MYOFIBRILLOGENESIS IN LIVING CARDIAC MUSCLE CELLS

心脏形态和功能的核心是肌原纤维，它提供细胞骨架结构和心肌细胞特征的收缩力。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相关的肉瘤蛋白的质粒、肽片段的显微注射和荧光标记的细胞骨架蛋白，以及使用定量光学技术，分析活心肌细胞内肌原纤维的形成。第二种方法侧重于对组装肌原纤维的z -小体和z -带中相互作用蛋白的分子分析。第一个具体目的是分析在正常扩散条件下，细胞分裂后和肥大期间，活心肌细胞培养中肌合成亚基组装成肌原纤维的情况。第二个具体目标是确定活心肌细胞中肌原纤维的形成及其向成熟肌原纤维的转化是否需要非肌球蛋白II，其功能通过g蛋白的Rho家族激活。第三个具体目的是分析α -肌动蛋白在肌原纤维前z小体形成中的作用，以及在活的心肌细胞成熟肌原纤维中z小体融合形成的血管蛋白的募集和作用。第四个具体目标是使用GFP靶向测定来确定titin的n端哪些亚结构域可以定位到活心肌细胞的z带。我们将探讨这些不同的titin结构域在将沿海蛋白靶向到z波段中的作用。蛋白质-蛋白质相互作用测定将用于鉴定与titin的z波段区域相互作用的z波段和沿海蛋白。先进的光学方法与分子生物学技术相结合，将允许在单个活的大鼠新生和鸡胚胎心肌细胞中直接测试关于肌原纤维形成的假设。这些方法将对活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.