Dynamics of Proteins in the A-bands of Cardiac Muscle

心肌 A 带中蛋白质的动力学

基本信息

批准号：
7385985
负责人：
Joseph William Sanger
金额：
$ 33.79万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
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/7385985
关键词：
Binding Biochemical Biological Birds Cardiac Cardiac Myocytes Cells Complex Coupled Disease Doctor of Philosophy Fibrinogen Filament Fluorescence Fluorescence Recovery After Photobleaching Fluorescent Dyes GTP-Binding Proteins Goals Green Fluorescent Proteins Hypertrophic Cardiomyopathy Image Label Lead Life Light Link Maintenance Methods Microinjections Microscopy Modeling Molecular Muscle Muscle Cells Muscle Fibers Muscle Proteins Mutate Mutation Myocardium Myofibrillogenesis Myofibrils Myopathy Myosin ATPase Myosin Regulatory Light Chains Myosin Rod Myosin Type II Numbers Optical Methods Optics Pathologic Processes Plasmids Production Property Protein Array Protein Dynamics Protein Family Protein Isoforms Proteins RNA Interference Rate Recombinants Recovery Recruitment Activity Research Personnel Role Sarcomeres Staging Techniques Testing Time Transact Transfection Work cellular engineering citrate carrier connectin genetic analysis insight interest member photoactivation prevent protein protein interaction research study

项目摘要

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）：刺激对心肌肌原纤维形成和维持的重新兴趣的因素之一是将疾病与肉瘤蛋白突变联系起来的遗传分析。该提案的重点集中在A波段的形成和维护上，A波段是心脏肉瘤的稳定性和收缩的肌纤维的关键区域。所提出的实验将结合对经历肌原纤维发生的活细胞中某些A波段蛋白的时间和空间组织的分析。我们提出了一个三步模型，用于在肌肉细胞中形成肌原纤维：前纤维至新生的肌原纤维以使成熟的肌原纤维。我们的第一个工作假设是，随着从重叠阵列中的肌肉肌球蛋白II分子从重叠的阵列重新调整以形成成熟的肌纤维中的A频带，它们与肌球蛋白的细胞质池的交换由于增加的结合相互作用而减少了组装蛋白之间的结合相互作用。突变的蛋白质可能改变了导致肌原纤维不稳定性的动力学。我们的第二个工作假设是，Titin在A波段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。该提案中有三个具体的目标。第一个具体目的是分析活细胞中四种A波段蛋白（肌肉肌球蛋白II重链，必不可少的和调节的轻链，C蛋白）的动力学，因为肌原纤维在活的鸟类心肌细胞中从头组装DE。 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.第三个特定目标是；为了测试钛和titin的A频段区域的假设，可以防止非肌肉肌球蛋白II和肌肉肌球蛋白LL的共同组装。有效防止两种肌球蛋白同工型共聚的钛的最小结构域将与源自TITIN的A波段结构域得出的重组钛片片段鉴定，这些片段构成了其与肌肉肌球蛋白II重链肌肉肌球蛋白II重链的光合霉素区域的结合。先进的成像方法与分子生物学和生化技术相结合，应为生活心肌细胞中肌纤维组装中的基本和病理过程提供新的见解。