Dynamics of Proteins in the A-bands of Cardiac Muscle

描述（由申请人提供）：引起对心肌中肌原纤维形成和维持重新产生兴趣的因素之一是将疾病与肌合成蛋白突变联系起来的遗传分析。本建议的重点是a带的形成和维持，a带是肌原纤维的一个关键区域，对心肌肌节的稳定和收缩起作用。拟议的实验将结合分析肌原纤维形成活细胞中选定的a带蛋白的时空组织和动态特性。我们提出了肌原纤维在肌肉细胞中形成的三步模型：前肌原纤维到新生肌原纤维到成熟肌原纤维。我们的第一个工作假设是，当新生肌原纤维中的肌球蛋白II分子从重叠阵列重新排列到成熟肌原纤维中形成a带时，由于组装蛋白之间的结合相互作用增加，它们与肌球蛋白细胞质池的交换减少。突变的蛋白质可能改变了导致肌原纤维不稳定的动力学。我们的第二个工作假设是，titin在a -band形成中的作用之一是防止非肌肌球蛋白II与肌肌球蛋白II共聚合。这项建议有三个具体目标。第一个具体目标是分析活细胞中肌原纤维在活禽心肌细胞中重新组装时四个a带蛋白（肌肌球蛋白II重链，必需和调节轻链，c蛋白）的动力学。第二个特定目的是验证假设，即已知参与肥厚性心肌病的肌球蛋白II重链和g蛋白突变分子的表达将具有不同于野生型a带蛋白的动态特性，并将改变a带稳定性或诱导交易心肌细胞中的肌原纤维紊乱。第三个具体目标是；以验证titin和titin的a带区域阻止非肌凝蛋白II和肌凝蛋白II共组装的假设。有效防止两种肌球蛋白异构体共聚的最小肌凝蛋白结构域将被鉴定为来自肌凝蛋白A带结构域的重组肌凝蛋白片段，该结构域负责与肌凝蛋白II重链的轻肌凝蛋白区域结合。先进的成像方法与分子生物学和生化技术相结合，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。

DESCRIPTION (provided by applicant): One of the factors that has spurred renewed interest in myofibril formation and maintenance in cardiac muscles is the genetic analyses linking diseases with mutations in sarcomeric proteins. The emphasis in this proposal focuses on the formation and maintenance of the A-band, a key region of the myofibril for the stability and contraction of cardiac sarcomeres. The proposed experiments will combine analyses of the temporal and spatial organization and dynamic properties of selected A-band proteins in live cells undergoing myofibrillogenesis. We have proposed a three-step model for the formation of myofibrils in muscle cells: premyofibrils to nascent myofibrils to mature myofibrils. Our first working hypothesis is that as muscle myosin II molecules in nascent myofibrils realign from an overlapping array to form A-bands in mature myofibrils, their exchange with a cytoplasmic pool of myosin is reduced due to increased binding interactions between the assembling proteins. Proteins that are mutated may have altered dynamics that lead to myofibril instability. Our second working hypothesis is that one of titin's roles in the formation of A-bands is to prevent non-muscle myosin II from co-polymerizing with muscle myosin II. There are three specific aims in this proposal. The first specific aim is to analyze the dynamics of four A-band proteins (muscle myosin II heavy chains, essential and regulatory light chains, C-Proteins) in live cells as myofibrils assemble de novo in living avian cardiomyocytes. The second specific Aim is to test the hypothesis that expression of mutated molecules of muscle myosin II heavy chains, and G-protein, that are known to be involved in Hypertrophic Cardiomyopathies, will have different dynamic properties from wild type A-band proteins and will alter A-band stability or induce myofibril disarray in transacted cardiomyocytes. The third specific aim is; to test the hypothesis that titin and A-band regions of titin prevent the co-assembly of non-muscle myosin II and muscle myosin ll. A minimal domain of titin effective in preventing copolymerization of the two myosin isoforms will be identified with recombinant titin fragments derived from the A-band domains of titin that are responsible for its binding to the light meromyosin regions of muscle myosin II heavy chains. The advanced imaging methods coupled with molecular biological and biochemical techniques should yield new insights into basic and pathologic processes in myofibril assembly in living cardiomyocytes.