DEVELOPMENT OF THIN FILAMENT REGULATION OF CONTRACTION

细丝收缩调节的发展

• 批准号: 2702072
• 负责人: SCOTT H BUCK
• 金额: $ 8.69万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2702072
• 关键词: SDS polyacrylamide gel electrophoresis; acidity /alkalinity; biomechanics; caffeine; calcium binding protein; cell growth regulation; chemical kinetics; gene expression; heart cell; heart contraction; histogenesis; laboratory rat; muscle cells; muscle contraction; muscle tension; myofibrils; myosins; point mutation; protein isoforms; protein reconstitution; protein structure function; stainings; troponin; western blottings

The overall goal of this project is to elucidate the ontogeny of regulation of cardiac contraction by developmental isoforms of troponin I (TnI) and troponin T (TnT). Contraction of vertebrate striated muscle is regulated by Ca2+ binding to troponin. Troponin is comprised of three components, two of which, TnI and TnT, undergo isoform switching during cardiac development. The central hypothesis of this proposal is that observed differences of contractile function comparing immature and mature heart are in part mediated by TnI and TnT developmental isoforms conferring significant effects on regulation of contraction by affecting Ca2+ sensitivity of tension, by affecting cooperative activation of thin filaments by bound cross-bridges, and by affecting cross-bridge interaction kinetics. Experiments to address the following specific aims will be performed: (Specific Aim #1) to identify TnT and TnI developmental isoforms in rat heart, (Specific Aim #2) to characterize the tension-pCa relationship during TnT and TnI isoform transitions at various developmental stages and in response to reconstitution with TnT and TnI developmental isoforms, (Specific Aim #3) to determine the effects of TnT and TnI developmental isoforms on activation of contraction by Ca2+ sensitizing agents and cooperative activation by strong binding myosin cross-bridges, and (Specific Aim #4) to determine possible roles of TnT and Tnl developmental isoforms in determining cross-bridge cycling kinetics. The relationship between TnI and TnT isoform expression (determined by SDS-PAGE, ultra-sensitive silver staining, and immunoblotting) and myofibrillar contractile behavior (tension-pCa relationship, cross-bridge cycling rate, cross-bridge number, force per cross-bridge, effect of the strong binding myosin S-1 derivative NEM-S1) will be assessed in native skinned trabeculae and single myocytes from rat heart at various developmental stages during TnT and TnT isoform transitions and in preparations in which native TnI and/or TnI is extracted and replaced with specific TnT and TnI developmental isoforms. Experiments in this proposal will elucidate specific roles of thin filament proteins TnI and TnT in the development of regulation of cardiac contraction. The applicant anticipates extending this unique multi-level approach to studies of cross-bridge kinetics during development utilizing caged compounds, to studies employing mutated TnI and TnT in order to determine site (or sites) conferring observed developmental changes, and to studies relating myofibrillar protein content and contractile dysfunction in congenital or acquired cardiomyopathies.

本项目的总体目标是阐明 肌钙蛋白I发育亚型对心脏收缩的调节 (TnI)和肌钙蛋白T（TnT）。脊椎动物横纹肌的收缩是 由Ca 2+与肌钙蛋白结合调节。肌钙蛋白由三个组成 组分，其中两个，TnI和TnT，在 心脏发育该提案的中心假设是， 观察到未成熟和成熟的收缩功能差异 心脏部分由TnI和TnT发育同种型介导 通过影响对收缩的调节产生显著影响， Ca 2+敏感性的张力，通过影响协同激活薄 通过约束横桥和影响横桥 相互作用动力学 为实现以下具体目标而进行的实验 将进行：（具体目标#1）以确定TnT和TnI发育 大鼠心脏中的亚型（具体目标#2），以表征张力-pCa 在不同温度下TnT和TnI亚型转换期间的关系 发育阶段和对TnT和TnI重建的反应 发育亚型，（具体目标#3）以确定TnT的影响 和TnI发育亚型对Ca ~（2+）激活收缩的影响 致敏剂和强结合肌球蛋白的协同激活 跨桥，和（具体目标#4），以确定TnT的可能作用 和Tnl发育亚型在确定跨桥循环中的作用 动力学 TnI与TnT亚型表达的关系 （通过SDS-PAGE、超灵敏银染色和 免疫印迹）和肌原纤维收缩行为（张力-pCa 关系，跨桥循环率，跨桥数，力每 交叉桥，强结合肌球蛋白S-1衍生物NEM-S1的作用） 将在来自大鼠的天然皮肤小梁和单个肌细胞中进行评估 在TnT和TnT同工型期间不同发育阶段的心脏 在天然TnI和/或TnI是 提取并替换为特异性TnT和TnI发育同种型。 本提案中的实验将阐明薄的具体作用 肌丝蛋白TnI和TnT在心脏发育调节中的作用 收缩。申请人预计将这种独特的多层次 研究开发过程中的跨桥动力学方法 笼状化合物，使用突变的TnI和TnT的研究， 确定赋予观察到的发育变化的位点，以及 与肌原纤维蛋白含量和收缩相关的研究 先天性或获得性心肌病的功能障碍。