Effects of Excessive Retinoic Acid on C2C12 Myogenesis

Abstract

Retinoic acid (RA) is a widely-used agent inducing cell differentiation. In order to study the effect of excessive RA on myogenesis, C2C12 cells were cultured in gradient concentrations of RA. In this study, the cell cycle arrested in G₀/G₁ phase when the concentration of RA was higher than 10 µM. Cell proliferation was inhibited by RA over 40 µM. Therefore, 1 and 10 µM RA were chosen to treat C2C12 cells under serum withdrawal, respectively, and then, myogenic regulatory factors (MRFs) were measured by real-time PCR. It was found that after RA treatment, Myf5 transcription was maintained in a lower level. The change curves of MyoD and Myogenin transcription were consistent with the vehicle control, but MRF4 transcription was higher than that of the vehicle control at day 6. Additionally, compared with 10 µM RA-treated cells, the elevated transcription of MyoD and Myogenin insisted longer and the transcription of MRF4 at 6 day was higher in 1 µM RA-treated cells. Myosin heavy chain (MyHC) was also calibrated by Western blot to analysis myogenic terminal differentiation. Our findings illustrated that MyHC protein level was obviously declined in 10 µM RA-treated cells. Finally, immunostaining revealed that a growing number of bifurcations in RA-treated myosin-positive cells were accompanied by longer and thinner cell bodies, especially in 10µM RA-treated cells. In conclusion, excessive RA could still induce myoblast myogenic differentiation. However, the maturation of myotubes might be discouraged by a higher-dose of RA.