Myofibrillogenesis in Living Cardiac Muscle Cells

描述(申请人提供)：肌原纤维的一个关键亚基，Z-带，串联连接肌节收缩单元，也是中间丝、胞囊和膜的附着点。这种肌原纤维复合体是如何组装的还不清楚。第一个具体目标是分析两种禽类培养系统中活细胞中Z带的组装情况：心脏前中胚层培养和10天胚胎心肌细胞培养，在这两种培养体系中，第一批肌原纤维形成第一个肌原纤维，而在10天胚胎心肌细胞培养中，新的肌原纤维在具有预先存在的肌原纤维的细胞中组装。第二个特定目的是测试突变的Z-带titin片段的表达将改变Z-带的稳定性，干扰α-肌动蛋白细丝的能力，和/或在转基因的心肌细胞中诱导肌原纤维紊乱。第三个具体目的是分析一种新的Z-带蛋白NSPI-1在成熟肌原纤维的Z-小体向Z-带的转变和Z-带结构的维持中的作用。NSPL-1可能是连接Z带、胞壁、中间丝和膜的一个潜在的重要分子。实验方法包括使用编码连接到肌节蛋白和突变结构域的绿色荧光蛋白组成员的质粒来转染心肌细胞。活细胞的图像将通过光学技术进行分析，如光漂白后荧光恢复(FRAP)、共聚焦和去卷积显微镜。这些先进的光学方法与分子和生化技术相结合，将被用来检验关于肌原纤维的形成以及Z-带突变的Titin片段对与其相互作用的蛋白质动力学的影响的假设。这些方法应该会对Z带在心肌细胞肌原纤维组装的基本和病理过程中的作用产生新的见解。

DESCRIPTION (provided by applicant): A key subunit of myofibrils, the Z-band, links the sarcomeric contractile units in series and also serves as attachment sites for the intermediate filaments, the costameres and membranes. How this myofibrillar complex is assembled is unknown. The first specific aim is to analyze the assembly of Z-bands in live cells in two avian culture systems: precardiac mesoderm cultures in which the first myofibrils form, and 10-day embryonic cardiomyocyte cultures in which new myofibrils assemble in cells with pre-existing myofibrils. The second specific aim is to test the idea that expression of a mutated Z -band titin fragment, corresponding to a mutation reported to be involved in Hypertrophic Cardiomyopathy, will alter Z-band stability, interfere with the ability of alpha-actinin to cross-link actin filaments, and/or induce myofibril disarray in transfected cardiomyocytes. The third specific aim is to analyze the role of a new Z-band protein, NspI-1, in the transition of Z-bodies to Z-bands of mature myofibrils and in the maintenance of Z-band structure. Nspl-1 may be a potentially important molecule for linking Z-bands, costameres, intermediate filaments, and membranes. The experimental approaches include the use of plasmids encoding members of the group of Green Fluorescent Proteins ligated to sarcomeric proteins and mutated domains for transfections of cardiomyocytes. Images of live cells will be analyzed by optical techniques, such as Fluorescence Recovery after Photobleaching (FRAP), confocal and deconvolution microscopy. These advanced optical methods coupled with molecular and biochemical techniques will be used to test hypotheses about the formation of myofibrils and the effects of mutant Z- band titin fragments on the dynamics of proteins interacting with them. These approaches should yield new insights into the role of the Z-band in basic and pathologic processes in myofibril assembly in cardiomyocytes.