Clostridium perfringens α-toxin inhibits myogenic differentiation of C2C12 myoblasts

Clostridium perfringens type A is the causative agent of clostridial myonecrosis, and alpha-toxin has been reported to be responsible for the pathogenesis. Recently, it was reported that regeneration of skeletal muscle after C. perfringens-induced muscle disorders is delayed, but the detailed mechanisms have not been elucidated. Here, we tested whether alpha-toxin impairs the differentiation of C2C12 myoblasts, a useful cell line to study muscle growth, maturation, and regeneration in vitro. alpha-Toxin dose-dependently inhibited myotube formation in C2C12 cultures after induction of their differentiation by horse serum. Also, immunoblot analysis revealed that alpha-toxin dose-dependently decreases the expressions of two skeletal muscle differentiation markers, myogenic differentiation 1 (MyoD) and myogenin. These results demonstrate that alpha-toxin impairs the myogenic differentiation of C2C12 myoblasts. To reveal the mechanism behind alpha-toxin-mediated impairment of myogenic differentiation, we focused on ceramide production since alpha-toxin is known to promote the formation of ceramide by its sphingomyelinase activity. Immunofluorescent analysis revealed that ceramide production is accelerated by treatment with alpha-toxin. Furthermore, a synthetic cell-permeable ceramide analog, C2-ceramide, inhibited myotube formation in C2C12 cells and decreased the expressions of MyoD and myogenin, suggesting that accelerated ceramide production is involved in the alpha-toxin-mediated blockage of myogenic differentiation. Together, our results illustrate that the impairment of myogenic differentiation by alpha-toxin might be crucial for the pathogenesis of C. perfringens to delay regeneration of severely damaged skeletal muscles. (C) 2020 Elsevier Ltd. All rights reserved.