Myofibril Assembly in Cultured Mouse Neonatal Cardiomyocytes

Abstract

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. © 2018 Wiley Periodicals, Inc.

Myofibril assembly and the roles of the ubiquitin proteasome system

“…Investigations of de novo myofibril assembly in several model systems: cultured cardiac and skeletal muscle cells isolated from chick, quail, and neonatal mouse cardiac and skeletal muscle, as well as in skeletal muscle in fixed and living zebrafish (Dabiri et al, 1997;Du, Sanger, Linask, & Sanger, 2003;Du, Sanger, & Sanger, 2008;Mittal, Sanger, & Sanger, 1987a;Sanger et al, 2002Sanger et al, , 2006Sanger et al, , 2017Sanger, Mittal, Pochapin, & Sanger, 1986;Sanger, Mittal, & Sanger, 1984;Sanger, Wang, Fan, White, & Sanger, 2010;Sanger, Wang, Holloway, Du, & Sanger, 2009;White et al, 2014White et al, , 2018 led us to propose a three-step model to describe the path of myofibrillogenesis from premyofibrils to nascent myofibrils to mature myofibrils (diagram in Figure 1) (Rhee et al, 1994;Sanger et al, 2017). In addition to recording changes in the appearance and arrangement of sarcomeric proteins in the three different stages of myofibril assembly, we…”

“…Understanding how the structural framework is assembled from sarcomeric proteins, and maintained to generate continual force provides the background for interpreting myopathies that can occur in the muscle. Studies of de novo assembly of myofibrils in vertebrate cross-striated muscles led us to propose that myofibril assembly follows a three-step sequence that begins with premyofibrils, progresses to formation of nascent myofibrils, and concludes with the formation of mature myofibrils (Figure 1), (Dabiri, Turnacioglu, Sanger, & Sanger, 1997;Rhee, Sanger, & Sanger, 1994;Sanger et al, 2017;Sanger, Sanger, & Franzini-Armstrong, 2004;White et al, 2014White et al, , 2018. Previous photobleaching fluorescently tagged sarcomeric proteins (fluorescence recovery after photobleaching [FRAP]) in cells undergoing myofibrillogenesis indicated that dynamic exchange of the proteins decreased as premyofibrils transformed to nascent myofibrils and then to mature myofibrils (Sanger et al, 2017;Wang et al, 2005;Wang, Dube, Mittal, Sanger, & Sanger, 2011).…”

Linking the Landscape of MYH9-Related Diseases to the Molecular Mechanisms that Control Non-Muscle Myosin II-A Function in Cells

“…Its levels are very low in mature neurons [23]. Both NMII-A and -B paralogs participate in the early stages of myofibrillogenesis [24,25], although their expression levels are also low in adult skeletal muscle [21] and absent in in mature cardiomyocytes [26] and the monkey cell line COS7 [27].…”

CAP2 is a regulator of actin pointed end dynamics and myofibrillogenesis in cardiac muscle

“…Since Cap2 -KO cells demonstrated delayed myofibrillogenesis, we investigated subcellular assemblies of known myofibril maturation markers in cardiomyocytes: specifically, α-SMA, desmin and nonmuscle myosin IIB, which are components of thin filaments, intermediate filaments and plasma membrane/pre-myofibrils, respectively 42 – 45 . Immunofluorescence microscopy demonstrated no notable difference in desmin and nonmuscle myosin IIB staining between WT and KO cardiomyocytes (Fig.…”