Role of Cardiac Troponin in Health and Disease

描述（由申请人提供）：心脏收缩和舒张动力学在生命过程中是连续调节的，在疾病中可以改变。众所周知，细胞内外因素都会影响心脏的收缩动力学。我们认为，可以影响这些动力学的一个未被重视的因素是肌钙蛋白C （TnC）的Ca2+交换率。本建议的目的是描述TnC在心肌收缩和松弛动力学中的作用。一般认为TnC与Ca2+迅速平衡，因此对心肌收缩或舒张动力学没有影响。然而，我们发表的和初步的数据表明，1)TnC Ca2+结合特性影响心肌收缩的速度，2)在更生理相关的生化系统（重构的细丝和肌原纤维）中，TnC的Ca2+解离并不快，与心肌松弛的速度相似。3)与心肌松弛加速或减慢相关的TnI和TnT的生理和病理生理改变同样加速或减慢Ca2+从TnC解离的速率，4)疾病相关蛋白的异常Ca2+结合特性可以通过特异性工程TnC在生化和生理系统中得到纠正。我将检验与心肌收缩和松弛动力学改变相关的TnI和TnT修饰改变Ca2+与TnC交换速率的假设。Ca2+交换率将在重建细丝和大鼠心室肌原纤维的生理相关生化系统中使用停流装置和我们的新型荧光TnC进行监测。我们将利用TnI和TnT构建物：1)模拟PKA和PKC磷酸化，2)与缺血再灌注损伤相关，3)与家族性肥厚、限制性和扩张性心肌病（分别为HCM、RCM和DCM）相关。特异性目的II将阐明和调节影响Ca2+与TnC交换速率的分子机制。我们假设有两个基本机制影响Ca2+与TnC交换的速率：1)TnC固有的Ca2+结合特性和2)TnC的调节结构域结合TnI的能力。我们已经开发了一种新的竞争分析和荧光共振能量转移（FRET）系统，可以探测关键的TnC-TnI相互作用。特异性Aim III将测试Ca2+与TnC交换速率影响心肌收缩和舒张速率的假设。笼状Ca2+螯合剂重氮-2的光解作用将诱导弛缓，而收缩动力学将通过测量力再开发的速率来评估。此外，我们将测试特异性工程TnC结构是否可以纠正与TnI和TnT修饰相关的疾病异常收缩动力学。获得的数据将阐明TnC在心肌收缩和舒张动力学中的作用，这将转化为新的假说驱动的治疗策略，以对抗收缩和舒张功能障碍。

DESCRIPTION (provided by applicant): Cardiac contraction and relaxation kinetics are modulated continuously during life and can be altered in disease. It is known that both external and intracellular factors impact the contractile kinetics of the heart. We believe an under-appreciated element that can influence these kinetics is the Ca2+ exchange rate of troponin C (TnC). The goal of this proposal is to delineate the role of TnC in cardiac muscle contraction and relaxation kinetics. It is generally assumed that TnC equilibrates rapidly with Ca2+ and thus has no influence on the kinetics of cardiac muscle contraction or relaxation. However, our published and preliminary data demonstrate that 1) TnC Ca2+ binding properties influence the rate of cardiac muscle contraction, 2) Ca2+ dissociation from TnC is not rapid in more physiologically relevant biochemical systems (reconstituted thin filaments and myofibrils) and is similar to the rate of cardiac muscle relaxation, 3) physiological and pathophysiological modifications of TnI and TnT associated with accelerated or slowed cardiac muscle relaxation likewise accelerate or slow the rates of Ca2+ dissociation from TnC, and 4) the aberrant Ca2+ binding properties of the disease associated proteins can be corrected in biochemical and physiological systems by specifically engineered TnCs. Specific Aim I will test the hypothesis that the rates of Ca2+ exchange with TnC are altered by TnI and TnT modifications that are associated with altered cardiac muscle contraction and relaxation kinetics. Ca2+ exchange rates will be monitored in physiologically relevant biochemical systems of reconstituted thin filaments and in rat ventricular myofibrils utilizing a stopped-flow apparatus and our novel fluorescent TnC. We will utilize TnI and TnT constructs that: 1) mimic PKA and PKC phosphorylation, 2) are associated with ischemia-reperfusion injury and 3) are associated with familial hypertrophic, restrictive and dilated cardiomyopathies (HCM, RCM and DCM, respectively). Specific Aim II will elucidate and modulate the molecular mechanism(s) that influence the rates of Ca2+ exchange with TnC. We hypothesize that there are two fundamental mechanisms that influence the rates of Ca2+ exchange with TnC: 1) the intrinsic Ca2+ binding properties of TnC and 2) the ability of the regulatory domain of TnC to bind TnI. We have developed a novel competition assay and a fluorescence resonance energy transfer (FRET) system that can probe the critical TnC-TnI interactions. Specific Aim III will test the hypothesis that the rates of Ca2+ exchange with TnC influences the rates of cardiac muscle contraction and relaxation. Relaxation will be induced by photolysis of the caged Ca2+ chelator diazo-2, whereas the kinetics of contraction will be assessed by measuring the rate of force redevelopment. Furthermore, we will test if specifically engineered TnC constructs can correct the aberrant contractile kinetics of the disease associated TnI and TnT modifications. The data obtained will elucidate the role of TnC in cardiac muscle contraction and relaxation kinetics, which will translate to novel hypothesis driven treatment strategies to combat systolic and diastolic dysfunction.