DEVELOPMENT OF THIN FILAMENT REGULATION OF CONTRACTION

这个项目的总体目标是阐明 发育中的肌钙蛋白I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 受钙离子与肌钙蛋白结合的调节。肌钙蛋白由三种物质组成 其中两个组分，TnI和TnT，在 心脏发育。这项提议的中心假设是 未成熟与成熟的收缩功能的观察差异 心脏部分受TnI和TnT发育异构体的调节 通过影响对收缩的调节产生显著影响 钙离子对张力的敏感性，通过影响Thin的协同激活 通过捆绑的交叉桥和影响交叉桥的长丝 相互作用动力学。旨在实现以下具体目标的实验 将执行：(具体目标1)确定TNT和TNI发展 大鼠心脏中的异构体，(特定目标2)以表征张力-PCA 不同TnT和TnI亚型转变过程中的关系 发展阶段以及对TNT和TnI重建的反应 发育异构体，(特定目标3)以确定TNT的影响 钙激活收缩的TnI发育异构体 增敏剂与强结合肌球蛋白的协同激活 跨越桥梁，以及(具体目标#4)确定TNT的可能作用 和TNL发育亚型在决定跨桥骑行中的作用 运动学。TnI与TnT异构体表达的关系 (通过SDS-PAGE、超敏感银染和 免疫印迹)和肌原纤维收缩行为(张力-PCA 关系、跨桥速度、跨桥次数、受力单位 强结合肌球蛋白S-1衍生物NEM-S1的跨桥效应 将在大鼠天然皮肤小梁和单个心肌细胞中进行评估 心脏在TnT和TnT亚型发育的不同阶段 过渡期和原生TnI和/或TnI为 提取并替换为特定的TnT和TnI发育异构体。 本提案中的实验将阐明Thin的具体作用 细丝蛋白TnI和TnT在心脏发育调控中的作用 收缩。申请人期望将这一独特的多层次 开发利用过程中跨桥动力学研究方法探讨 笼状化合物，用于使用突变的TnI和TNT的研究，以便 确定与观察到的发育变化有关的一个或多个地点，以及 与肌原纤维蛋白含量和收缩有关的研究 先天性或获得性心肌病的功能障碍。

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.