. We found that myostatin inhibition improved glucose disposal in obese high fat diet-fed mice alongside the induction of muscle hypertrophy but did not have an impact in chow-fed mice. This improvement was not associated with greater glucose transporter or peroxisome proliferator-activated receptor-γ coactivator-1α expression or 5' AMP-activated protein kinase activation as previously suggested. Instead, transcriptomic analysis suggested that the improvement in glucose disposal was associated with significant enrichment in genes involved in fatty acid metabolism and translation of mitochondrial genes. Thus, myostatin inhibition improves muscle insulin-stimulated glucose disposal in obese high fat diet-fed mice independent of muscle hypertrophy, potentially involving previously unidentified pathways.

Muscle Hyperplasia in Japanese Quail by Single Amino Acid Deletion in MSTN Propeptide.

Mutation in myostatin (MSTN), a negative regulator of muscle growth in skeletal muscle, resulted in increased muscle mass in mammals and fishes. However, MSTN mutation in avian species has not been reported. The objective of this study was to generate MSTN mutation in quail and investigate the effect of MSTN mutation in avian muscle growth. Recently, a new targeted gene knockout approach for the avian species has been developed using an adenoviral CRISPR/Cas9 system. By injecting the recombinant adenovirus containing CRISPR/Cas9 into the quail blastoderm, potential germline chimeras were generated and offspring with three base-pair deletion in the targeted region of the MSTN gene was identified. This non-frameshift mutation in MSTN resulted in deletion of cysteine 42 in the MSTN propeptide region and homozygous mutant quail showed significantly increased body weight and muscle mass with muscle hyperplasia compared to heterozygous mutant and wild-type quail. In addition, decreased fat pad weight and increased heart weight were observed in MSTN mutant quail in an age- and sex-dependent manner, respectively. Taken together, these data indicate anti-myogenic function of MSTN in the avian species and the importance of cysteine 42 in regulating MSTN function.

Myostatin inhibition in combination with antisense oligonucleotide therapy improves outcomes in spinal muscular atrophy.

BackgroundSpinal muscular atrophy (SMA) is caused by genetic defects in the survival motor neuron 1 (SMN1) gene that lead to SMN deficiency. Different SMN‐restoring therapies substantially prolong survival and function in transgenic mice of SMA. However, these therapies do not entirely prevent muscle atrophy and restore function completely. To further improve the outcome, we explored the potential of a combinatorial therapy by modulating SMN production and muscle‐enhancing approach as a novel therapeutic strategy for SMA.MethodsThe experiments were performed in a mouse model of severe SMA. A previously reported 25‐mer morpholino antisense oligomer PMO25 was used to restore SMN expression. The adeno‐associated virus‐mediated expression of myostatin propeptide was used to block the myostatin pathway. Newborn SMA mice were treated with a single subcutaneous injection of 40 μg/g (therapeutic dose) or 10 μg/g (low‐dose) PMO25 on its own or together with systemic delivery of a single dose of adeno‐associated virus‐mediated expression of myostatin propeptide. The multiple effects of myostatin inhibition on survival, skeletal muscle phenotype, motor function, neuromuscular junction maturation, and proprioceptive afferences were evaluated.ResultsWe show that myostatin inhibition acts synergistically with SMN‐restoring antisense therapy in SMA mice treated with the higher therapeutic dose PMO25 (40 μg/g), by increasing not only body weight (21% increase in male mice at Day 40), muscle mass (38% increase), and fibre size (35% increase in tibialis anterior muscle in 3 month female SMA mice), but also motor function and physical performance as measured in hanging wire test (two‐fold increase in time score) and treadmill exercise test (two‐fold increase in running distance). In SMA mice treated with low‐dose PMO25 (10 μg/g), the early application of myostatin inhibition prolongs survival (40% increase), improves neuromuscular junction maturation (50% increase) and innervation (30% increase), and increases both the size of sensory neurons in dorsal root ganglia (60% increase) and the preservation of proprioceptive synapses in the spinal cord (30% increase).ConclusionsThese data suggest that myostatin inhibition, in addition to the well‐known effect on muscle mass, can also positively influence the sensory neural circuits that may enhance motor neurons function. While the availability of the antisense drug Spinraza for SMA and other SMN‐enhancing therapies has provided unprecedented improvement in SMA patients, there are still unmet needs in these patients. Our study provides further rationale for considering myostatin inhibitors as a therapeutic intervention in SMA patients, in combination with SMN‐restoring drugs.

