Author： Shiraishi I. Simpson D.G. Carver W. Price R. Hirozane T. Terracio L. Borg T.K.
Publisher： Academic Press
ISSN： 0022-2828
Source： Journal of Molecular and Cellular Cardiology, Vol.29, Iss.8, 1997-08, pp. : 2041-2052
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Abstract
Vinculin is a cytoskeletal protein that is believed to be an essential component in the linkage of cytoskeletal actin filaments to the plasma membrane. To investigate the precise function of vinculin in the development of cardiac myofibrils, antisense oligodeoxynucleotides complementary to vinculin mRNA were used to perturb the expression of the protein during myofibril assembly and arrangement in mouse cardiac myocytes. Fetal (day 18-20 post-conception) mouse cardiac myocytes were isolated by collagenase digestion, separated by Percoll density gradient centrifugation, and plated on aligned collagen gels. By 72 h of culture, mouse myocytes displayed an elongated in vivo-like phenotype in parallel with the aligned fibrils of the collagen gels with polarized arrays of myofibrils. Two different antisense oligonucleotides (20-mer) altered the formation of the tissue-like phenotype of myocytes. These antisense oligonucleotides suppressed vinculin protein expression at 43.5±26.8% and 48.7±20.9% when compared to myocytes that were not treated. Examination of these myocytes by confocal scanning laser and transmission electron microscopy revealed a disruption of the aligned in vivo-like phenotype, assembly of thick and thin filaments, and formulation of Z-bands. Random sequence 20-mer oligonucleotides used as controls had little detectable effect on vinculin protein expression (94.2±14.8%), cell shape, normal alignment or assembly of myofibrils. These results indicate that vinculin is a critical cytoskeletal component, that functions in the determination of cell shape and the arrangement and organization of developing myofibrils.
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