ROLE OF M-LINE PROTEINS IN SARCOMERIC TITIN ASSEMBLY DURING CARDIAC MYOFIBRILLOGENESIS

A rat polyclonal anti-M-line protein antiserum and three mouse monoclo nal anti-titin antibodies (E2, F3, and A12) were used to study the spa tiotemporal relationship between M-line proteins and titin during myof ibril assembly in cultured chicken cardiomyocytes by immunofluorescenc e microscopy. In day 2 cultures, M-line proteins and titin were detect ed as punctate staining in most cardiomyocytes, which possessed many n onstriated 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 developmen t progressed, M-line proteins were registered in periodic pattern in t he mid-A band. In cardiomyocytes from day 5 cultures, the titin bands were separated by an unstained region, and achieved their adult double t pattern. Thus, the organization of titin in the sarcomere appears to occur later than that of M-line proteins in the M-line. Our morpholog ical data indicate that the early registration of M-line proteins in p rimitive myofibrils may guide titin filament alignment via interaction between M-line proteins and titin. In order to investigate the role o f M-line proteins in the assembly of titin filaments, anti-M-line prot ein or anti-titin antibodies were introduced into cultured cardiomyocy tes by electroporation to functionally bind the respective proteins, a nd the profile of myofibril assembly was examined. Cardiomyocytes from day 2-3 cultures with incorporated anti-M-line protein antibodies bec ame shrunk. and exhibited defective myofibrillar assembly, as shown by the failure of titin to assemble into a typical sarcomeric pattern. I ncorporation of anti-titin antibody E2, which recognizes the M-line en d domain of titin, resulted in the failure of M-line proteins organize d into the M-line structure, as shown by random, sporadic staining wit h anti-M-line protein antibody. These studies confirm the essential ro le of M-line proteins in the organization of titin filaments in the sa rcomere and that the interaction between titin and M-line proteins is crucial to the formation of the M-line structure. (C) 1998 Wiley-Liss, Inc.