Abstract
Skeletal muscle mass and activity are primordial for homeostasis and are related to important functions such as breathing, locomotion and glucose metabolism. This tissue is susceptible to lesion from mechanical‐chemical injuries and also from the mechanical stress generated by contractions. Fortunately, skeletal muscle is highly capable of regeneration, although the cellular‐molecular mechanisms involved are still not properly understood. TRB3 is a gene that codes the homonymous protein often studied for its involvement in insulin resistance by inhibiting Akt2 activity. Because it has also been shown that TRB3 overexpression can decrease myotube formation, we decided to evaluate the TRB3 expression during skeletal muscle regeneration and also in myogenesis using C2C12‐derived myotubes as a model. We cultured C2C12 cells using DMEM 10% FBS with antibiotics as growth medium. Cells at ~80% confluence were induced to differentiation into myotubes with DMEM 2% for 2 and 4 days. Cells were then harvested and total RNA and protein were isolated for further analysis by using RT‐PCR and Western blot. Also, 16 adult male C57Bl mice (~8 weeks, ~30g) were housed in 12/12h light/dark regimen with food and water ad libitum and divided into Control and tibialis anterior‐injured by cardiotoxin (CTX, 10uM) for 1, 3 and 10 days. Statistical analyzes were conducted using t‐student test in comparison with Control Group (not injured mice) or one‐way ANOVA for nonparametric data with Dunns post‐hoc for C2C12 samples analysis, expressed in Mean +− SD and p≤0.05. All experiments were approved by the Institute of Biomedical Sciences Animal Experimentation Office Committee (6085260718). We observed elevated levels of TRB3 mRNA at 2 and 4 days of C2C12 differentiation (~2.5 fold for both time points, p<0.05). Interestingly, in spite of a clear rise in TRB3 mRNA, AKT2 (a target for TRB3) mRNA is also upregulated in differentiated C2C12 cells (~3.5 fold 4 days after differentiation, p<0.05). Likewise, GSK3b mRNA levels are elevated 4 days after differentiation (2.5 fold, p<0.05). In the regeneration model we also observed increased TRB3 mRNA levels in all time points evaluated (1 day~19 fold 3 days~9 fold and 10 days~5 fold, p<0.05). Accordingly, we preliminarily observed increased levels of TRB3 10 days after injury by Western blot. These results suggests that TRB3 might be involved in the early and late phases of the myogenic‐regenerative process and functional experiments involving manipulated TRB3 levels are ongoing.Support or Funding InformationFAPESP, CAPES, CNPq
Published Version
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