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不断调节，并可在疾病中改变。已知外部和细胞内因素都影响心脏的收缩动力学。我们认为，一个低估的因素，可以影响这些动力学是钙离子交换率的肌钙蛋白C（TnC）。该提案的目标是描绘TnC在心肌收缩和舒张动力学中的作用。一般认为TnC与Ca2+迅速平衡，因此对心肌收缩或舒张的动力学没有影响。然而，我们发表的和初步的数据表明：1）TnC的Ca 2+结合特性影响心肌收缩的速率，2）Ca 2+从TnC解离在生理相关的生化系统中并不迅速（重构的细丝和肌原纤维）并且类似于心肌松弛的速率，3）与加速或减慢心肌松弛相关的TnI和TnT的生理和病理生理学改变同样加速或减慢Ca 2+从TnC解离的速率，和4）疾病相关蛋白的异常Ca 2+结合特性可以在生物化学和生理系统中通过特异性工程化的TnC来校正。具体目标I将检验以下假设：与TnC的Ca2+交换速率通过与心肌收缩和舒张动力学改变相关的TnI和TnT修饰而改变。Ca2+交换率将监测生理相关的生化系统的重构细丝和大鼠心室肌原纤维利用停流装置和我们的新的荧光TnC。我们将利用TnI和TnT构建体：1）模拟PKA和PKC磷酸化，2）与缺血再灌注损伤相关，3）与家族性肥厚性、限制性和扩张性心肌病（分别为HCM、RCM和DCM）相关。具体目标II将阐明和调节影响Ca 2+与TnC交换速率的分子机制。我们假设有两种基本机制影响Ca 2+与TnC的交换速率：1）TnC的固有Ca 2+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。具体目标III将检验Ca2+与TnC交换的速率影响心肌收缩和舒张速率的假设。通过笼状Ca2+螯合剂重氮-2的光解诱导松弛，而收缩的动力学将通过测量力再发展的速率来评估。此外，我们将测试特异性工程化的TnC构建体是否可以纠正与疾病相关的TnI和TnT修饰的异常收缩动力学。获得的数据将阐明TnC在心肌收缩和舒张动力学中的作用，这将转化为新的假设驱动的治疗策略，以对抗收缩和舒张功能障碍。 公共卫生相关性：这项研究的意义是多方面的，将：1）提供对控制心脏细丝的Ca2+结合和交换动力学的机制的洞察，2）提供对这些机制在生理和病理生理状态下如何改变的更清楚的理解，3）确定合理工程化的TnC构建体是否可以纠正异常的生化和生理Ca2 +细丝的Ca2+结合和交换动力学，4）直接测试细丝的Ca2+结合和交换动力学是否调节心肌收缩和舒张的速率，和5）促进靶向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.