Vitamin C is essential for proper myogenic differentiation.

BACKGROUND

Vitamin C (VC) is naturally present in the blood and skeletal muscles. However, conventional myoblast culture media typically lack VC. Because VC exists in skeletal muscle, VC-supplemented media should better represent physiological conditions than VC-deficient media. Therefore, we used a VC-supplemented culture medium to examine the effects of VC deficiency on myogenic differentiation.

METHODS

Mouse C2C12 myoblasts were cultured in VC-supplemented or VC-free differentiation medium (DM), with the medium replaced every 24 h to preserve the efficacy of VC.

RESULTS

First, we confirmed that VC was reliably taken up by the C2C12 cells. We assessed the expression of muscle regulatory factors during myogenic differentiation. The expression levels of late-stage differentiation markers, including myogenin (MyoG), myomaker (Mymk), myosin heavy chain 1 (Myh1), and Myh4 were elevated in VC-free DM during the early stages of myogenic differentiation. In contrast, the expression levels of terminal myogenic markers in mature myofibrils, such as troponin I slow skeletal muscle (Tnni1) and troponin I fast skeletal muscle (Tnni2), increased in cells differentiated in VC-free DM but were lower than those in cells differentiated in VC-supplemented DM. The diameters of the differentiated myotubes were smaller in VC-free DM than in VC-supplemented DM. The levels of 5-hydroxymethylcytosine (5-hmC), a product of the VC-dependent DNA demethylation enzyme ten-eleven translocation (Tet), were markedly lower in VC-free DM.

CONCLUSION

These results suggest that VC modulates myogenic differentiation.