STUDIES ON THE FUNCTION OF RHO-A PROTEIN IN CARDIAC MYOFIBRILLOGENESIS

The aim of this study was to provide morphological evidence for the pr esence of rho A protein in developing cardiomyocytes and to investigat e its possible role in myofibrillogenesis. Immunostaining with a monoc lonal anti-rho antibody gave a diffuse pattern in the cytosol of cultu red cardiomyocytes. Introduction of C3 exoenzyme into the cells by ele ctroporation was used to inactivate rho A protein by ADP-ribosylation. An immunostaining with anti-vinculin, anti-talin, and anti-integrin a ntibodies showed the focal adhesions in electroporation control cardio myocytes to be evenly distributed in the ventral sarcolemma; the costa meric structure was also detected using these antibodies. In contrast, in C3 exoenzyme treated cells, focal adhesions were disassembled and costamere were absent; in addition, beta-actin-positive, non-striated fibrils were lost and assembly of M-protein, titin, and a-actinin into myofibrils was poor, as shown by diffuse and filamentous staining pat tern. C3 exoenzyme treatment had a less marked effect on mature cardio myocytes than on immature cells; in this case, cells became distorted and few myofibrils were seen. The intensity of anti-phosphotyrosine an tibody staining of the focal adhesion was also decreased or diffuse in C3 exoenzyme-treated cardiomyocytes, suggesting dephosphorylation of focal adhesion components. We therefore conclude that small G protein rho A plays an important role in myofibril assembly in cardiomyocytes. (C) 1997 Wiley-Liss, Inc.