Vitamin D Exerts Anti-proliferative and Modulatory effects in the Differentiation of Skeletal Muscle Cells (#39)
Background An association between vitamin D deficiency and muscle weakness has been reported for decades. However, the mechanisms are unclear. Whether vitamin D exerts direct effects or indirect effects (e.g. via calcium and phosphate homeostasis) remains unclear. Central to this question is the debate about whether vitamin D receptor (VDR) is expressed in muscle and regulates transcription at this site.
Methods C2C12 cells, an established cell culture model of skeletal muscle and VDR knockout (VDRKO) mice were studied.
Results Using a highly-specific VDR antibody, we detected VDR protein in contractile C2C12 myotubes. Following 1,25(OH)2D treatment, VDR mRNA increased in a dose-dependent fashion. The maximum increase was 10-fold following 24hrs of 10-7M 1,25(OH)2D, p<0.005. We examined the differentiation of C2C12 cells treated ±1,25(OH)2D or vehicle. At 72 hours, 1,25(OH)2D dose-dependently reduced myoblast number due to an anti-proliferative effect. Flow cytometric analysis of cell proliferation (BrdU and 7AAD) showed 82% vs. 72% of cells in resting phase of the cell cycle, p<0.005. Continued treatment with 1,25(OH)2D delayed transformation of C2C12 myoblasts to myotubes and reduced their number. This coincided with down-regulation of myogenic transcription factors including myf5, myogenin, desmin and myoD.
However, despite delayed proliferation and lower absolute number of myotubes, by day 10, cells treated with 1,25(OH)2D were markedly larger (1.8-fold increase in cross-sectional area, p<0.005). Myostatin, a negative regulator of muscle mass displayed pronounced down-regulation (>10-fold, p<0.005).
In vivo, vitamin D receptor knockout mice (VDRKO) display lighter muscles, whether expressed as weight or as proportion of body weight (gastrocnemius: 0.50% vs. 0.57%, p=0.01), smaller muscle fibres and reduced grip strength vs. wild-types (1.8-fold decrease, p<0.005).
Conclusions VDR was present in C2C12 cells, and responded to 1,25(OH)2D. Treatment increased muscle fibre size. These results suggest that vitamin D is an important regulator of muscle differentiation and size.