Myofibril Assembly in Cultured Mouse Neonatal Cardiomyocytes

The formation of myofibrils was analyzed in neonatal mouse cardiomyocytes grown in culture and stained with fluorescent antibodies directed against myofibrillar proteins. The cardiomyocyte cultures also were exposed to siRNA probes to test the role of nonmuscle myosin IIB expression in the formation of myofibrils. In culture, new myofibrils formed in the spreading cell margins surrounding contractile myofibrils previously assembled in utero. Observations indicated that assembly of mature myofibrils occurred in three-stages, as previously reported in cultured mouse skeletal muscle. Premyofibrils, characterized by minisarcomeres with nonmuscle myosin IIB and muscle-specific alpha-actinin bound to actin filaments, formed in the first stage; followed by nascent myofibrils, the second stage when muscle myosin II and titin were first detected. In the mature myofibril stage muscle myosin II filaments aligned in periodic A-Bands; late assembling proteins, including myomesin and telethonin, were integrated in the sarcomeres, and nonmuscle IIB was absent from the sarcomeres. Treatment of the cultured neonatal cardiomyocytes with gene-specific siRNAs for nonmuscle myosin IIB, led to a marked decrease in the formation of premyofibrils, and subsequently of mature myofibrils. Anat Rec, 301:2067-2079, 2018.