Dynamics of Proteins in the A-bands of Cardiac Muscle

描述（由申请人提供）：促使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节蛋白突变联系起来的遗传分析。在这个建议的重点集中在A带的形成和维护，一个关键区域的肌原纤维的稳定性和收缩的心脏肌节。拟议的实验将结合联合收割机的时间和空间的组织和动态特性的选择A-带蛋白在活细胞进行肌原纤维生成的分析。我们提出了一个肌原纤维在肌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，由于新生肌原纤维中的肌肉肌球蛋白II分子从重叠阵列重新排列形成成熟肌原纤维中的A带，它们与肌球蛋白的细胞质池的交换由于组装蛋白之间的结合相互作用增加而减少。突变的蛋白质可能具有导致肌原纤维不稳定的改变的动力学。我们的第二个工作假设是肌联蛋白在A带形成中的作用之一是防止非肌肉肌球蛋白II与肌肉肌球蛋白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四个A带蛋白（肌球蛋白II重链，必需和调节轻链，C-蛋白）在活细胞中的肌原纤维从头组装活禽心肌细胞的动力学。第二个具体目的是检验以下假设：已知参与肥厚性心肌病的肌肉肌球蛋白II重链和G蛋白的突变分子的表达将具有与野生型A带蛋白不同的动力学性质，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：为了检验肌联蛋白和肌联蛋白的A带区域阻止非肌肉肌球蛋白II和肌肉肌球蛋白II的共组装的假设。 一个最小的结构域的肌联蛋白有效地防止两种肌球蛋白亚型的共聚将被确定与重组肌联蛋白片段衍生自肌联蛋白的A带结构域，负责其结合到轻肌球蛋白区域的肌肉肌球蛋白II重链。先进的成像方法加上分子生物学和生物化学技术应该产生新的见解的基本和病理过程中的肌原纤维组装在活的心肌细胞。

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.