Determinants of Cardiac Thin Filament Regulation

心脏细丝调节的决定因素

基本信息

批准号：
7685309
负责人：
Robert J. Barsotti
金额：
$ 39.63万
依托单位：
PHILADELPHIA COLLEGE OF OSTEOPATHIC MED
依托单位国家：
美国
项目类别：
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/7685309
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 Regulation Relative (related person)Relaxation Reporting Resolution Rhodamine Rhodamines Role Shapes Site Skin Solutions Striated Muscles Structure System Testing Therapeutic Thin Filament 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

项目摘要

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+ 与细丝调节单元结合引发的，该细丝调节单元由 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) 和 1原肌球蛋白 (Tm) 和 7 种肌动蛋白。心脏通常在次最大激活水平下发挥作用，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准确描述 Ca2+ 结合如何导致调节复合物的协同相互作用、这些过程的速率如何控制紧张时间过程以及 Tn 突变如何改变这一机制并导致心肌病。拟议的研究将测试以下假设：心脏细丝和 [Ca2+] 的激活状态处于快速平衡状态，并且调节单元不是单独发挥作用，而是作为一个整体发挥作用。因此，在次最大[Ca2+]激活期间和松弛期间，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 突变增加 Ca2+- 张力敏感性的作用是通过改变细丝激活和松弛动力学以及跨桥力学介导的。为了检验这些假设，我们将通过结合到特定位点的罗丹明探针同时监测张力和 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) 来测量，即探针方向（探针角度）相对于细丝轴和角色散的变化。带皮心脏小梁的内源性 cTnC 或 Tn 将被交换为标记的 cTnC 和具有非活性 TnC Ca2+ 调节位点的新型标记 Tn 复合物。该复合物模仿无 Ca2+ 的调节单元，而附加的探针可以监测 Tn 结构变化，该变化仅通过其与邻近调节单元的相互作用而产生，而不是由 Ca2+ 的直接结合引起。通过在含有不同 [Ca2+] 的溶液中孵育以及通过新型笼状 Ca2+ 的激光光解使 [Ca2+] 快速跃升来引发激活。笼状 Ca2+ 螯合剂 diazo-2 的光解会引发弛豫。该项目的长期目标是了解心肌细丝调节机制，从而为明确评估 Tn 突变的作用并开发治疗由此产生的心肌病的治疗方式奠定基础。