Effects of exosome-mediated delivery of myostatin propeptide on functional recovery of mdx mice

Duchenne muscular dystrophy (DMD) is a devastating disorder caused by loss of functional dystrophin protein, resulting in muscle wasting. Enhancing muscle growth by inhibiting myostatin, a growth factor negatively regulating skeletal muscle mass, is a promising approach to slow disease progression. Direct administration of myostatin propeptide, a natural inhibitor of mature myostatin, has shown limited efficacy probably due to low serum stability. Here, we demonstrate that serum stability, delivery efficiency and efficacy of propeptide can be significantly enhanced by anchoring propeptide to the surface of exosomes by fusing the inhibitory domain of myostatin propeptide into the second extracellular loop of CD63 (EXOpro). Repeated administrations of EXOpro accelerated muscle regeneration and growth, resulting in significantly increased muscle mass and functional rescue without any detectable toxicity in mdx mice. Importantly, EXOpro partially rehabilitated bone structure and promoted bone regeneration in mdx mice. Our findings demonstrate that anchoring to exosomes increased delivery and serum stability of propeptide and augmented the inhibitory efficacy of myostatin propeptide and thus provide a delivery platform for propeptide-based intervention in DMD.

A high-content cytokine screen identifies myostatin propeptide as a positive regulator of primitive chronic myeloid leukemia cells.

Aberrantly expressed cytokines in the bone marrow (BM) niche are increasingly recognized as critical mediators of survival and expansion of leukemic stem cells. To identify regulators of primitive chronic myeloid leukemia (CML) cells, we performed a high-content cytokine screen using primary CD34+ CD38low chronic phase CML cells. Out of the 313 unique human cytokines evaluated, 11 were found to expand cell numbers ≥2-fold in a 7-day culture. Focusing on novel positive regulators of primitive CML cells, the myostatin antagonist myostatin propeptide gave the largest increase in cell expansion and was chosen for further studies. Herein, we demonstrate that myostatin propeptide expands primitive CML and normal BM cells, as shown by increased colony-forming capacity. For primary CML samples, retention of CD34-expression was also seen after culture. Furthermore, we show expression of MSTN by CML mesenchymal stromal cells, and that myostatin propeptide has a direct and instant effect on CML cells, independent of myostatin, by demonstrating binding of myostatin propeptide to the cell surface and increased phosphorylation of STAT5 and SMAD2/3. In summary, we identify myostatin propeptide as a novel positive regulator of primitive CML cells and corresponding normal hematopoietic cells.

Identification of the minimum region of flatfish myostatin propeptide (Pep45-65) for myostatin inhibition and its potential to enhance muscle growth and performance in animals.

