Clostridium perfringens type A is the causative agent of clostridial myonecrosis, and α-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 α-toxin impairs the differentiation of C2C12 myoblasts, a useful cell line to study muscle growth, maturation, and regeneration in vitro. α-Toxin dose-dependently inhibited myotube formation in C2C12 cultures after induction of their differentiation by horse serum. Also, immunoblot analysis revealed that α-toxin dose-dependently decreases the expressions of two skeletal muscle differentiation markers, myogenic differentiation 1 (MyoD) and myogenin. These results demonstrate that α-toxin impairs the myogenic differentiation of C2C12 myoblasts. To reveal the mechanism behind α-toxin-mediated impairment of myogenic differentiation, we focused on ceramide production since α-toxin is known to promote the formation of ceramide by its sphingomyelinase activity. Immunofluorescent analysis revealed that ceramide production is accelerated by treatment with α-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 α-toxin-mediated blockage of myogenic differentiation. Together, our results illustrate that the impairment of myogenic differentiation by α-toxin might be crucial for the pathogenesis of C. perfringens to delay regeneration of severely damaged skeletal muscles.

A型产气荚膜梭状芽胞杆菌是梭菌性肌坏死的病原体，据报道α-毒素是其致病原因。最近，据报道，产气荚膜梭菌诱导的肌肉疾病后骨骼肌的再生被延迟，但是详细的机制尚未阐明。在这里，我们测试了α毒素是否会破坏C2C12成肌细胞的分化，C2C12成肌细胞是研究体外肌肉生长，成熟和再生的有用细胞系。在马血清诱导分化后，α-毒素剂量依赖性地抑制了C2C12培养物中肌管的形成。另外，免疫印迹分析显示，α-毒素剂量依赖性地降低了两种骨骼肌分化标记物，即肌源性分化1（MyoD）和肌生成素的表达。这些结果表明，α-毒素会损害C2C12成肌细胞的成肌分化。为了揭示α-毒素介导的肌原性分化受损的机理，我们集中于神经酰胺的产生，因为已知α-毒素通过其鞘磷脂酶活性促进神经酰胺的形成。免疫荧光分析表明，用α-毒素处理可促进神经酰胺的产生。此外，合成的可透过细胞的神经酰胺类似物C ₂-神经酰胺抑制C2C12细胞中的肌管形成并降低MyoD和肌生成素的表达，这表明加速的神经酰胺产生与α-毒素介导的肌原性分化的阻断有关。总之，我们的结果表明，α-毒素对肌源性分化的损害可能对产气荚膜梭菌的发病机理至关重要，以延迟严重受损的骨骼肌的再生。