POTENTIAL OF MYOSTATIN INHIBITION TO AMELIORATE AGE-ASSOCIATED MUSCLE DECLINE

Abstract

The degenerative loss of muscle mass and physical function with progressive age is defined as sarcopenia, leading to a pronounced decrease in the quality of life (physical disability, chronic disease, and mortality risk). Myostatin (MSTN), also called growth and differentiation factor 8, has been shown to be a powerful negative regulator of skeletal muscle mass suggesting that MSTN inhibition would be a potential strategy to ameliorate sarcopenia. The objective of this study was to produce a recombinant MSTN propeptide (MSTNpro), a strong MSTN inhibitor, to investigate the effects of MSTNpro on muscle cell proliferation and differentiation, then to investigate whether MSTN suppression with the in-house laboratory-developed recombinant MSTNpro has an effect on age-associated muscle decline in mice. Flatfish MSTNpro amino acid sequence 45-100 (fMSTNpro45-100) and mouse immunoglobulin fragment Fc (mFc) was assembled into a pMAL-c5X vector to form an expression plasmid. The plasmid fusion protein construct (MBP-fMSTNpro45-100mFc) was transformed into E. coli for protein expression. Protein expression was optimally carried out at 20°C for 8 hours. The MBP-fMSTNPro45-100mFc protein was purified from the supernatant of lysed cultures by a combination of amylose and Protein A affinity chromatography. 33 ug/mL of MBP-fMSTNPro45-100mFc was purified by amylose affinity chromatography then protein recovery was 22 ug/mL with subsequent purification by Protein A chromatography. The purified MBP-fMSTNPro45-100mFc protein suppressed MSTN activity with a bioactive potency comparable to a commercial MSTN propeptide in a pGL-(CAGA)12-luciferase reporter assay. Removal of MBP drastically lowered the MSTN-inhibitory capacity of the truncated form of MBP-fMSTNPro45-100mFc, indicating that MBP fusion potentiated the bioactivity of MSTNPro45-100mFc.Proliferating and differentiated C2C12 cells were treated with MBP-fMSTNPro45-100mFc. Expression of genes and proteins involved in myoblast proliferation and differentiation was examined using real time quantitative PCR (qPCR) and Western blot analysis. In pre-differentiated C2C12 cell cultures, treatment with MBP-fMSTNPro45-100mFc resulted in higher gene expression of myosin heavy chain (MHC) and myogenin while muRF1 gene expression was lower. MSTN, MyoD, mrf4, myf5, Akt, and myomaker gene expression levels were not significantly different in the proliferating myoblast cells. In differentiated C2C12 cell cultures, treatment with MBP-fMSTNPro45-100mFc resulted in greater gene expression of MHC, MSTN, myoMaker and MyoD. Myogenin, mrf4, myf5, and murf1 gene expression decreased in the treated cell cultures. Akt gene expression levels were comparable in the differentiated myoblast cell cultures. In pre-differentiated C2C12 cell cultures, treated cultures showed a significant decrease in Smad2 phosphorylation in ECL western blots while phosphorylated Akt was not apparent. The treated cell cultures showed a greater amount pAkt and myogenin. In differentiated C2C12 cell cultures, treated cultures showed a significant decrease in Smad2 phosphorylation. The treated cell cultures showed a greater amount pAkt and myoD. SDS-PAGE gels of differentiated C2C12 cell cultures showed a greater amount of MHC at the 250 kDa range. The data demonstrates MBP-fMSTNPro45-100mFc suppresses MSTN signaling in proliferating and differentiating C2C12 myoblasts and MSTN inhibition increases the proliferation and differentiation of C2C12 cells. The animal studies showed that MBP-fMSTNPro45-100mFc administration had no effects on body and in muscle mass in adult aging mice regardless of gender, but increased grip strength. A significant increase in spleen weight was observed in mice treated with MBP-fMSTNPro45-100mFc while weights of white and brown adipose tissue, liver, and kidney were not affected by the treatment. ELISA result showed the presence of antibodies against MBP and MBP-fMSTNPro45-100mFc in the serum of treated mice, suggesting that the MSTN-inhibitory capacity of MBP-fMSTNPro45-100mFc was neutralized by the immune response. In summary, the study found that 1) MBP-fMSTNPro45-100mFc, a recombinant protein produced in E. coli, has a strong MSTN-inhibitory capacity, 2) Removal of MBP, a fusion partner, drastically lowers the MSTN-inhibitory capacity of MBP-fMSTNPro45-100mFc, indicating that MBP fusion potentiated the bioactivity of MSTNPro45-100mFc, 3) MBP-fMSTNPro45-100mFc enhances the proliferation and differentiation of C2C12 myoblasts, supporting the in vitro result of MBP-fMSTNPro45-100mFc’s capacity to suppress MSTN, and indicating MBP-fMSTNPro45-100mFc is an excellent agent to suppress MSTN activity in vitro, 4) MBP-fMSTNPro45-100mFc did not increase muscle mass in adult aging mice, 5) MBP-fMSTNPro45-100mFc induced an immune reaction, suggesting that the MSTN-inhibitory capacity of the MSTNpro was neutralized by the immune response, indicating that MBP-fused recombinant MSTNPro is not a good reagent to suppress MSTN activity in vivo.