Myostatin (MSTN) negatively regulates skeletal muscle growth, and its activity is inhibited by the binding of MSTN propeptide (MSTNpro), the N-terminal domain of proMSTN that is proteolytically cleaved from the proMSTN. Partial sequences from the N-terminal side of MSTNpro have shown to be sufficient to inhibit MSTN activity. In this study, to determine the minimum size of flatfish MSTNpro for MSTN inhibition, various truncated forms of flatfish MSTNpro with N-terminal maltose binding protein (MBP) fusion were expressed in E. coli and purified. MSTNpro regions consisting of residues 45–68, -69, and -70 with MBP fusion suppressed MSTN activity with a potency comparable to that of full-sequence flatfish MSTNpro in a pGL3-(CAGA)12-luciferase reporter assay. Even though the MSTN-inhibitory potency was about 1,000-fold lower, the flatfish MSTNpro region containing residues 45–65 (MBP-Pro45-65) showed MSTN-inhibitory capacity but not the MBP-Pro45-64, indicating that the region 45–65 is the minimum domain required for MSTN binding and suppression of its activity. To examine the in vivo effect of MBP-fused, truncated flatfish MSTNpro, MBP-Pro45-70-His6 (20 mg/kg body wt) was subcutaneously injected 5 times for 14 days in mice. Body wt gain and bone mass were not affected by the administration. Grip strength and swimming time were significantly enhanced at 7 d after the administration. At 14 d, the effect on grip strength disappeared, and the extent of the effect on swimming time significantly diminished. The presence of antibody against MBP-Pro45-70-His6 was observed at both 7 and 14 d after the administration with the titer value at 14 d being much greater than that at 7 d, suggesting that antibodies against MBP-Pro45-70-His6 neutralized the MSTN-inhibitory effect of MBP-Pro45-70-His6. We, thus, examined the MSTN-inhibitory capacity and in vivo effect of flatfish MSTNpro region 45–65 peptide (Pep45-65-NH2), which was predicted to have no immunogenicity in silico analysis. Pep45-65-NH2 suppressed MSTN activity with a potency similar to that of MBP-Pro45-65 but did not suppress GDF11, or activin A. Pep45-65-NH2 blocked MSTN-induced Smad2 phosphorylation in HepG2 cells. The administration of Pep45-65 (20 mg/kg body wt, 5 times for 2 weeks) increased the body wt gain with a greater gain at 14 d than at 7 d and muscle wt. Grip strength and swimming time were also significantly enhanced by the administration. Antibody titer against Pep45-65 was not detected. In conclusion, current results indicate that MSTN-inhibitory proteins with heterologous fusion partner may not be effective in suppressing MSTN activity in vivo due to an immune response against the proteins. Current results also show that the region of flatfish MSTNpro consisting of 45–65 (Pep45-65) can suppress mouse MSTN activity and increase muscle mass and function without invoking an immune response, implying that Pep45-65 would be a potential agent to enhance skeletal muscle growth and function in animals or to treat muscle atrophy caused by various clinical conditions.

Recombinant porcine myostatin propeptide generated by the Pichia pastoris elevates myoblast growth and ameliorates high-fat diet-induced glucose intolerance

Myostatin (MSTN) was identified as a negative regulator of skeletal muscle growth. MSTN inhibition by myostatin propeptide (MSPP) increased skeletal muscle mass, myofiber growth and muscle force. Thus, this study was designed to produce wild-type porcine MSPP (WT-MSPP) and its mutated form (D75A-MSPP) in yeast Pichia pastoris and to investigate its potential enhancement of myoblast growth and differentiation. In an in vitro study, C2C12 myoblasts were treated with the purified WT-MSPP or D75A-MSPP (10 μg/mL) in either a regular culture medium or in a differentiation medium for 72 h. In an animal trial, post-weaning C57BL/6 mice fed with a high-fat diet (HFD) were administered WT-MSPP or D75A-MSPP for 6 weeks. The results showed that C2C12 myoblasts treated with the purified WT-MSPP or D75A-MSPP could dramatically promote cell proliferation. Both myoD and myogenin were significantly increased (p < .05) after WT-MSPP or D75A-MSPP treatment. D75A-MSPP was particularly more effective than WT-MSPP in promoting myotube formation (p < .05). The post-weaning mice treated with D75A-MSPP significantly increased both body and muscle weights compared with the mock and WT-MSPP groups (p < .05). Furthermore, the mice treatment with D75A-MSPP could prevent increased glucose injection from inducing glucose elevation. Our data indicated that a mutant-type MSPP (D75A-MSPP) was superior to WT-MSPP in effectively enhancing myofiber growth due to the highly resistant to proteolytic cleavage by the bone morphogenetic protein-1/tolloid (BMP-1/TLD) and thus has potential applications for clinical muscle wasting diseases or for increasing muscle mass in meat-producing animals.

