Cardiac Troponin in Health and Disease

描述（由申请人提供）：拟议研究的总体目标是阐明肌钙蛋白（Tn）在健康和疾病中调节心肌收缩的分子机制。目前的提案将确定cTnT、cTnI和cTnC突变对心肌生化、收缩和电生理特性的影响，这些突变已知会导致家族性肥厚性心肌病（FHC或HCM）、扩张型心肌病（DCM）和限制性心肌病（RCM）。将产生表达cTn亚基的敲入小鼠，这些亚基含有已知导致人类HCM， DCM和RCM的突变，并将研究这些心脏病状态的形态学和体外和体内特性。具体目标1：在HCM、DCM和RCM小鼠模型中研究肌钙蛋白介导的遗传疾病的生理后果。我们建议利用以下敲入小鼠：(A) HCM: cTnI-R21C和cTnT-R92W；(B) DCM: cTnI- K183和cTnT-R141W； (C) RCM: cTnI- k178e和cTnI- r145w。我们将进行：i)生化表征；ii)纤维研究（建立Ca2+对atp酶/力和gapp的敏感性）；iii)力和细胞内[Ca2+]瞬态；iv)组织分析；生理和电生理表征。具体目的2：阐明肌钙蛋白C，一种分子Ca2+开关在HCM、DCM和RCM中的作用。本研究提出了以下突变型cTnC敲入小鼠：cTnC- s37g (HCM), cTnC- f20q （DCM）和cTnC- v44q （RCM）。如果，正如我们假设的那样，cTnC最终负责由cTnC或TnT和/或TnI突变引起的HCM、DCM和RCM的钙依赖性表型特性，人们可以预期，在cTnC中制造这些敲入突变，改变其Ca2+结合亲和力和/或其他肌肉收缩特性，将在敲入小鼠中产生与人类相似的表型。具体目标3：分析目标1和目标2中的生理测量值，提出HCM、RCM和DCM机制的统一理论。将提出一个或多个关于HCM、DCM和RCM机制的综合理论。我们将根据不同的心肌病对数据进行评估和分组。结果将进一步分析定义HCM、RCM和DCM特征的相关性。我们的多维方法将允许阐明负责特定肌病的机制，并确定导致人类恶性表型和SCD的特定突变的严重程度。这些研究对于理解这些基因结构变化对心肌的影响以及它们如何导致三种不同的疾病状态至关重要。

DESCRIPTION (provided by applicant): The overall goal of the proposed studies is to elucidate the molecular mechanisms involved in the regulation of cardiac muscle contraction by troponin (Tn) in health and disease. The current proposal will determine the effect of mutations in cTnT, cTnI and cTnC, known to cause familial hypertrophic cardiomyopathy (FHC or HCM), dilated cardiomyopathy (DCM) and restrictive cardiomyopathy (RCM) on the biochemical, contractile and electrophysiological properties of cardiac muscle. Knock-in mice will be generated expressing cTn subunits that contain mutations known to cause HCM, DCM and RCM in man and the morphological and in vitro and in vivo properties of these cardiac disease states will be investigated. The following Specific Aims will be pursued: SPECIFIC AIM 1: PHYSIOLOGICAL CONSEQUENCES OF TROPONIN - MEDIATED GENETIC DISORDERS STUDIED IN THE HCM, DCM, AND RCM MOUSE MODELS. We propose to utilize the following knock-in mice: (A) HCM: cTnI-R21C and cTnT-R92W; (B) DCM: cTnI- K183 and cTnT-R141W and (C) RCM: cTnI-K178E and cTnI-R145W. We will perform: i) Biochemical characterization; ii) Fiber studies (to establish Ca2+ sensitivity of ATPase/force and gapp); iii) Force and intracellular [Ca2+] transients; iv) Tissue analysis; and v) Physiological and electrophysiological characterization. SPECIFIC AIM 2: ELUCIDATE THE ROLE THAT TROPONIN C, A MOLECULAR Ca2+ SWITCH PLAYS IN HCM, DCM AND RCM. The following mutant cTnC knock-in mice are proposed for this study: cTnC-S37G (HCM), cTnC-F20Q (DCM) and cTnC-V44Q (RCM). If, as we hypothesize, cTnC is ultimately responsible for the calcium dependent phenotypic properties underlying HCM, DCM and RCM that are caused by the mutations in either cTnC or TnT and/or TnI, one would expect that making these knock-in mutations in cTnC that alter its Ca2+ binding affinity and/or other contractile properties of muscle, would produce phenotypes in proposed knock-in mice that are similar to those seen in man. SPECIFIC AIM 3: ANALYSIS OF THE PHYSIOLOGICAL MEASUREMENTS IN AIM 1 and 2 WILL BE UTILIZED TO PROPOSE UNIFYING THEORIES OF MECHANISMS RESPONSIBLE FOR HCM, RCM AND DCM. A comprehensive theory or theories regarding the mechanisms responsible for HCM, DCM and RCM will be proposed. We will evaluate the data and group the results according to various cardiomyopathies. The results will be further analyzed for correlations that define characteristics of HCM, RCM and DCM. Our multidimensional approach will allow elucidation of the mechanisms that are responsible for specific myopathies and the determination of the severity of specific mutations that cause malignant phenotypes and SCD in man. These studies will be critical in understanding the effect these genetic structural changes have on cardiac muscle and how they might lead to the three distinctive disease states.