Role of Cardiac Troponin in Health and Disease

描述(申请人提供)：心脏收缩和松弛动力学在生命中不断调节，并可以在疾病中改变。众所周知，外部因素和细胞内因素都会影响心脏的收缩动力学。我们认为，肌钙蛋白C的钙交换率(TNC)是影响这些动力学的一个被低估的因素。这项提议的目的是描述TNC在心肌收缩和松弛动力学中的作用。一般认为，TNC与钙离子迅速平衡，因此对心肌收缩或松弛的动力学没有影响。然而，我们已发表的和初步的数据表明，1)TNC的钙结合特性影响心肌收缩的速率，2)在更生理相关的生化系统(重组的细丝和肌原纤维)中，TNC的钙解离并不迅速，并且与心肌松弛的速率相似，3)TnI和TnT的生理和病理生理修饰与心肌松弛加速或减慢相关，同样加速或减缓TNC的钙解离速率，以及4)疾病相关蛋白的异常的钙结合特性可以通过特定的工程TNC在生化和生理系统中纠正。具体目的I将验证这样的假设，即与TNC的钙交换速率被TnI和TNT修饰改变，这与心肌收缩和松弛动力学改变有关。利用停流仪和我们的新型荧光TNC，将监测重组细丝和大鼠心室肌原纤维的生理相关生化系统中的CA2交换率。我们将利用TnI和TnT结构：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、限制性和扩张型心肌病(分别为HCM、RCM和DCM)相关。专一目的II将阐明和调控影响钙离子交换速率的分子机制(S)。我们假设，影响与TNC的钙交换速率的基本机制有两个：1)TNC固有的钙结合特性；2)TNC调节域与TnI结合的能力。我们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。《特定目的III》将验证与TNC的钙交换速率影响心肌收缩和松弛速率的假设。钙离子螯合剂重氮-2的光解将引起松弛，而收缩的动力学将通过测定力的再发展速率来评估。此外，我们将测试特殊设计的TNC结构是否可以纠正疾病相关的TnI和TnT修饰的异常收缩动力学。获得的数据将阐明TNC在心肌收缩和松弛动力学中的作用，这将转化为新的假说驱动的治疗策略，以对抗收缩和舒张期功能障碍。 与公共卫生相关：这项研究的意义是多方面的，将：1)提供控制心脏细丝钙结合和交换动力学的机制；2)更清楚地了解这些机制在生理和病理生理状态下如何改变；3)确定合理设计的TNC结构是否可以纠正细丝异常的生化和生理钙结合和交换动力学；4)直接测试细丝的钙结合和交换动力学是否调节心肌收缩和松弛的速率；5)促进针对心脏收缩和舒张功能障碍的治疗剂的设计。

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. PUBLIC HEALTH RELEVANCE: The significance of this research is multifold and will: 1) provide insight into the mechanisms that control the Ca2+ binding and exchange kinetics of the cardiac thin filament, 2) provide a clearer understanding of how these mechanisms are altered in physiological and pathophysiological states, 3) determine if rationally engineered TnC constructs can correct the aberrant biochemical and physiological Ca2+ binding and exchange kinetics of the thin filament, 4) directly test whether the Ca2+ binding and exchange kinetics of the thin filament modulate the rates of cardiac muscle contraction and relaxation and 5) facilitate the design of therapeutic agents targeted against cardiac systolic and diastolic dysfunctions.