Stage-dependent doxorubicin toxicity impairs C2C12 myogenesis via AMPK/AKT-FOXO signaling and is attenuated by metformin.
Doxorubicin (DOX) can compromise skeletal muscle, but whether toxicity depends on myogenic stage is unclear. C2C12 myoblasts were exposed to non‑cytotoxic DOX (0.025-0.1μg/mL) before differentiation (24h) or after 3 days of differentiation (48h), with or without metformin (Met; 0.1-2mM). Pre‑differentiation DOX markedly impaired later myotube formation, whereas post‑differentiation DOX disrupted myotube maturation. Both regimens reduced myogenin and myosin heavy chain expression and suppressed AMP‑activated protein kinase (AMPK) and AKT phosphorylation. DOX activated a FOXO transcription factor-dependent catabolic program, decreasing inhibitory phosphorylation of FOXO3a (and FOXO1 post‑differentiation) and increasing MAFbx and MuRF1. Met co‑treatment partially preserved myotube integrity and myogenic markers, reactivated AMPK, partially recovered AKT signaling, and attenuated FOXO-MAFbx/MuRF1 induction. These findings define stage-dependent DOX myotoxicity and suggest that Met-associated AMPK reactivation may partially mitigate DOX-induced myogenic dysfunction under defined in vitro conditions.