Studies on the function of rho A protein in cardiac myofibrillogenesis.
The aim of this study was to provide morphological evidence for the presence of rho A protein in developing cardiomyocytes and to investigate its possible role in myofibrillogenesis. Immunostaining with a monoclonal anti-rho antibody gave a diffuse pattern in the cytosol of cultured cardiomyocytes. Introduction of C3 exoenzyme into the cells by electroporation was used to inactivate rho A protein by ADP-ribosylation. An immunostaining with anti-vinculin, anti-talin, and anti-integrin antibodies showed the focal adhesions in electroporation control cardiomyocytes to be evenly distributed in the ventral sarcolemma; the costameric 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 alpha-actinin into myofibrils was poor, as shown by diffuse and filamentous staining pattern. C3 exoenzyme treatment had a less marked effect on mature cardiomyocytes than on immature cells; in this case, cells became distorted and few myofibrils were seen. The intensity of anti-phosphotyrosine antibody 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.
Wang
S M
SM
Department of Anatomy, College of Medicine, National Taiwan University, Taipei, ROC.smwang@ntumc1.mc.ntu.edu.tw
Tsai
Y J
YJ
Jiang
M J
MJ
Tseng
Y Z
YZ
D036002:ADP Ribose Transferases / Q000378:metabolism; D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001905:Botulinum Toxins; D002454:Cell Differentiation; D002645:Chickens; D064211:Connectin; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins / Q000502:physiology*; D016023:Integrins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D009206:Myocardium / Q000166:cytology*; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D016608:Talin / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism; D020742:rhoA GTP-Binding Protein
D000199:Actins; D000911:Antibodies, Monoclonal; D064211:Connectin; D016023:Integrins; D009124:Muscle Proteins; D010750:Phosphoproteins; D016608:Talin; D000185:Actinin; D016596:Vinculin; D019000:Phosphotyrosine; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011494:Protein Kinases; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein