All-trans retinoic acid regulates sheep primary myoblast proliferation and differentiation in vitro

Vitamin A and its metabolite, retinoic acid (RA), play key roles in cell differentiation and organ morphogenesis. The objective of this study was to investigate the effect of RA on sheep primary myoblast proliferation and differentiation. Sheep primary myoblasts were isolated and treated with all-trans retinoic acid (ATRA, 10 nM and 100 nM) and vehicle. The results showed that 10 nM ATRA sufficiently inhibited cell proliferation, which might be through downregulation of cyclin D1 (P < 0.05) and cyclin-dependent kinase 4 (P < 0.05) and proliferating cell nuclear antigen protein (P < 0.05) abundance. Moreover, compared with control cells, both 10 nM and 100 nM ATRA promoted myotube formation and increased fusion index (P < 0.05), which was associated with elevated myogenin mRNA content (P < 0.05). As expected, both myogenin (P < 0.01) and myosin heavy chain (P < 0.05) protein levels were increased by ATRA. Interestingly, ATRA treatment increased H3K4me3 and decreased H3K27me3 enrichment in the myogenin promoter region (P < 0.05). Meanwhile, 100 nM ATRA stimulated 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-deoxyglucose uptake (P < 0.05) and upregulated glucose transporter 4 expression at both mRNA and protein levels (P < 0.05). Although ATRA did not alter p38 content, phospho-p38 content was increased (P < 0.01). In addition, ATRA treatment activated the mTOR signaling pathway (P < 0.05). Taken together, these results demonstrated that ATRA plays an important role in regulating sheep myoblast proliferation and myogenic differentiation and suggested vitamin A as a potential target for manipulating muscle growth efficiency in sheep industry.