Pretreatment of rAAV-Mediated Expression of Myostatin Propeptide Lowers Type 2 Diabetes Incidence inC57BL/6Mice on a High-Fat Diet

Myostatin, a negative modulator of muscle growth, has been considered as a potential target for the treatment of type 2 diabetes (T2D). In previous work, it was found that myostatin inhibition by adeno-associated virus (AAV)-mediated gene delivery of myostatin propeptide (MPRO) could improve muscle mass and achieve therapeutic effects on glucose regulation and lipid metabolism in db/db mice. This study investigated whether pre-intervention of rAAV-mediated expression of MPRO could lower the incidence of T2D. Three-week-old male C57BL/6 mice were randomly divided into saline control, rAAV-GFP, and rAAV-MPRO groups, all of which were fed on a high-fat diet. It was observed that pre-intervention of rAAV-MPRO prevented high-fat diet-induced hyperglycemia and hyperlipidemia. It also improved glucose tolerance, downregulated serum insulin levels, and facilitated the growth of skeletal muscle and fat redistribution, with no significant difference in serum free fatty acid levels and body weight, which ultimately reduced the incidence of T2D. In addition, pretreatment of rAAV-MPRO in C2C12 cells increased insulin-stimulated glucose uptake, as well as glycogen synthesis under insulin resistance conditions induced by free fatty acids, with no significant difference in insulin-stimulated glucose oxidation. Finally, the study demonstrated that improved glucose metabolism by rAAV-MPRO pretreatment might be due to the activation of the PI3K/Akt/GSK3β pathway and spurring Glut4 transposition from the cytoplasm to the cytomembrane in C2C12 cells. Based on these findings, MPRO is most likely to be a new method for the prevention of T2D.

Rapamycin suppresses postnatal muscle hypertrophy induced by myostatin-inhibition accompanied by transcriptional suppression of the Akt/mTOR pathway

Myostatin (MSTN) is a well-known negative growth factor of muscle mass, and studies have shown that MSTN-inhibition would be a potential strategy to treat muscle atrophy seen in various clinical conditions. Recent studies suggest that MSTN-inhibition induces skeletal muscle hypertrophy through up-regulation of the anabolic Akt/mTOR pathway. Therefore, it was hypothesized that the muscle hypertrophy induced by MSTN-inhibition would be suppressed by the administration of rapamycin (RAP), a mTOR suppressor. A MSTN transgenic mouse strain (MSTN-pro), which is characterized by a postnatal hyper-muscularity due to MSTN inhibition through transgenic overexpression of MSTN propeptide, was used in producing experimental animals. Five-week-old male heterozygous MSTN-pro mice and wild-type littermates were administered with 0 or 3 mg/kg body weight of RAP intraperitoneally every other day for 4 weeks. The effects of RAP on muscle growth, mRNA abundance of signaling components of the Akt/mTOR pathway, and myogenic regulatory factors (MyoD, Myf5, MyoG, and Mrf4) were examined in comparison to wild-type mice. Body weight gain of MSTN-pro mice was significantly greater than that of wild-type mice. RAP suppressed body weight gain and muscle mass in both MSTN-pro and wild-type mice. The extent of both body weight and muscle mass suppression was significantly greater in MSTN-pro mice than in wild-type mice. Real-time qPCR analysis showed that mRNA abundance of the signaling molecules of the Akt/mTOR pathway, including Akt, p70S6K, and 4E-BP1, were significantly higher in MSTN-pro mice. RAP treatment decreased mRNA abundance of Akt, p70S6K and 4E-BP1 only in MSTN-pro mice. mRNA abundances of MyoD and MyoG were not affected by MSTN suppression or RAP treatment. mRNA abundance of Myf5 was decreased by RAP, but not affected by MSTN suppression. mRNA abundance of Mrf4 was decreased by MSTN suppression. RAP treatment decreased mRNA abundance of Mrf4 only in wild type mice. Results of this study indicate that transcriptional regulation of signaling components of the Akt/mTOR pathway and myogenic regulatory transcription factor Mrf4 is involved in the enhancement of skeletal muscle mass induced by MSTN suppression.

