In vitro mechanical stretch inhibits differentiation of mouse myoblast C2C12 cells without sustained eIF2α...

Mechanical stretch critically influences skeletal muscle physiology, yet its role in myoblast differentiation and the associated molecular mechanisms have not been fully clarified. This study investigated the effects of uniaxial cyclic mechanical stretch (UnCyMSt) on differentiation of mouse myoblast C2C12 cells, focusing particularly on the potential involvement of eukaryotic initiation factor 2 alpha (eIF2α), a key regulator maintaining muscle stem cell quiescence. To apply mechanical stretch, C2C12 cells were cultured on polydimethylsiloxane surfaces covalently immobilized with collagen (Col-GA-PDMS), ensuring stable cell adhesion under UnCyMSt, whereas cells cultured on physically adsorbed collagen surfaces (Col-PDMS) detached under similar conditions. Under differentiation conditions, UnCyMSt markedly inhibited myoblast differentiation, as evidenced by suppressed expression of the differentiation marker myogenin. Additionally, stretched cells aligned perpendicular to the direction of mechanical stretch application. Given the established role of phosphorylated eIF2α (p-eIF2α) in maintaining myoblast quiescence, we investigated whether UnCyMSt inhibits differentiation by modulating eIF2α phosphorylation at serine 51. UnCyMSt did not prevent the progressive dephosphorylation of eIF2α during differentiation induction. Correspondingly, expression levels of activating transcription factor 4 (ATF4), downstream of p-eIF2α, also decreased under UnCyMSt. Our results demonstrate that UnCyMSt inhibits C2C12 myoblast differentiation without sustained phosphorylated eIF2α, suggesting the involvement of alternative mechanosensitive signaling pathways. These findings provide new insights into mechanical regulation of muscle differentiation and highlight the need for further exploration into stretch-responsive molecular mechanisms influencing myogenesis.