VINCULIN IS AN ESSENTIAL COMPONENT FOR NORMAL MYOFIBRILLAR ARRANGEMENT IN FETAL MOUSE CARDIAC MYOCYTES

Vinculin is a cytoskeletal protein that is believed to be an essential component in the linkage of cytoskeletal actin filaments to the plasm a membrane. To investigate the precise function of vinculin in the dev elopment of cardiac myofibrils, antisense oligodeoxynucleotides comple mentary to vinculin mRNA were used to perturb the expression of the pr otein during myofibril assembly and arrangement in mouse cardiac myocy tes. Fetal (day 18-20 post-conception) mouse cardiac myocytes were iso lated by collagenase digestion, separated by Percoll density gradient centrifugation. and plated on aligned collagen gels, By 72 h of cultur e, mouse myocytes displayed an elongated in vivo-like phenotype in par allel with the aligned fibrils of the collagen gels with polarized arr ays of myofibrils. Two different antisense oligonucleotides (20-mer) a ltered the formation of the tissue-like phenotype of myocytes. These a ntisense oligonucleotides suppressed vinculin protein expression at 43 .5 +/- 26.8% and 48.7 +/- 20.9% when compared to myocytes that were no t treated. Examination of these myocytes by confocal scanning laser an d transmission electron microscopy revealed a disruption of the aligne d in vivo-like phenotype, assembly of thick and thin filaments, and fo rmulation 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 myofibril s. These results indicate that vinculin is a critical cytoskeletal com ponent, that functions in the determination of cell shape and the arra ngement and organization of developing myofibrils. (C) 1997 Academic P ress Limited.