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.
Wang
S M
SM
Department of Anatomy, College of Medicine, National Taiwan University, Taipei, ROC. smwang@ha.mc.ntu.edu.tw
Lo
M C
MC
Shang
C
C
Kao
S C
SC
Tseng
Y Z
YZ
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D002645:Chickens; D002856:Chromatophores / Q000276:immunology; D064211:Connectin; D018274:Electroporation; D019085:Fluorescent Antibody Technique, Direct; D006321:Heart / Q000196:embryology; D007106:Immune Sera; D051379:Mice; D009124:Muscle Proteins / Q000276:immunology / Q000378:metabolism*; D009194:Myeloma Proteins; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D009210:Myofibrils / Q000187:drug effects / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012518:Sarcomeres / Q000378:metabolism*
D000906:Antibodies; D064211:Connectin; D007106:Immune Sera; D009124:Muscle Proteins; D009194:Myeloma Proteins; C085016:multiple myeloma M-proteins; D011494:Protein Kinases