Effect of myostatin inhibitor (myostatin pro-peptide) microinjection on in vitro maturation and subsequent early developmental stages of buffalo embryo

Expression of myostatin (MSTN, also known as growth differentiation factor 8, GDF8) was recently detected in cumulus-oocytes complexes (COCs), however little is known about its role in in vitro maturation (IVM) and fertilization (IVF) in large animals. Therefore, this study was designed to investigate the effect of MSTN inhibition on IVM of buffalo oocytes through investigation of IVM efficiency and expression of some specific genes in COCs from IVM till subsequent developmental stages following IVF. To reach this goal, we prepared a construct of adeno-associated virus (AAV) carrying MSTN pro-peptides (AAV-MSTNP) to inhibit MSTN. Over-expression of MSTNP was verified by upregulated expression of MSTNP and downregulated expression of the TGFβ receptor ActRIIb, the TGFβ signal transducer SMAD2 in COCs using qPCR. Microinjection of AAV-MSTNP to oocytes before IVM yielded a significant decrease in maturation rate as revealed by less cumulus cells expansion, fewer oocytes reaching metaphase II, and downregulation of cumulus expansion-related genes pentraxin 3 (Ptx3) and prostaglandin-endoperoxide synthase 2 (Ptgs2) as compared to the control and vehicle groups. These changes were also accompanied by elevated intracellular reactive oxygen species (ROS), upregulated expression of the apoptotic Bax gene, reduced antioxidant enzymes (SOD, CAT, GPX) activities, and downregulated expression of the antioxidant gene nuclear factor erythroid 2 like 2 (Nrf2), and the anti-apoptotic gene Bcl2 in COCs after IVM. Overexpression of MSTN inhibitor, MSTNP, also inhibited GDF9 and BMP15 genes expression in COCs. Additionally, both the fertilization efficiency and cleavage and blastocyst rates were significantly lower in MSTNP group than in the control and vehicle groups. The obtained data suggest an important role for MSTN during IVM and the subsequent developmental stages probably through, at least in part, inhibition of ROS production and apoptosis and modulation of IVM-related gene expression in COCs.

Maltose binding protein-fusion enhances the bioactivity of truncated forms of pig myostatin propeptide produced in E. coli.

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth. MSTN propeptide (MSTNpro) inhibits MSTN binding to its receptor through complex formation with MSTN, implying that MSTNpro can be a useful agent to improve skeletal muscle growth in meat-producing animals. Four different truncated forms of pig MSTNpro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in E. coli, and purified by the combination of affinity chromatography and gel filtration. The MSTN-inhibitory capacities of these proteins were examined in an in vitro gene reporter assay. A MBP-fused, truncated MSTNpro containing residues 42–175 (MBP-Pro42-175) exhibited the same MSTN-inhibitory potency as the full sequence MSTNpro. Truncated MSTNpro proteins containing either residues 42–115 (MBP-Pro42-115) or 42–98 (MBP-Pro42-98) also exhibited MSTN-inhibitory capacity even though the potencies were significantly lower than that of full sequence MSTNpro. In pull-down assays, MBP-Pro42-175, MBP-Pro42-115, and MBP-Pro42-98 demonstrated their binding to MSTN. MBP was removed from the truncated MSTNpro proteins by incubation with factor Xa to examine the potential role of MBP on MSTN-inhibitory capacity of those proteins. Removal of MBP from MBP-Pro42-175 and MBP-Pro42-98 resulted in 20-fold decrease in MSTN-inhibitory capacity of Pro42-175 and abolition of MSTN-inhibitory capacity of Pro42-98, indicating that MBP as fusion partner enhanced the MSTN-inhibitory capacity of those truncated MSTNpro proteins. In summary, this study shows that MBP is a very useful fusion partner in enhancing MSTN-inhibitory potency of truncated forms of MSTNpro proteins, and MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity.

