NAD+ subcellular partitioning mediated by miR-183 and miR-96 regulates muscle stem cell differentiation

The intracellular abundance of NAD+, a vital metabolic cofactor, critically influences muscle stem cell (MuSC) function. However, the spatial regulation of NAD+ and its impact on MuSC function remain unclear. In this study, we demonstrated that the loss of miR-183 and miR-96 leads to inefficient skeletal muscle regeneration upon injury and triggers premature differentiation of MuSC-derived primary myoblasts. The underlying mechanism involves miRNA-mediated regulation through targeting SLC25A51, a mitochondrial transporter for NAD+ that elevates mitochondrial NAD+ while reducing cytoplasmic NAD+ levels. Our results suggest that the reduction in cytoplasmic NAD+ diminishes SIRT1-mediated deacetylation, increasing H4K16ac at the promoters of myogenic genes to promote differentiation. Concurrently, the mitochondrial NAD+ accumulation stimulates the tricarboxylic acid cycle, leading to elevated levels of ATP and citrate. These metabolites allosterically activate the ACLY pathway, which in turn increases acetyl-CoA production, thereby supplying acetyl groups for H4K16ac. Furthermore, SIRT3 knockdown impaired myogenic differentiation and attenuated the increased levels of both ATP and acetyl-CoA in miR-183/96-deficient cells, suggesting that the elevated mitochondrial NAD+ also enhances differentiation via SIRT3-mediated regulation of mitochondrial metabolism and acetyl-CoA production. Our work establishes miR-183 and miR-96 as critical regulators of epigenetic-metabolic networks that influence MuSC differentiation through subcellular partitioning of NAD+, ensuring proper regeneration timing.