Cardiac Troponin in Health and Disease

描述（由申请方提供）：拟定研究的总体目标是阐明健康和疾病中肌钙蛋白（Tn）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型心肌病（FHC或HCM）、扩张型心肌病（DCM）和限制型心肌病（RCM）。将产生表达cTn亚基的敲入小鼠，所述cTn亚基含有已知在人类中引起HCM、DCM和RCM的突变，并且将研究这些心脏疾病状态的形态学和体外和体内特性。将追求以下特定目的：特定目的1：在HCM、DCM和RCM小鼠模型中研究的肌钙蛋白介导的遗传疾病的生理后果。我们建议使用以下基因敲入小鼠：（A）HCM：cTnI-R21 C和cTnT-R92 W;（B）DCM：cTnI-K183和cTnT-R141 W和（C）RCM：cTnI-K178 E和cTnI-R145 W。我们将执行：i）生物化学表征; ii）纤维研究（以建立ATP酶/力和gapp的Ca 2+敏感性）; iii）力和细胞内[Ca 2 +]瞬变; iv）组织分析;和v）生理学和电生理学表征。具体目的2：阐明肌钙蛋白C，一种分子钙离子在HCM，DCM和RCM中的作用。本研究拟使用以下突变cTnC敲入小鼠：cTnC-S37 G（HCM）、cTnC-F20 Q（DCM）和cTnC-V44 Q（RCM）。如果如我们假设的那样，cTnC最终负责由cTnC或TnT和/或TnI中的突变引起的HCM、DCM和RCM的钙依赖性表型性质，则可以预期在cTnC中产生这些敲入突变，改变其Ca 2+结合亲和力和/或肌肉的其他收缩性质，将产生的表型，在拟议的敲入小鼠，是类似于那些在人身上看到的。具体的目的3：分析的目的1和2中的生理测量将被利用，以提出支持理论的机制负责HCM，RCM和DCM。一个全面的理论或理论负责HCM，DCM和RCM的机制将被提出。我们将评估数据并根据各种心肌病对结果进行分组。将进一步分析结果，以确定HCM、RCM和DCM特征的相关性。我们的多维方法将允许阐明的机制，负责特定的肌病和确定的严重程度的特定突变，导致恶性表型和SCD在man. These研究将是至关重要的了解这些遗传结构的变化对心肌的影响，以及它们如何可能导致三个不同的疾病状态。

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.