Adeno-associated virus-mediated expression of myostatin propeptide improves the growth of skeletal muscle and attenuates hyperglycemia in db/db mice.

Inhibition of myostatin, a negative growth modulator for muscle, can functionally enhance muscle mass and improve glucose and fat metabolism in myostatin propeptide (MPRO) transgenic mice. This study was to investigate whether myostatin inhibition by adeno-associated virus (AAV)-mediated gene delivery of MPRO could improve muscle mass and achieve therapeutic effects on glucose regulation and lipid metabolism in the db/db mice and the mechanisms involved in that process. Eight-week-old male db/db mice were administered saline, AAV-GFP and AAV-MPRO/Fc vectors and monitored random blood glucose levels and body weight for 36 weeks. Body weight gain was not different during follow-up among the groups, but AAV-MPRO/Fc vectors resulted high level of MPRO in the blood companied by an increase in skeletal muscle mass and muscle hypertrophy. In addition, AAV-MPRO/Fc-treated db/db mice showed significantly lower blood glucose and insulin levels and significantly increased glucose tolerance and insulin sensitivity compared with the control groups (P<0.05). Moreover, these mice exhibited lower triglyceride (TG) and free fatty acid (FFA) content in the skeletal muscle, although no difference was observed in fat pad weights and serum TG and FFA levels. Finally, AAV-MPRO/Fc-treated mice had enhanced insulin signaling in the skeletal muscle. These data suggest that AAV-mediated MPRO therapy may provide an important clue for potential clinical applications to prevent type II diabetes, and these studies confirm that MPRO is a therapeutic target for type II diabetes.

Myostatin propeptide mutation of the hypermuscular Compact mice decreases the formation of myostatin and improves insulin sensitivity.

The TGFβ family member myostatin (growth/differentiation factor-8) is a negative regulator of skeletal muscle growth. The hypermuscular Compact mice carry the 12-bp Mstn(Cmpt-dl1Abc) deletion in the sequence encoding the propeptide region of the precursor promyostatin, and additional modifier genes of the Compact genetic background contribute to determine the full expression of the phenotype. In this study, by using mice strains carrying mutant or wild-type myostatin alleles with the Compact genetic background and nonmutant myostatin with the wild-type background, we studied separately the effect of the Mstn(Cmpt-dl1Abc) mutation or the Compact genetic background on morphology, metabolism, and signaling. We show that both the Compact myostatin mutation and Compact genetic background account for determination of skeletal muscle size. Despite the increased musculature of Compacts, the absolute size of heart and kidney is not influenced by myostatin mutation; however, the Compact genetic background increases them. Both Compact myostatin and genetic background exhibit systemic metabolic effects. The Compact mutation decreases adiposity and improves whole body glucose uptake, insulin sensitivity, and 18FDG uptake of skeletal muscle and white adipose tissue, whereas the Compact genetic background has the opposite effect. Importantly, the mutation does not prevent the formation of mature myostatin; however, a decrease in myostatin level was observed, leading to altered activation of Smad2, Smad1/5/8, and Akt, and an increased level of p-AS160, a Rab-GTPase-activating protein responsible for GLUT4 translocation. Based on our analysis, the Compact genetic background strengthens the effect of myostatin mutation on muscle mass, but those can compensate for each other when systemic metabolic effects are compared.

