BDM (2,3-butanedione monoxime), an inhibitor of myosin-actin interaction, suppresses myofibrillogenesis in skeletal muscle cells in culture

During the initial phase of myofibrillogenesis in developing muscle cells, the majority of thin filaments lie parallel to, and exhibit correct polarit y and spatial position with thick filaments, as in mature myofibrils. Since myosin is known to function as an accelerator of actin polymerization in v itro, it has been postulated that myosin-actin interaction is important in the initial phase of myofibrillogenesis. To clarify further the role of act in-myosin interaction in myofibril formation during development, BDM (2,3-b utanedione 2-monoxime), an inhibitor of myosin ATPase, was applied to prima ry cultures of skeletal muscle to inhibit myosin activity during myofibrill ogenesis, and myofibril formation was examined. When 10 mM BDM was added to the myotubes just after fusion and the cultures were maintained for a furt her 4 days, cross-striated myofibrils were scarcely observed by fluorescenc e microscopy when examined by staining with antibodies to actin, myosin, tr oponin and a-actinin, whereas in the control myotubes not exposed to BDM, t ypical sarcomeric structures were detected. Electron microscopy revealed a disorganized arrangement of myofilaments and incomplete sarcomeric structur es in the BDM-treated myotubes. Thus, formation of cross-striated myofibril s was remarkably suppressed in the BDM-treated myotubes, When the myotubes cultured in BDM-containing media were transferred to control media, sarcome ric structures were formed in 2-3 days, suggesting that the inhibitory effe ct of BDM on myotubes is reversible. These results suggest that actin-myosi n interaction plays a critical role in the early process of myofibrillogene sis.