Urolithin A enhances mitochondrial biogenesis-related markers and maximal respiratory capacity during C2C12 differentiation

Abstract

Introduction Skeletal muscle differentiation in the C2C12 myoblast model requires extensive mitochondrial remodeling to meet rising bioenergetic demands through coordinated changes in biogenesis, dynamics, and respiratory adaptation. Urolithin A (UA), a gut microbiota-derived metabolite of ellagitannins, improves mitochondrial health, but its role in late-stage myogenic differentiation remains unclear. Methods C2C12 myotubes were treated with UA (2 μM) for 72 h during late-stage differentiation (days 3–6). Mitochondrial signaling, respiratory capacity, myogenic morphology, and ultrastructure were assessed by Western blot, high-resolution respirometry, hematoxylin–eosin staining, and transmission electron microscopy. Results UA was non-cytotoxic and increased AMPKα phosphorylation and PGC-1α expression, whereas TOM20, MFN2, and OPA1 were unchanged. Mitophagy/autophagy-related markers (p-ULK1, p62, BNIP3L/NIX, LC3-II/I) were not altered, indicating no detectable changes in steady-state autophagy under the conditions tested. UA selectively increased OXPHOS Complex I and II abundance and enhanced maximal uncoupled respiration, and was associated with increased myotube diameter and myogenic marker abundance. No overt ultrastructural differences were observed by electron microscopy. Discussion These findings suggest that UA promotes mitochondrial functional adaptation during myogenic differentiation, with accompanying changes in myogenic phenotype, without clear evidence of altered steady-state mitophagy/autophagy markers or mitochondrial morphology.