Wang S M, Tsai Y J, Jiang M J, Tseng Y Z
Department of Anatomy, College of Medicine, National Taiwan University, Taipei,
J Cell Biochem. 1997 Jul 1;66(1):43-53.
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.
本研究的目的是为发育中的心肌细胞中存在rho A蛋白提供形态学证据，并研究其在肌原纤维形成中的可能作用。用单克隆抗rho抗体进行免疫染色，在培养的心肌细胞胞质溶胶中呈现弥漫性模式。通过电穿孔将C3外切酶导入细胞，用于通过ADP-核糖基化使rho A蛋白失活。用抗纽蛋白、抗踝蛋白和抗整合素抗体进行免疫染色显示，电穿孔对照心肌细胞中的粘着斑均匀分布在腹侧肌膜；使用这些抗体也检测到了肌小节结构。相比之下，在C3外切酶处理的细胞中，粘着斑被分解且不存在肌小节；此外，β-肌动蛋白阳性的非横纹肌原纤维消失，M蛋白、肌联蛋白和α-辅肌动蛋白组装到肌原纤维中的情况较差，表现为弥漫性和丝状染色模式。C3外切酶处理对成熟心肌细胞的影响比对未成熟细胞的影响小；在这种情况下，细胞变形，可见的肌原纤维很少。在C3外切酶处理的心肌细胞中，粘着斑的抗磷酸酪氨酸抗体染色强度也降低或呈弥漫性，提示粘着斑成分去磷酸化。因此，我们得出结论，小G蛋白rho A在心肌细胞的肌原纤维组装中起重要作用。
