Abstract
Dystrophies are characterized by progressive skeletal muscle degeneration and weakness as consequence of their molecular abnormalities. Thus, new drugs for restoring skeletal muscle deterioration are critically needed. To identify new and alternative compounds with a functional role in skeletal muscle myogenesis, we screened a library of pharmacologically active compounds and selected the small molecule 6-bromoindirubin-3′-oxime (BIO) as an inhibitor of myoblast proliferation. Using C2C12 cells, we examined BIO’s effect during myoblast proliferation and differentiation showing that BIO treatment promotes transition from cell proliferation to myogenic differentiation through the arrest of cell cycle. Here, we show that BIO is able to promote myogenic differentiation in damaged myotubes in-vitro by enriching the population of newly formed skeletal muscle myotubes. Moreover, in-vivo experiments in CTX-damaged TA muscle confirmed the pro-differentiation capability of BIO as shown by the increasing of the percentage of myofibers with centralized nuclei as well as by the increasing of myofibers number. Additionally, we have identified a strong correlation of miR-206 with BIO treatment both in-vitro and in-vivo: the enhanced expression of miR-206 was observed in-vitro in BIO-treated proliferating myoblasts, miR-206 restored expression was observed in a forced miR-206 silencing conditions antagomiR-mediated upon BIO treatment, and in-vivo in CTX-injured muscles miR-206 enhanced expression was observed upon BIO treatment. Taken together, our results highlight the capacity of BIO to act as a positive modulator of skeletal muscle differentiation in-vitro and in-vivo opening up a new perspective for novel therapeutic targets to correct skeletal muscle defects.
Highlights
Dystrophies are characterized by progressive skeletal muscle degeneration and weakness as consequence of their molecular abnormalities
Muscle differentiation is usually induced by growing C2C12 cells up to 70% confluence in Growth Medium (GM) and by shifting the cells to Differentiation Medium (DM) consisting of Dulbecco’s Modified Eagle Medium (DMEM) plus adult Horse Serum (HS)
To validate the screening results, BIO was dissolved in GM medium and its activity was examined in comparison with GM, DM or Vehicle (DMSO) for 24 and 48 h by Cell Titer-Glo and Cell Toxicity assays performed on C2C12 cells to evaluate cell proliferation and viability upon BIO treatment
Summary
Dystrophies are characterized by progressive skeletal muscle degeneration and weakness as consequence of their molecular abnormalities. Western blot analysis was performed on C2C12 cells treated with BIO dissolved in GM medium (3 μM) or Vehicle (DMSO) in GM condition comparing their expression with DM treatment only. QRT-PCR assay was performed to evaluate CyclinD1 and MyoD gene expression level in C2C12-BIO in GM medium treated cells for 24 and 48 h.
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