DEVELOPMENT OF THIN FILAMENT REGULATION OF CONTRACTION

细丝收缩调节的发展

• 批准号: 2211182
• 负责人: SCOTT H BUCK
• 金额: $ 8.44万
• 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 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2211182
• 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.

该项目的总体目标是阐明个体发育 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 受 Ca2+ 与肌钙蛋白结合的调节。肌钙蛋白由三部分组成 其中两个成分 TnI 和 TnT 在 心脏发育。该提案的中心假设是 观察到比较未成熟和成熟的收缩功能的差异 心脏部分由 TnI 和 TnT 发育亚型介导 通过影响对收缩调节产生显着影响 Ca2+ 对张力的敏感性，通过影响薄层的协同激活 通过束缚横桥和影响横桥来形成细丝 相互作用动力学。 为实现以下具体目标而进行的实验 将执行：（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）表征张力-PCA TnT 和 TnI 亚型转变期间的关系 发育阶段以及对 TnT 和 TnI 重建的反应 发育亚型（具体目标 #3）以确定 TnT 的效果 Ca2+ 激活收缩时的 TnI 和 TnI 发育亚型 敏化剂和强结合肌球蛋白的协同激活 跨桥，以及（具体目标#4）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 和 TnT 同工型表达之间的关系 （通过 SDS-PAGE、超灵敏银染法和 免疫印迹）和肌原纤维收缩行为（张力-PCA 关系、过桥循环率、过桥次数、每次受力 跨桥，强结合肌球蛋白 S-1 衍生物 NEM-S1 的作用） 将在大鼠的天然皮肤小梁和单个肌细胞中进行评估 TnT 和 TnT 同工型期间处于不同发育阶段的心脏 天然 TnI 和/或 TnI 的转变和制剂 提取并替换为特定的 TnT 和 TnI 发育亚型。 该提案中的实验将阐明薄层的具体作用 丝状蛋白 TnI 和 TnT 在心脏调节发展中的作用 收缩。申请人期望扩展这种独特的多层次 开发过程中跨桥动力学的研究方法 笼状化合物，利用突变的 TnI 和 TnT 进行研究 确定赋予观察到的发育变化的位点（或多个位点），以及 与肌原纤维蛋白含量和收缩力相关的研究 先天性或获得性心肌病的功能障碍。