MLC2V AND 2A IN CARDIAC DEVELOPMENT AND DISEASE

MLC2V 和 2A 在心脏发育和疾病中的作用

基本信息

批准号：
6527489
负责人：
Ju Chen
金额：
$ 26.6万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-09-05 至 2004-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6527489
关键词：
biotechnology congenital heart disorder disease /disorder model gene mutation gene targeting genetically modified animals heart function heart metabolism hypertrophic myocardiopathy isozymes laboratory mouse model design /development molecular pathology myosins phosphorylation

项目摘要

DESCRIPTION (the applicant's description verbatim): Utilizing a combination of genetic engineering in mouse model systems and miniaturized physiological technology, the proposed studies will test two principal hypotheses concerning MLC2v function: 1) MLC2a cannot functionally substitute for MLC2v in ventricular cardiac muscle; 2) Phosphorylation of MLC2v plays an important role in ventricular papillary muscle and cardiac function, and impairment of MLC2v phosphorylation will result in FHC. The overall goals of the proposal are to understand the functional differences between two cardiac MLC2 isoforms, MLC2v and MLC2a and the functional role of MLC2v phosphorylation. We hope to gain insight into mechanisms by which mutations in MLC2v cause cardiomyopathy and the roles of environment, physical state, and genetic background in the incomplete penetrance and variable phenotype of this disease. We will achieve these goals by creating several gene targeted mouse lines through knock-in, and site-specific mutagenesis followed by comprehensive histological, biochemical, and physiological analysis of the resulting cardiac phenotypes. Accordingly, the Specific Aims are: 1. To investigate the functional equivalence of MLC2a and MLC2v by examining the ability of MLC2a to rescue the C2v null mutant phenotype. This will be performed by knocking the MLC2a cDNA into the MLC2v endogenous locus thereby deleting MLC2v and leaving MILC2a under the control of the endogenous MLC2v promoter. 2. To understand the functional significance of MLC2v phosphorylation and to determine whether elimination of MLC2v phosphorylation is sufficient to induce a form of hypertrophic cardiomyopathy. Mouse lines will be generated in which the phosphorylation site(s) (Ser 15 or Ser 15 plus Ser 14) of the endogenous MLC2v gene have been mutated to Ala. 3. To understand the mechanism by which MLC2v mutations cause familial hypertrophic cardiomyopathy (FHC) with middle left ventricular chamber thickening. A mouse model will be created by introducing a Glu22Lys mutation into the ALC2v gene (A Glu22Lys mutation in human MLC2v causes familial hypertrophic cardiomyopathy). For each mouse model proposed in Specific Aims 1-3, comprehensive physiological assessment of cardiac function, and detailed biochemical and biophysical analyses of resulting cardiac muscle phenotypes will be performed.

描述（申请人的逐字描述）：利用以下内容的组合小鼠模型系统中的基因工程和小型化生理学技术，拟议的研究将检验两个主要假设： MLC2v 功能： 1) MLC2a 不能在功能上替代 MLC2v 心室心肌； 2）MLC2v的磷酸化发挥重要作用心室乳头肌和心脏功能以及 MLC2v 损伤磷酸化会导致 FHC。该提案的总体目标是了解两种心脏 MLC2 异构体 MLC2v 之间的功能差异 MLC2a 和 MLC2v 磷酸化的功能作用。我们希望能够收获深入了解 MLC2v 突变导致心肌病的机制环境、身体状态和遗传背景在其中的作用这种疾病的不完全外显率和可变表型。我们将实现通过基因敲入创建多个基因靶向小鼠品系来实现这些目标，以及位点特异性诱变，然后进行全面的组织学、生化、以及由此产生的心脏表型的生理分析。因此，具体目标是： 1. 调查功能通过检查 MLC2a 救援能力来确定 MLC2a 和 MLC2v 的等效性 C2v 无效突变体表型。这将通过敲入 MLC2a cDNA 来完成进入 MLC2v 内源基因座，从而删除 MLC2v 并留下 MILC2a 内源MLC2v启动子的控制。 2. 理解函数式 MLC2v 磷酸化的意义并确定是否消除 MLC2v 磷酸化足以诱导某种形式的肥大心肌病。将产生其中磷酸化的小鼠系内源 MLC2v 基因的位点（Ser 15 或 Ser 15 加 Ser 14）已被 3.了解MLC2v突变引起的机制伴有中左心室的家族性肥厚型心肌病 (FHC) 增稠。将通过引入 Glu22Lys 突变来创建小鼠模型进入 ALC2v 基因（人类 MLC2v 中的 Glu22Lys 突变会导致家族性遗传肥厚型心肌病）。对于特定目标中提出的每个小鼠模型 1-3、心功能综合生理评估，详细对所得心肌表型进行生化和生物物理分析将被执行。