Myotube-specific vulnerability to clozapine

Clozapine (CLZ), an atypical antipsychotic used in treatment-resistant schizophrenia, has a limited clinical application due to numerous serious adverse reactions. This study focused on myotoxicity, a rare and poorly understood side effect of the drug. CLZ concentrations within the therapeutic or supratherapeutic range caused time- and concentration-dependent toxicity in C2C12-derived myotubes, as demonstrated by the loss of muscle-specific morphological (elongation and multinucleation) and biochemical (i.e., ryanodine receptor activity, ATP levels, creatine kinase activity, and the expression levels of myogenin and myosin heavy chain) markers. Specific apoptotic features (e.g., nuclear fragmentation, nuclear clusters) were detected in myotubes, identified using various criteria. Apoptosis was also found in mouse primary myotubes. Interestingly, even under conditions in which CLZ caused massive myotube loss, mononuclear reserve cells remained viable and retained the ability to proliferate in 10 % fetal bovine serum (FBS) medium or differentiate in 1 % FBS medium. In addition, isolated reserve cells and pure C2C12 myoblasts were resistant to CLZ (in 10 % FBS medium). Finally, albumin was used to simulate CLZ protein binding in myotube cultures in a medium containing 1 % FBS, achieving levels equivalent to those in 10 % FBS medium, without significant differences in toxicity outcomes. Thus, even in the presence of 10 % FBS, free CLZ is continuously made available to, and accumulated within, myotubes. In conclusion, relevant concentrations of CLZ selectively induce apoptosis in myotubes without causing toxic effects in proliferating myoblasts or mononuclear reserve cells.