Separating myoblast differentiation from muscle cell fusion using IGF-I and the p38 MAP kinase inhibitor SB202190

The p38 MAP kinases play critical roles in skeletal muscle biology, but the specific processes regulated by these kinases remain poorly defined. Here we find that activity of p38 alpha/beta is important not only in early phases of myoblast differentiation, but also in later stages of myocyte fusion and myofibrillogenesis. By treatment of C2 myoblasts with the promyogenic growth factor insulin-like growth factor (IGF)-I, the early block in differentiation imposed by the p38 chemical inhibitor SB202190 could be overcome. Yet, under these conditions, IGF-I could not prevent the later impairment of muscle cell fusion, as marked by the nearly complete absence of multinucleated myofibers. Removal of SB202190 from the medium of differentiating myoblasts reversed the fusion block, as multinucleated myofibers were detected several hours later and reached similar to 90% of the culture within 30 h. Analysis by quantitative mass spectroscopy of proteins that changed in abundance following removal of the inhibitor revealed a cohort of upregulated muscle-enriched molecules that may be important for both myofibrillogenesis and fusion. We have thus developed a model system that allows separation of myoblast differentiation from muscle cell fusion and should be useful in identifying specific steps regulated by p38 MAP kinase-mediated signaling in myogenesis.