Role of M‐line proteins in sarcomeric titin assembly during cardiac myofibrillogenesis

A rat polyclonal anti‐M‐line protein antiserum and three mouse monoclonal anti‐titin antibodies (E2, F3, and A12) were used to study the spatiotemporal relationship between M‐line proteins and titin during myofibril assembly in cultured chicken cardiomyocytes by immunofluorescence microscopy. In day 2 cultures, M‐line proteins and titin were detected as punctate staining in most cardiomyocytes, which possessed many nonstriated fibrils. At a late stage (day 3 cultures), M‐line proteins were incorporated into dot‐like structures along nonstriated fibrils, while titin staining was continuous on these structures. As development progressed, M‐line proteins were registered in periodic pattern in the mid‐A band. In cardiomyocytes from day 5 cultures, the titin bands were separated by an unstained region, and achieved their adult doublet pattern. Thus, the organization of titin in the sarcomere appears to occur later than that of M‐line proteins in the M‐line. Our morphological data indicate that the early registration of M‐line proteins in primitive myofibrils may guide titin filament alignment via interaction between M‐line proteins and titin. In order to investigate the role of M‐line proteins in the assembly of titin filaments, anti‐M‐line protein or anti‐titin antibodies were introduced into cultured cardiomyocytes by electroporation to functionally bind the respective proteins, and the profile of myofibril assembly was examined. Cardiomyocytes from day 2–3 cultures with incorporated anti‐M‐line protein antibodies became shrunk, and exhibited defective myofibrillar assembly, as shown by the failure of titin to assemble into a typical sarcomeric pattern. Incorporation of anti‐titin antibody E2, which recognizes the M‐line end domain of titin, resulted in the failure of M‐line proteins organized into the M‐line structure, as shown by random, sporadic staining with anti‐M‐line protein antibody. These studies confirm the essential role of M‐line proteins in the organization of titin filaments in the sarcomere and that the interaction between titin and M‐line proteins is crucial to the formation of the M‐line structure. J. Cell. Biochem. 71:82–95, 1998. © 1998 Wiley‐Liss, Inc.