Dynamics of Proteins in the A-bands of Cardiac Muscle

心肌 A 带中蛋白质的动力学

• 批准号: 7189050
• 负责人: Joseph William Sanger
• 金额: $ 34.88万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7189050
• 关键词: RNA interference; asymmetric septal hypertrophy; cardiac myocytes; cardiogenesis; developmental genetics; fluorescence recovery after photobleaching; gene mutation; hypertrophic myocardiopathy; molecular /cellular imaging; molecular assembly /self assembly; muscle proteins; myocardium; myofibrils; myosins; polymerization; protein binding; protein isoforms; protein structure function; quail; sarcomeres; tissue /cell culture

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.

描述(由申请人提供)：引起人们对心肌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。本提案的重点是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着关键作用。拟议的实验将结合对肌原纤维形成过程中活细胞中选定的A-带蛋白的时间和空间组织和动态特性的分析。我们提出了一个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再到成熟的肌原纤维。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分子从重叠的阵列重新排列到成熟的肌原纤维中形成A-带时，由于组装蛋白之间结合作用的增加，它们与细胞质肌球蛋白池的交换减少。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。这一提议有三个具体目的。第一个特殊目的是分析活细胞中四种A-带蛋白(肌肉肌球蛋白II重链、必需和调节轻链、C-蛋白)在活细胞中的动力学。第二个特殊目的是验证一种假设，即已知与肥厚性心肌病有关的肌肉肌球蛋白II重链和G-蛋白的突变分子的表达将具有不同于野生型A-带蛋白的动态特性，并将改变A-带的稳定性或在处理的心肌细胞中诱导肌原纤维紊乱。第三个具体目标是：检验肌球蛋白和肌球蛋白A带区域阻止非肌肉肌球蛋白II和肌肉肌球蛋白II共同组装的假设。有效防止两种肌球蛋白亚型共聚的最小肌球蛋白结构域将与来自肌球蛋白A带结构域的重组肌球蛋白片段进行鉴定，这些重组肌球蛋白片段负责其与肌肉肌球蛋白II重链的轻质肌球蛋白区域的结合。先进的成像方法与分子生物学和生化技术相结合，应该会对活着的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。