Early incorporation of obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis |
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Authors: | Andrei B Borisov Marina G Martynova Mark W Russell |
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Institution: | (1) Division of Pediatric Cardiology, Congenital Heart Center, Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI 48109, USA;(2) Cellular Cardiology Research Group, Laboratory of Cell Morphology, Institute of Cytology RAS, 194064 St Petersburg, Russia |
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Abstract: | Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this
study was to investigate the process of assembly of obscurin into nascent sarcomeres during the transition from non-striated
myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes.
Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of obscurin in
the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral
fusion of nascent sarcomere bundles. We found that obscurin rapidly and precisely accumulated in the middle of the A-band
regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern
of sarcomeric α-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern
of obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal
Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of obscurin in the areas of the terminal
Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many
non-striated fibers with very low concentration of obscurin were already immunopositive for sarcomeric actin and myosin. This
suggests that obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison
of obscurin and titin localization in these areas showed that obscurin assembly into the A-bands occurred soon after or concomitantly
with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin
filaments into the “open” half-assembled sarcomeres in the areas of the terminal Z–I structures and at the lateral surfaces
of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed,
terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the
completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in
immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned
pattern. The sarcoplasmic reticulum, the organelle known to interact with obscurin, assembled around each new sarcomere. These
results suggest that obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent
sarcomeres and in the establishment of the registered pattern of newly assembled myofibrils and the sarcoplasmic reticulum
at advanced stages of myofibrillogenesis.
This paper is dedicated to the memory of Professor Pavel P. Rumyantsev (1927–1988), a pioneer in studies of cardiac muscle
differentiation, who is a lasting inspiration to all who worked with him. |
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Keywords: | Cardiac myocytes Myofibrillogenesis Myosin Obscurin Sarcomere Sarcoplasmic reticulum Z-disks |
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