Molecular Mechanism of Dilated Cardiomyopathy

扩张型心肌病的分子机制

基本信息

批准号：
8426856
负责人：
BRADLEY K MCCONNELL
金额：
$ 45.12万
依托单位：
UNIVERSITY OF HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-17 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8426856
关键词：
ATP phosphohydrolase Animals Binding Biochemical Body Weight Cardiac Cardiac Myosins Cardiac Output Cardiomegaly Complex Control Groups Cyclic AMP-Dependent Protein Kinases Development Dilated Cardiomyopathy Disease Dissociation EFRAC Echocardiography Engineering Exposure to Fibrosis Fluorescent Probes Functional disorder Gene Targeting Goals Health Heart Heart failure Histology Knock-in Mouse Lead Left Link Measures Microfilaments Molecular Monitor Morphology Mus Muscle Cells Mutation Myocardium Myofibrils Myosin ATPase Myosin Heavy Chains N Domain Nature Pathogenesis Phenotype Phosphorylation Physiological Property Proteins Research Role Students Techniques Technology Testing Thin Filament Troponin C Ventricular Weight Western Blotting Wild Type Mouse career design graduate student insight interstitial mortality mouse model mutant novel public health relevance reconstitution response undergraduate student

项目摘要

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.

描述（申请人提供）：扩张型心肌病（DCM）是一种以左心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，30-40% 的 DCM 是由细胞骨架和肌节蛋白突变引起的。尽管大多数已知的与 DCM 相关的肌节蛋白突变使心肌对 Ca2+ 不敏感，但仍缺乏对这些突变如何导致 DCM 发展的了解。然而，心肌肌钙蛋白 C (cTnC) 的 Ca2+ 敏感性在引发 DCM 发病机制中的作用仍存在争议。该项目的目标是确定我们是否可以在 cTnC 中产生自然界中未发现的突变，然后将其引入小鼠心肌中以重建 DCM 表型。为了实现这一目标，我们设计了一种 cTnC 突变体，该突变体可降低 Ca2+ 敏感性并加快 Ca2+ 从重构细丝中解离的速度。然后，我们通过基因打靶技术将Ca2+脱敏cTnC突变引入小鼠心肌，产生了新型Tnnc1敲入小鼠模型。新型敲入小鼠模型将使我们能够评估 cTnC 对 Ca2+ 脱敏在触发 DCM 发病机制中的作用。为了评估 cTnC 的 Ca2+ 敏感性在 DCM 发展中的作用，拟议项目将利用以下目标：具体目标 1：检查 Ca2+ 脱敏（包括 DCM 相关）cTnC 突变对重建细丝对cTnI 磷酸化后的 Ca2+。该目标将检验以下假设：Ca2+ 脱敏（包括 DCM 相关）cTnC 突变会减弱蛋白激酶 A (PKA) 磷酸化 cTnI 对 Ca2+ 结合以及与重建细丝交换的影响。我们将评估降低 cTnC 的 Ca2+ 敏感性对经 PKA 磷酸化 cTnI 后重建的细丝对 Ca2+ 的响应的影响。具体目标 2：确定降低 cTnC 的 Ca2+ 敏感性是否会导致 DCM 的发病机制。这一目标将检验以下假设：降低 cTnC 的 Ca2+ 敏感性（通过加速 Ca2+ 解离速率）会导致 DCM 的发生。两个实验组（杂合子和纯合敲入小鼠）的结果将与对照组（野生型小鼠）的结果进行比较。预计 D73N cTnC 突变对纯合子小鼠的影响比对杂合子小鼠更严重。具体目标 3：确定降低 cTnC 的 Ca2+ 敏感性对收缩功能的影响。该目标将检验以下假设：降低 cTnC 的 Ca2+ 敏感性（通过加速 Ca2+ 解离速率）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。还将利用从心脏分离的心室肌细胞在细胞水平上评估收缩功能杂合子、纯合子和野生型小鼠。拟议的研究将为 DCM 的分子机制提供更深入的了解。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Desensitizing mouse cardiac troponin C to calcium converts slow muscle towards a fast muscle phenotype.

使小鼠心肌肌钙蛋白 C 对钙脱敏可将慢肌转化为快肌表型。

DOI：
发表时间：
2018-10
期刊：
影响因子：
0
作者：
Tikunova, Svetlana;Belevych, Natalya;Doan, Kelly;Reiser, Peter J
通讯作者：
Reiser, Peter J

Molecular and functional consequences of mutations in the central helix of cardiac troponin C.

心肌肌钙蛋白 C 中央螺旋突变的分子和功能后果。

DOI：
发表时间：
2014-04-15
期刊：
影响因子：
3.9
作者：
Swindle, Nicholas;Albury, Acchia N J;Baroud, Belal;Burney, Maryam;Tikunova, Svetlana B
通讯作者：
Tikunova, Svetlana B

C.B-17 SCID mice develop epicardial calcinosis with unaltered cardiac function.

C.B-17 SCID 小鼠出现心外膜钙质沉着，但心功能未改变。

DOI：
发表时间：
2019-02
期刊：
影响因子：
2.9
作者：
Raghunathan, Suchi;Reynolds, Corey L;Schwartz, Robert J;Stewart, M David;McConnell, Bradley K
通讯作者：
McConnell, Bradley K