Activin Receptor Type IIB Inhibition Improves Muscle Phenotype and Function in a Mouse Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a devastating neurodegenerative disorder that causes progressive muscle atrophy and weakness. Using adeno-associated virus-mediated gene transfer, we evaluated the potential to improve skeletal muscle weakness via systemic, postnatal inhibition of either myostatin or all signaling via the activin receptor type IIB (ActRIIB). After demonstrating elevated p-SMAD3 content and differential content of ActRIIB ligands, 4-week-old male C/C SMA model mice were treated intraperitoneally with 1x1012 genome copies of pseudotype 2/8 virus encoding a soluble form of the ActRIIB extracellular domain (sActRIIB) or protease-resistant myostatin propeptide (dnMstn) driven by a liver specific promoter. At 12 weeks of age, muscle mass and function were improved in treated C/C mice by both treatments, compared to controls. The fast fiber type muscles had a greater response to treatment than did slow muscles, and the greatest therapeutic effects were found with sActRIIB treatment. Myostatin/activin inhibition, however, did not rescue C/C mice from the reduction in motor unit numbers of the tibialis anterior muscle. Collectively, this study indicates that myostatin/activin inhibition represents a potential therapeutic strategy to increase muscle mass and strength, but not neuromuscular junction defects, in less severe forms of SMA.

Myostatin propeptide gene delivery by gene gun ameliorates muscle atrophy in a rat model of botulinum toxin-induced nerve denervation

AimMuscle atrophy is a common symptom after nerve denervation. Myostatin propeptide, a precursor of myostatin, has been documented to improve muscle growth. However, the mechanism underlying the muscle atrophy attenuation effects of myostatin propeptide in muscles and the changes in gene expression are not well established. We investigated the possible underlying mechanisms associated with myostatin propeptide gene delivery by gene gun in a rat denervation muscle atrophy model, and evaluated gene expression patterns. Main methodsIn a rat botulinum toxin-induced nerve denervation muscle atrophy model, we evaluated the effects of wild-type (MSPP) and mutant-type (MSPPD75A) of myostatin propeptide gene delivery, and observed changes in gene activation associated with the neuromuscular junction, muscle and nerve. Key findingsMuscle mass and muscle fiber size was moderately increased in myostatin propeptide treated muscles (p<0.05). And enhancement of the gene expression of the muscle regulatory factors, neurite outgrowth factors (IGF-1, GAP43) and acetylcholine receptors was observed. Our results demonstrate that myostatin propeptide gene delivery, especially the mutant-type of MSPPD75A, attenuates muscle atrophy through myogenic regulatory factors and acetylcholine receptor regulation. SignificanceOur data concluded that myostatin propeptide gene therapy may be a promising treatment for nerve denervation induced muscle atrophy.

Effect of MSTN propeptide protein on the growth and development of Altay lamb muscle.

Prokaryotic expression technology was used to express maltose-binding protein binding myostatin (MSTN) propeptide fusion protein. Six disease-free Altay lambs were used in this study. The right leg gastrocnemii were injected with MSTN recombinant propeptide protein. The left leg gastrocnemii (the control group) were injected with the same dose of phosphate based saline. The lambs were fed during four months under the same conditions and then slaughtered. Gastrocnemius samples were hematoxylin-eosin stained and the size of the muscle fibers was measured. A real-time polymerase chain reaction (RT-PCR) showed that single gastrocnemius cells in the experimental group had an average area of 1163.01 µm(2), while it was 845.09 µm(2) in the control group (P < 0.05). This indicates that the MSTN propeptide biological agents had an inhibitory effect on MSTN. In order to reveal its mechanism, RT-PCR was conducted to detect the expression of the differentiation-associated genes MyoD, Myf5, Myogenin, p21, and Smad3. The results showed that, in the MSTN propeptide biological agent injected group, expression levels of MSTN, Smad3, and p21 were lower than the control group, while Myf5, MyoD, and Myogenin were higher compared to the control group. This indicates that, when expression of the MSTN gene was inhibited, muscle cell differentiation and growth can be promoted by Smad3 up-regulated expression of Myf5, MyoD, and Myogenin.

Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice.

Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity.

Functional verification of a porcine myostatin propeptide mutant.

Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells.