Eid H, Chen J H, de Bold A J
University of Ottawa Heart Institute, Research Centre, Ontario, Canada.
Ann N Y Acad Sci. 1995 Mar 27;752:192-201. doi: 10.1111/j.1749-6632.1995.tb17422.x.
Adult rat ventricular myocytes assume after 2 weeks in culture a flattened spread morphology and a loss in organized myofibrils. This sequence of phenotypic changes is accompanied by the reexpression of the fetal gene program. Although different signal transduction pathways were recently shown to be involved in cell growth and differentiation, not much is known about tyrosine kinase activation and cardiac myocyte differentiation. We investigated whether the tyrosine kinase signal transduction pathway is involved in the dedifferentiation of adult rat ventricular myocytes in long-term culture using a specific inhibitor of tyrosine phosphorylation, genistein. For this experiment, adult rat ventricular myocytes were cultured as previously described and incubated in culture medium containing different concentrations of genistein (10-250 microM). After 24 hr of incubation and in a concentration-dependent manner genistein prevented cell spreading. However, at high concentration, cells detached from the plates (10% to 100 microM and 95% at 250 microM). The effect of genistein on adult rat ventricular myocyte phenotype in culture was investigated by examining the expression of total actins and alpha-smooth muscle actin and alpha-sarcomeric actin in cells after 6 days of incubation with and without genistein. Myofibrillar proteins were extracted and separated by gel electrophoresis. Expression of alpha-smooth muscle actin and alpha-sarcomeric actin was determined by Western blotting using specific antibodies. While there was an increase in the amount of total actins and no change in the amount of alpha-sarcomeric actin in the cells exposed to genistein, the amount of alpha-smooth muscle actin decreased with increasing concentrations of genistein reaching undetectable levels at 100 microM. These results demonstrate that genistein inhibits cell spreading and the reexpression of alpha-smooth muscle actin in adult rat ventricular myocytes in culture in a dose-dependent manner, therefore, inhibiting the process of dedifferentiation.
成年大鼠心室肌细胞在培养2周后呈现扁平伸展的形态，且有组织的肌原纤维消失。这一系列表型变化伴随着胎儿基因程序的重新表达。尽管最近有研究表明不同的信号转导途径参与细胞生长和分化，但关于酪氨酸激酶激活与心肌细胞分化的了解还不多。我们使用酪氨酸磷酸化的特异性抑制剂染料木黄酮，研究酪氨酸激酶信号转导途径是否参与成年大鼠心室肌细胞在长期培养中的去分化过程。在本实验中，成年大鼠心室肌细胞按先前描述的方法进行培养，并在含有不同浓度染料木黄酮（10 - 250微摩尔）的培养基中孵育。孵育24小时后，染料木黄酮以浓度依赖的方式阻止细胞伸展。然而，在高浓度时，细胞从平板上脱落（10%至100微摩尔时，250微摩尔时为95%）。通过检测在有和没有染料木黄酮孵育6天后细胞中总肌动蛋白、α - 平滑肌肌动蛋白和α - 肌节肌动蛋白的表达，研究染料木黄酮对培养的成年大鼠心室肌细胞表型的影响。提取肌原纤维蛋白并通过凝胶电泳进行分离。使用特异性抗体通过蛋白质免疫印迹法测定α - 平滑肌肌动蛋白和α - 肌节肌动蛋白的表达。在暴露于染料木黄酮的细胞中，总肌动蛋白量增加，α - 肌节肌动蛋白量没有变化，而α - 平滑肌肌动蛋白量随着染料木黄酮浓度的增加而减少，在100微摩尔时达到不可检测水平。这些结果表明，染料木黄酮以剂量依赖的方式抑制培养的成年大鼠心室肌细胞的细胞伸展和α - 平滑肌肌动蛋白的重新表达，因此抑制去分化过程。
