Dynamics of obscurin localization during differentiation and remodeling of cardiac myocytes: obscurin as an integrator of myofibrillar structure.
Obscurin is a newly identified giant muscle protein whose functions remain to be elucidated. In this study we used high-resolution confocal microscopy to examine the dynamics of obscurin localization in cultures of rat cardiac myocytes during the assembly and disassembly of myofibrils. Double immunolabeling of neonatal and adult rat cells for obscurin and sarcomeric alpha-actinin, the major protein of Z-lines, demonstrated that, during myofibrillogenesis, obscurin is intensely incorporated into M-band areas of A-bands and, to a lesser extent, in Z-lines of newly formed sarcomeres. Presarcomeric structural precursors of myofibrils were intensely immunopositive for alpha-actinin and, unlike mature myofibrils, weakly immunopositive or immunonegative for obscurin. This indicates that most of the obscurin assembles in developing myofibrils after abundant incorporation of alpha-actinin and that massive integration of obscurin occurs at more advanced stages of sarcomere assembly. Immunoreactivity for obscurin in the middle of A-bands and in Z-lines of sarcomeres bridged the gaps between individual bundles of newly formed myofibrils, suggesting that this protein appears to be directly involved in their primary lateral connection and registered alignment into larger clusters. Close sarcomeric localization of obscurin and titin suggests that they may interact during myofibril assembly. Interestingly, the laterally aligned striated pattern of obscurin formed at a stage when desmin, traditionally considered as a molecular linker responsible for the lateral binding and stabilization of myofibrils at the Z-bands, was still diffusely localized. During the disassembly of the contractile system in adult myocytes, disappearance of the cross-striated pattern of obscurin preceded the disorganization of registered alignment and intense breakdown of myofibrils. The cross-striated pattern of desmin typical of terminally differentiated myocytes disappeared before or simultaneously with obscurin. During redifferentiation, as in neonatal myocytes, sarcomeric incorporation of obscurin closely followed that of alpha-actinin and occurred earlier than the striated arrangement of desmin intermediate filaments. The presence of obscurin in the Z-lines and its later assembly into the A/M-bands indicate that it may serve to stabilize and align sarcomeric structure when myosin filaments are incorporated. Our data suggest that obscurin, interacting with other muscle proteins and possibly with the sarcoplasmic reticulum, may have a role as a flexible structural integrator of myofibrils during assembly and adaptive remodeling of the contractile apparatus.
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI 48109, USA. aborisov@umich.edu
Andrei B
AB
Borisov
Aikaterini
A
Kontrogianni-Konstantopoulos
Robert J
RJ
Bloch
Margaret V
MV
Westfall
Mark W
MW
Russell
D000199:Actins / Q000378:metabolism; D000375:Aging / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation; D002478:Cells, Cultured; D003893:Desmin / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009124:Muscle Proteins / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D009210:Myofibrils / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats
D000199:Actins; D003893:Desmin; D020662:Guanine Nucleotide Exchange Factors; D009124:Muscle Proteins; C503780:Obscn protein, rat