Determinants of Cardiac Thin Filament Regulation

描述（由申请方提供）：心肌收缩由Ca 2+结合至细丝调节单位启动，细丝调节单位由3个肌钙蛋白（Tn）亚基组成：肌钙蛋白C（TnC）、肌钙蛋白I（TnI）、肌钙蛋白T（TnT）、1个原肌球蛋白（Tm）和7个肌动蛋白。心脏通常在次最大激活水平下发挥功能，其中协作激活机制最明显，因此是收缩性的重要决定因素。这些研究的长期目标是精确描述Ca 2+结合如何导致调节复合物的合作相互作用，这些过程的速率如何控制张力时间过程以及Tn突变如何改变这种机制并导致心肌病。拟议的研究将测试的假设，即激活状态的心脏细丝和[Ca 2 +]是在快速平衡和监管单位的功能，而不是单独的，但作为一个整体。因此，在次最大[Ca 2 +]激活期间，以及在松弛期间，细丝激活的程度和张力时间过程主要由正跨桥反馈及其动力学决定。我们还假设，cTnI突变的作用，增加Ca 2+敏感性的张力介导的改变细丝激活和松弛动力学和跨桥力学。为了验证这些假设，我们将监测张力同时通过罗丹明探针绑定到特定网站的Tn亚基的构象变化。Tn构象和迁移率的变化将使用荧光偏振（FP）作为探针方向（探针角度）相对于细丝轴和角色散的变化进行测量。将皮肤化心脏小梁的内源性cTnC或Tn交换为标记的cTnC和具有失活的TnC Ca 2+调节位点的新型标记的Tn复合物。该复合物模拟无钙调节单元，而所附的探针允许监测仅通过其与相邻调节单元的相互作用而不是直接结合Ca 2+产生的Tn结构变化。通过在含有不同[Ca 2 +]的溶液中孵育并通过激光光解新的笼状Ca 2+使[Ca 2 +]快速跳跃来引发激活。通过笼状Ca 2+螯合剂重氮-2的光解将引起弛豫。该项目的长期目标是了解心肌中细丝调节的机制，从而为明确评估Tn突变的作用和开发治疗方法以治疗由此产生的心肌病奠定基础。

DESCRIPTION (provided by applicant): Cardiac muscle contraction is initiated by Ca2+ binding to the thin filament regulatory unit comprised of 3 troponin (Tn) subunits: troponin C (TnC), troponin I (TnI), troponin T (TnT), and 1 tropomyosin (Tm) and 7 actins. The heart normally functions at submaximal activation levels where cooperative activation mechanisms are most pronounced and thus are a significant determinant of contractility. The long term goal of these studies is to provide a precise description of how Ca2+ binding leads to the cooperative interaction of regulatory complexes, how the rate of these processes control the tension time course and how Tn mutation alter this mechanism and leads to cardiomyopathies. The proposed studies will test the hypotheses that the activation state of the cardiac thin filament and [Ca2+] are in rapid equilibrium and regulatory units function, not individually, but as an ensemble. Thus, during activation at submaximal [Ca2+], and during relaxation, the extent of thin filament activation and the tension time course are dictated primarily by positive cross-bridge feedback and their kinetics. We also hypothesize that the action of cTnI mutations that increase Ca2+- sensitivity of tension are mediated by altered thin filament activation and relaxation kinetics and cross-bridge mechanics. To test these hypotheses, we will monitor tension simultaneously with conformational changes of Tn subunits via a rhodamine probe bound to specific sites. Alterations in Tn conformation and mobility will be measured using fluorescence polarization (FP) as changes in probe orientation (probe angle) with respect to the thin filament axis and angular dispersion. Endogenous cTnC or Tn of skinned cardiac trabeculae will be exchanged for labeled cTnC and a novel labeled Tn complex with an inactive TnC Ca2+regulatory site. This complex mimics a Ca2+-free regulatory unit while the attached probe allows monitoring of Tn structural changes arising solely through its interactions with neighboring regulatory units and NOT from direct binding of Ca2+. Activation will be elicited by incubation in solutions containing varying [Ca2+] and via rapid jumps in [Ca2+] by laser photolysis of a novel caged Ca2+. Relaxation will be elicited by photolysis of caged-Ca2+ chelator, diazo-2. The long term goal of this project is to understand the mechanism of thin filament regulation in cardiac muscle and thus form the foundation from which the action of Tn mutations can be definitively assessed and therapeutic modalities developed to treat the resulting cardiomyopathies.