Non‐muscle myosin IIA is required for skeletal muscle myoblast fusion

Myoblast fusion is an essential and early step required for the generation of multinucleated muscle fibers during skeletal muscle development and for repairing damaged muscle. Although the molecular mechanisms governing myoblast fusion are not fully understood, a number of studies have suggested that the actomyosin‐cytoskeleton may have an important role. The objective of these studies was to test the hypothesis that non‐muscle (NM) myosin II has a critical role in myoblast fusion and differentiation . Using an in vitro differentiation system for the skeletal muscle cell line, L6, we show that inhibition nonmuscle myosin heavy chain II (NM‐MHC‐II) ATPase activity by blebbistatin (10uM) blocks myoblast fusion. Overexpression of a dominant negative, unphosphorylatable myosin II regulatory light chain dramatically attenuated myoblast fusion. Knockdown of NM‐MHC‐II expression by siRNA showed that only NM‐MHC‐IIA but not NM‐MHC‐IIB inhibited myoblast fusion. RT‐PCR results indicated depletion of NM‐MHC‐IIA ablated or attenuated myoD, myogenin and skeletal muscle α‐actin expression. In addition, studies using electron microscopy have shown that depletion of NM‐MHC‐IIA appears to alter vesicle “pairing” along the myoblast fusion sites. In summary, these studies demonstrate that NM‐MHC‐IIA plays a critical role in myoblast fusion and differentiation and suggests that NM‐MHC‐IIA is involved in transporting vesicles harboring proteins essential for myoblast fusion to those sites in differentiating skeletal muscle myoblasts.