Molecular Mechanism of Dilated Cardiomyopathy

描述(申请人提供)：扩张型心肌病(DCM)是一种以左室扩张和收缩功能障碍为特征的心肌疾病。据信，在所有病例中，有30%-40%的DCM是由细胞骨架和肌瘤蛋白的突变引起的。尽管大多数已知的与DCM相关的肌瘤蛋白突变会使心肌对钙离子不敏感，但对这些突变如何导致DCM的发展尚缺乏了解。然而，心肌肌钙蛋白C(CTNC)的钙敏感性在DCM发病机制中的作用仍存在争议。该项目的目的是确定我们是否可以在cTNC中产生自然界中没有发现的突变，然后将其引入小鼠心肌中，以重建DCM的表型。为了实现这一目标，我们设计了一种cTNC突变体，它能够降低重组细丝对钙的敏感性，并加快钙离子的解离速度。然后，我们通过基因打靶技术将钙离子脱敏的cTNC突变引入到小鼠心肌中，建立了一种新的Tnnc1敲入小鼠模型。这一新的基因敲入小鼠模型将使我们能够评估cTNC对钙离子脱敏在触发DCM发病机制中的作用。为了评价cTNC对钙的敏感性在扩张型心肌病发生发展中的作用，建议的项目将利用以下目标：具体目标1：检测钙减敏(包括DCM连锁的)cTNC突变对重组细丝对钙的反应对cTnI磷酸化的影响。这一目标将验证一种假设，即钙减敏(包括DCM连锁的)cTNC突变减弱了蛋白激酶A(PKA)对cTnI的磷酸化对钙结合和与重组细丝交换的影响。我们将评估降低cTNC对Ca~(2+)敏感性对重组细丝对Ca~(2+)的反应的影响，以期通过PKA将cTnI磷酸化。特异性目标2：确定cTNC对钙的敏感性降低是否导致DCM的发病。这一目的将验证以下假设：降低cTNC对钙的敏感性(通过加快钙解离速度)会导致DCM的发生。来自两个试验组(杂合子和纯合子敲入鼠)的结果将与对照组(野生型鼠)的结果进行比较。D73N cTNC突变对纯合子小鼠的后果预计比对杂合子小鼠更严重。具体目标3：确定降低cTNC钙敏感性对收缩功能的影响。这一目标将检验这样一种假设，即降低cTNC对钙的敏感性(通过加快钙解离速度)会导致收缩功能障碍。将使用超声心动图在整个动物水平上评估收缩功能。收缩功能也将利用从心脏分离的心室肌细胞在细胞水平上进行评估 杂合子、纯合子和野生型小鼠。这项研究将对扩张型心肌病的分子机制提供更深入的认识。

DESCRIPTION (provided by applicant): Dilated cardiomyopathy (DCM) is a disease of cardiac muscle characterized by left ventricular dilation and systolic dysfunction. DCM is believed to be caused by mutations in cytoskeletal and sarcomeric proteins in 30- 40 % of all cases. Understanding how these mutations lead to the development of DCM is lacking, although most known DCM-linked mutations of sarcomeric proteins desensitize cardiac muscle to Ca2+. However, the role of Ca2+ sensitivity of cardiac troponin C (cTnC) in triggering the pathogenesis of DCM is a matter of ongoing debate. The objective of the proposed project is to determine whether we can generate a mutation in cTnC not discovered in nature and then introduce it into mouse myocardium to recreate the phenotype of DCM. In order to achieve this objective, we designed a cTnC mutant that led to reduced Ca2+ sensitivity and faster rate of Ca2+ dissociation from reconstituted thin filaments. We then introduced the Ca2+ desensitizing cTnC mutation into mouse myocardium via gene targeting technology, generating a novel Tnnc1 knock-in mouse model. The novel knock-in mouse model will enable us to evaluate the role of desensitizing cTnC to Ca2+ in triggering the pathogenesis of DCM. To evaluate the role of Ca2+ sensitivity of cTnC in the development of DCM, the proposed project will utilize the following aims: Specific Aim 1: Examine the effect of Ca2+ desensitizing (including DCM-linked) cTnC mutations on the response of reconstituted thin filaments to Ca2+ upon cTnI phosphorylation. This aim will test the hypothesis that Ca2+ desensitizing (including DCM-linked) cTnC mutations diminish the effect of phosphorylation of cTnI by protein kinase A (PKA) on Ca2+ binding and exchange with reconstituted thin filaments. We will evaluate the effect of decreasing Ca2+ sensitivity of cTnC on the response of reconstituted thin filaments to Ca2+ upon cTnI phosphorylation by PKA. Specific Aim 2: Determine whether decreasing the Ca2+ sensitivity of cTnC leads to pathogenesis of DCM. This aim will test the hypothesis that decreasing the Ca2+ sensitivity of cTnC (by accelerating the rate of Ca2+ dissociation) leads to the development of DCM. The results from two experimental groups (heterozygous and homozygous knock-in mice) will be compared to those for control group (wild-type mice). The consequences of the D73N cTnC mutation are expected to be more severe for homozygous mice than for heterozygous mice. Specific Aim 3: Determine the effect of decreasing the Ca2+ sensitivity of cTnC on contractile function. This aim will test the hypothesis that decreasing the Ca2+ sensitivity of cTnC (by accelerating the rate of Ca2+ dissociation) leads to systolic dysfunction. Systolic function will be assessed at the whole animal level using echocardiography. Systolic function will also be assessed at the cellular level utilizing ventricular myocytes isolated from the hearts of heterozygous, homozygous and wild- type mice. The proposed research will provide a deeper insight into the molecular mechanism of DCM.