Impaired xCT-mediated cystine uptake drives serine and proline metabolic reprogramming and mitochondrial fission in skeletal muscle cells.

Muscle satellite cell (MuSC) proliferation is tightly regulated by redox homeostasis and nutrient availability, which are often disrupted in muscular pathologies. Beyond its role in maintaining cellular redox homeostasis, this study identified a key metabolic role for cystine/glutamate antiporter xCT in proliferating MuSCs. We investigated the impact of impaired xCT-mediated cystine import in Slc7a11 MuSCs isolated from mice that harbor a mutation in the SLC7A11 gene, which encodes xCT. We used complementary approaches to study how disrupted cystine import affects glutathione (GSH) redox, cellular bioenergetics, mitochondrial dynamics, and metabolism. Oxygen consumption rates of Slc7a11 MuSCs were lower, indicative of compromised mitochondrial oxidative capacity. This was accompanied by a fragmented mitochondrial network associated with OPA1 cleavage and redox-sensitive DRP1 oligomerization. Metabolomic profiling revealed a distinct metabolic signature in Slc7a11 MuSCs, manifested by major differences in BCAAs, pyrimidines, cysteine, methionine, and GSH. Despite lower overall bioenergetic flux, stable-isotope tracing analyses (SITA) showed that xCT deficiency increased glucose uptake, channeling glucose-derived carbons into de novo serine biosynthesis to fuel cysteine production via the transsulfuration pathway, partially compensating for disrupted GSH redox. Furthermore, xCT deficiency triggered upregulated pyrroline-5-carboxylate synthase (P5CS)-mediated proline reductive biosynthesis. By directing glutamate into proline synthesis, MuSCs apparently downregulate oxidative phosphorylation (OXPHOS) and regulate intracellular glutamate levels in response to impaired cystine/glutamate antiporter function. Our findings highlight the roles of xCT in regulating redox balance and metabolic reprogramming in proliferating MuSCs, providing insights that may inform therapeutic strategies for muscular and redox-related pathologies.