Regulatory role of vitamin D3 on myogenesis and fibrogenesis under Vdr gene silencing and TGF-β1 stimulation in skeletal muscle cells

Vitamin D deficiency is associated with a decline in muscle function and an increasing risk of muscle injury in athletes and the elderly. Nevertheless, how vitamin D₃ and vitamin D receptor (VDR) regulate skeletal muscle cells under profibrotic factor stimulation that could be pronounced during repetitive muscle damage have not been elucidated. Therefore, this study aimed to investigate the regulatory role of cholecalciferol (D₃), calcidiol (25D₃), calcitriol (1,25D₃), and the effect of Vdr gene suppression under TGF-β1 stimulation in C2C12 mouse skeletal muscle cells. All forms of vitamin D₃ exerted antifibrotic effects under TGF-β1 stimulation by suppression of COL1A1; however, D₃ preserves this effect without negative impact on myogenesis. Moreover, LC-MS/MS-based proteomics analysis revealed that myoblast fusion protein and mitochondrial regulation were altered following Vdr knockdown. These changes were associated with exacerbation of α-SMA expression in TGF-β1-treated cells and suggested VDR modulates fibrogenesis in skeletal muscle cells regardless of ligand binding. Under TGF-β1 stimulation, antifibrotic effects of 1,25D₃ but not D₃ were diminished after Vdr knockdown, supporting that D₃ action is largely mediated independently of VDR. Collectively, understanding antifibrotic effects of vitamin D₃ is beneficial for providing a strategy of vitamin D supplementation to counteract fibrosis development after muscle injury.