Determinants of Cardiac Thin Filament Regulation

心脏细丝调节的决定因素

• 批准号: 7379827
• 负责人: Robert J. Barsotti
• 金额: $ 39.16万
• 依托单位: PHILADELPHIA COLLEGE OF OSTEOPATHIC MED
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7379827
• 关键词: ATP phosphohydrolase; Actins; Actomyosin Adenosinetriphosphatase; Address; Affect; Affinity; Area; Behavior; Binding; Binding Sites; C-terminal; Cardiac; Cardiac Muscle Contraction; Cardiomyopathies; Chelating Agents; Communication; Complex; Couples; Coupling; Data; Dependence; Development; Dissociation; Equilibrium; Feedback; Fiber; Fluorescence Polarization; Foundations; Generations; Goals; Heart; Human; Individual; Iodoacetamide; Isometric Exercise; Kinetics; Knowledge; Label; Lasers; Link; Measures; Mechanics; Mediating; Modality; Modification; Molecular Conformation; Monitor; Mutation; Myocardium; Nature; Orthovanadate; Pathway interactions; Phosphorylation; Play; Population; Process; Property; Protein Conformation; Proteins; Rate; Regulation; Relative (related person); Relaxation; Reporting; Resolution; Rhodamine; Rhodamines; Role; Shapes; Site; Skin; Solutions; Striated Muscles; Structure; System; Testing; Therapeutic; Thin Filament; Thinking; Time; Tropomyosin; Troponin; Troponin C; Troponin I; Troponin T; Work; analog; base; clinical phenotype; design; diazo-2; monomer; mutant; novel; photolysis; research study; retinal rods; tetramethylrhodamine; tool

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.

描述（由申请人提供）：心肌收缩是由Ca2+结合到由3个肌钙蛋白（Tn）亚基组成的细丝调节单元引起的：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。心脏通常在亚最大激活水平上工作，在那里合作激活机制最为明显，因此是收缩性的重要决定因素。这些研究的长期目标是提供Ca2+结合如何导致调节复合物的合作相互作用的精确描述，这些过程的速率如何控制张力时间过程以及Tn突变如何改变这一机制并导致心肌病。提出的研究将验证心脏细丝和[Ca2+]的激活状态处于快速平衡和调节单元功能的假设，不是单独的，而是作为一个整体。因此，在亚最大值[Ca2+]激活期间和弛豫期间，细丝的激活程度和张力时间过程主要由正交叉桥反馈及其动力学决定。我们还假设cTnI突变增加Ca2+张力敏感性的作用是通过改变细丝激活和弛豫动力学和跨桥力学介导的。为了验证这些假设，我们将通过与特定位点结合的罗丹明探针同时监测张力与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来测量。剥皮心脏小梁的内源性cTnC或Tn将交换为标记的cTnC和具有非活性TnC Ca2+调节位点的新型标记Tn复合物。该复合体模拟Ca2+自由调节单元，而附着的探针允许监测仅通过其与邻近调节单元的相互作用而不是直接结合Ca2+而引起的Tn结构变化。激活将通过在含有不同[Ca2+]的溶液中孵育和通过激光光解新的笼状Ca2+在[Ca2+]中的快速跳跃引起。笼状ca2 +螯合剂重氮-2的光解作用可引起弛豫。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引起的心肌病奠定基础。