PSVII-20 - Effects of MBP-fMSTNPro45-100mFc, a myostatin inhibitory protein, on myoblast proliferation and differentiation

Abstract: Myostatin (MSTN) is a powerful negative regulator of skeletal muscle mass, suggesting that MSTN inhibition would be a potential strategy to improve muscle growth in animals or to ameliorate muscle atrophy occurring in various conditions. The objective of this study was to produce a bioactive recombinant MSTN inhibitor and to investigate its effect on myoblast proliferation and differentiation. A truncated form of flatfish MSTN propeptide (MSTNpro) containing N-terminal maltose-binding protein (MBP) and C-terminal mouse immunoglobulin Fc fragment (MBP-fMSTNpro45-100mFc) was produced in E. coli. MBP-fMSTNPro45-100mFc protein was purified from the supernatant of lysed cultures by a combination of amylose and Protein A affinity chromatography. The purified MBP-fMSTNPro45-100mFc suppressed MSTN activity with bioactivity comparable to a commercial mice MSTNpro in a pGL3 (CAGA)₁₂-luciferase reporter assay. Removal of MBP from the MBP-fMSTNPro45-100mFc drastically lowered the MSTN-inhibitory capacity, indicating that MBP fusion potentiated the bioactivity of MSTNPro45-100mFc. Proliferating and differentiated C₂C₁₂ cells were treated with MBP-fMSTNPro45-100mFc. The expression of genes and proteins involved in myoblast proliferation and differentiation was examined using qPCR and Western blot analysis. The phosphorylation of Smad2, a downstream effector for MSTN signaling, was significantly (P < 0.01) suppressed by MBP-fMSTNPro45-100mFc in the proliferating and differentiating C₂C₁₂ cells, demonstrating the MSTN-inhibitory capacity of MBP-fMSTNPro45-100mFc in myoblasts. When the proliferation was examined using the MTT assay, MBP-fMSTNPro45-100mFc treatment increased the proliferation of C₂C₁₂ cells in a dose-dependent manner during the 6 days of treatment. In support of the MTT assay result, H&E staining showed a greater number of cells in the C₂C₁₂ culture treated with 100 nM MBP-fMSTNPro45-100mFc as compared with the control culture with no MBP-fMSTNPro45-100mFc. In pre-differentiated C₂C₁₂ cell cultures, treatment with MBP-fMSTNPro45-100mFc significantly (P < 0.05) increased the mRNA expression of myosin heavy chain and myogenin but had no significant effect on the mRNA expression of MSTN, MyoD, Mrf4, Myf5, Akt, and myomaker. The abundance of myogenin protein and phosphorylated Akt in the myoblast culture treated with MBP-fMSTNPro45-100mFc was significantly (P < 0.01) greater than that of the control myoblast culture. In differentiated C₂C₁₂ cell cultures, MBP-fMSTNPro45-100mFc significantly (P < 0.01) increased myotube formation as compared with C₂C₁₂ myoblasts differentiated without MBP-fMSTNPro45-100mFc. In agreement with the above result, SDS-PAGE analysis showed that the differentiated myoblast culture treated with MBP-fMSTNPro45-100mFc had a 2.4-fold greater abundance of myosin as compared to the control myoblast culture after 5-day treatment. In summary, the study found that MBP-fMSTNPro45-100mFc produced in E. coli, had a strong MSTN-inhibitory capacity in vitro, and MBP fusion potentiated the bioactivity of MSTNPro45-100mFc. MBP-fMSTNPro45-100mFc enhanced the proliferation and differentiation of C₂C₁₂ myoblasts, supporting the in vitro result of MBP-fMSTNPro45-100mFc’s capacity to suppress MSTN, thus indicating that MBP-fMSTNPro45-100mFc is potentially an excellent agent to suppress MSTN activity in vivo.