MYOFIBRILLOGENESIS IN LIVING CARDIAC MUSCLE CELLS

心脏的形态和功能的中心是肌原纤维，它提供了心肌细胞特有的细胞骨架结构和收缩力量。实验方法的第一个策略是通过转染编码与绿色或蓝色荧光蛋白相连的肌节蛋白的质粒，显微注射多肽片段和荧光标记的细胞骨架蛋白，结合使用定量光学技术来分析活的心肌细胞内肌原纤维的形成。第二种方法侧重于对组装的肌原纤维的Z-小体和Z-带中相互作用的蛋白质进行分子分析。第一个具体目的是分析在正常扩张条件下、胞质分裂后和肥大期间活的心肌细胞培养中肌节亚基组装成肌原纤维的情况。第二个特定目的是确定在活的心肌细胞中前肌原纤维的形成和它们向成熟的肌原纤维的转化是否需要非肌肉肌球蛋白II，其功能是通过Rho家族的G-蛋白激活的。第三个具体目的是分析α-肌动蛋白在前肌原纤维Z-小体形成中的作用，以及纽蛋白在活体心肌细胞中Z-小体融合形成成熟肌原纤维的胞核Z-带中的募集和作用。第四个特定目的是使用GFP靶向试验来确定Titin N-末端的哪些亚域可以定位于活的心肌细胞的Z-带。我们将探索这些不同的titin结构域在将胞核蛋白靶向Z-带方面的作用。蛋白质-蛋白质相互作用分析将被用来识别与Titin的Z-带区域相互作用的Z-带和胞核蛋白质。先进的光学方法与分子生物学技术相结合，将使有关肌原纤维形成的假说可以直接在单个活着的大鼠、新生大鼠和鸡胚胎心肌细胞中进行测试。这些方法应该会对活的心肌细胞中肌原纤维组装的基本和病理过程产生新的见解。

Central to the form and function of the heart are the myofibrils that provide the cytoskeletal structure and the contractile force that characterize cardiomyocytes. The first strategy of the experimental approaches is on analyzing myofibril formation inside living cardiomyocytes via the transfections of plasmids encoding sarcomeric proteins linked to Green or Blue Fluorescent Proteins, the microinjection of peptide fragments and fluorescently labeled cytoskeletal proteins coupled to the use of quantitative optical techniques. The second approach is focused on a molecular analysis of interacting proteins in the Z-bodies and Z-bands of the assembling myofibrils. The first specific aim is to analyze the assembly of sarcomeric subunits into myofibrils in cultures of living cardiomyocytes under normal spreading conditions, after cytokinesis and during hypertrophy. The second specific aim is to determine if the formation of premyofibrils and their transformation to mature myofibrils in living cardiomyocytes requires non-muscle myosin II whose function is activated through the Rho family of G-Proteins. The third specific aim is to analyze the role of alpha-actinin in the formation of the Z-bodies in premyofibrils and the recruitment and role of vinculin in the fusion of the Z-bodies to form the costameric Z-bands of mature myofibrils in living cardiomyocytes. The fourth specific aim is to use a GFP targeting assay to determine which subdomains of the N-terminus of titin can localize to the Z-bands of living cardiac muscle cells. We will probe the role of these different titin domains in targeting costameric proteins to the Z-band. Protein-protein interaction assays will be used to identify the Z-band and costameric proteins, that interact with the Z-band regions of titin. The advanced optical methods coupled with molecular biological techniques will allow hypotheses about the formation of myofibrils to be tested directly in single living rat neonatal and chick embryonic cardiomyocytes. These approaches should yield new insights into basic and pathologic processes in myofibril assembly in living cardiomyocytes.