Metformin Partially Attenuates Simvastatin‐Induced Myotoxic Responses in <scp>C2C12</scp> Myotubes Through Metabolic Adaptation

Metformin is the first-line therapy for type 2 diabetes mellitus and is commonly co-administered with statins for cardiovascular risk reduction. However, statins can cause statin-associated muscle symptoms, while metformin itself exerts complex effects on skeletal muscle. Because both drugs influence cellular energy metabolism and stress-response pathways in skeletal muscle, their combined effects on muscle cells warrant investigation. C2C12 myotubes were treated with metformin (50 or 1000 μM) in the absence or presence of simvastatin (10 μM) for 24 h. Myotube morphology, differentiation, and fusion indices, myoblast proliferation, and expression of atrophy-, stress-, and metabolism-related genes were assessed. Phosphorylation of key metabolic and anabolic signaling proteins (AMPK/ACC and Akt/mTOR-p70S6K) was analyzed. Mitochondrial respiration was measured using Seahorse respirometry, and mitochondrial network organization was quantified by live-cell imaging. Simvastatin significantly reduced myotube diameter (p < 0.0001), impaired myogenic progression in differentiated myotubes (differentiation index, p < 0.0001; fusion index, p = 0.0152), and inhibited myoblast proliferation (p = 0.003). Simvastatin increased the atrophy markers (Trim63, Fbxo32), stress marker (Perk), and concurrently suppressed myogenic (Myod) and anabolic (p-p70s6k/p70s6k) activity. Simvastatin also induced a broad suppression of mitochondrial and glycolytic metabolism, accompanied by reduced expression of the metabolic genes (Glut4, Hk2) and disruption of mitochondrial network connectivity. Co-exposure with metformin significantly attenuated simvastatin-induced effects, increasing myotube diameter (1.43-fold at low dose, p = 0.0223, and 1.48-fold at high dose, p = 0.0131), differentiation index (low dose: 1.63-fold; high dose: 1.80-fold; both p < 0.0001), and fusion index (low dose: 1.35; high dose: 1.50-fold; both p < 0.01). Compared with simvastatin alone, co-treatment with high-dose metformin increased AMPK and ACC phosphorylation and further suppressed mTOR signaling without amplifying atrophy-related gene expression. Despite deeper suppression of metabolic parameters (routine respiration, ATP production, Hk2 expression), metformin preserved mitochondrial network structure, increased Ppargc1a expression, and reduced cellular stress markers (Hri, Perk, Atf4). Simvastatin induced metabolic suppression, mitochondrial dysfunction, and atrophy-related responses in skeletal muscle cells. Metformin partially attenuated these alterations by preserving myotube structural integrity and reducing cellular stress signaling despite further metabolic suppression. These findings suggest that metformin may promote adaptive metabolic responses that enhance cellular resilience during simvastatin-induced metabolic stress.