MYOFIBRILLOGENESIS IN LIVING CARDIAC MUSCLE CELLS

心脏的形式和功能的中心是肌原纤维，其提供细胞骨架结构和表征心肌细胞的收缩力。 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连接的肌节蛋白的质粒，显微注射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心肌细胞内的肌原纤维形成。 第二种方法是集中在相互作用的蛋白质的Z-体和Z-带的组装肌原纤维的分子分析。 第一个具体的目的是分析组装成肌原纤维的肌节亚基在正常的扩展条件下，细胞质分裂后和肥大的活心肌细胞的文化。 第二个具体目标是确定前肌原纤维的形成及其在活心肌细胞中向成熟肌原纤维的转化是否需要非肌肉肌球蛋白II，其功能通过G蛋白的Rho家族激活。 第三个具体目标是分析α-辅肌动蛋白在前肌原纤维中Z体形成中的作用，以及黏着斑蛋白在Z体融合以形成活心肌细胞中成熟肌原纤维的肋Z带中的募集和作用。第四个具体目标是使用GFP靶向测定来确定肌联蛋白的N-末端的哪些亚结构域可以定位于活心肌细胞的Z-带。 我们将探讨这些不同的肌联蛋白结构域在靶向costameric蛋白的Z带的作用。 蛋白质-蛋白质相互作用测定将用于鉴定与肌联蛋白的Z带区域相互作用的Z带和costameric蛋白。 先进的光学方法加上分子生物学技术将允许有关肌原纤维的形成的假设进行测试，直接在单个活的大鼠新生儿和鸡胚心肌细胞。 这些方法应该产生新的见解，在活心肌细胞的肌原纤维组装的基本和病理过程。

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.