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Understanding Molecular Basis of Cardiac Calcium Channelopathy

了解心脏钙离子通道病的分子基础

基本信息

批准号：
9241887
负责人：
LouJin Song
金额：
$ 2.07万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9241887
关键词：
Action Potentials Adverse effects Affect Arrhythmia Binding Binding Sites Biochemical Biological Biological Assay CDK5 gene Calcium Calcium Channel Cardiac Cardiac Myocytes Cells Chemicals Co-Immunoprecipitations Consensus Sequence Consumption Cyclin A Cyclin-Dependent Kinase 5 Cyclin-Dependent Kinase Inhibitor Cyclin-Dependent Kinase Inhibitor 3 Data Set Disease Dominant-Negative Mutation Environment Foundations Future Gene Expression Gene Expression Profiling Generations Genetic Goals Heart Human In Vitro Kinetics L Forms L-type calcium channel alpha(1C)Long QT Syndrome Measures Missense Mutation Modeling Molecular Mutation Neonatal Optics Pathogenesis Pathway interactions Patients Pharmaceutical Preparations Phenotype Phosphorylation Phosphotransferases Physiological Physiology Play Protein Kinase Proteins Protocols documentation Regulation Reporting Research Research Proposals Risk Role Site-Directed Mutagenesis Skin Specificity Stem cells Sudden Death Syndactyly Tachycardia Techniques Technology Testing Therapeutic Therapeutic Effect Timothy syndrome Transcript United States Ventricular Ventricular Tachycardia Western Blotting analog base cardiogenesis congenital heart disorder electrical property experimental study fetal gain of function mutation heart function imaging system induced pluripotent stem cell inhibitor/antagonist innovation insight mRNA Expression mechanical properties new therapeutic target novel novel therapeutic intervention novel therapeutics optical imaging patch clamp protein expression public health relevance roscovitine small hairpin RNA therapeutic target tool transcription activator-like effector nucleases voltage voltage clamp

项目摘要

DESCRIPTION (provided by applicant): Cardiac calcium channels play important roles in heart development and function. Missense mutations in calcium channel alpha1c subunit have been reported to be associated with several types of cardiac arrhythmias including long QT syndrome (LQTS) and lethal tachycardia. Previously, we have used human induced pluripotent stem cells (iPSCs) to generate human cardiomyocytes from LQTS patients and characterized those human cardiomyocytes. Our previous study demonstrated that the gain-of-function mutation (G406R) affected calcium channel inactivation, action potentials, calcium handling and contraction in human cardiomyocytes. We found that roscovitine (Ros), a cyclin-dependent kinase (cdk) inhibitor, could rescue the phenotypes in LQTS cardiomyocytes. However, how Ros restores physiological functions in the patient cardiomyocytes remains elusive. My preliminary results using Ros analogs and cdk inhibitors suggested that cdk5/p35 pathway could be involved in the molecular bases of cardiac calcium channelopathy caused by the G406R mutation in alpha1c subunit of cardiac calcium channels. Gene expression analyses demonstrated that the mRNA expression of molecules in this kinase pathway significantly increased in the patient cardiomyocytes. However, there are no studies on the role of cdk5/p35 pathway in cardiac physiology and pathogenesis of cardiac arrhythmias. The goal of this research proposal is to examine the role of cdk5/p35 pathway in cardiac calcium channel regulation and in molecular mechanisms underlying cardiac arrhythmias using patient-specific iPSCs. The goal encompasses the following aims: Aim 1 is to validate cdk5/p35 pathway as novel therapeutic targets for LQTS and to test the hypothesis that cdk5/p35 inhibition rescues the phenotypes of patient-specific cardiomyocytes. The dominant negative mutants and short hairpin RNAs (shRNAs) that inhibit the target kinase molecules will be tested in the patient cardiomyocytes using the physiological recording with patch clamp and optical imaging. Aim 2 is to examine the role of cdk5/p35 pathway in the regulation of calcium channel functions. First, I will examine whether there is an increased protein expression of molecules involved in the kinase pathway in LQTS cardiomyocytes using western blot. Second, I will examine whether the kinase directly binds to the alpha1c subunit using biochemical assays and site-directed mutagenesis. Third, I will develop an in vitro kinase activity assay using purified alpha1c subunit and the kinase proteins to examine whether the kinase phosphorylates the calcium channel. Using this assay, I will also test Ros, the Ros analogs and cdk inhibitors to examine whether these small compounds inhibit the phosphorylation of cardiac calcium channels by the kinase. The proposed research will provide new insights into the molecular bases of cardiac calcium channel regulation. The study will also provide answers to the question whether inhibition of the kinase could be novel therapeutics for LQTS patients caused by calcium channelopathy.

描述（由申请人提供）：心脏钙通道在心脏发育和功能中发挥重要作用。据报道，钙通道 α1c 亚基的错义突变与多种类型的心律失常相关，包括长 QT 综合征 (LQTS) 和致命性心动过速。此前，我们使用人类诱导多能干细胞 (iPSC) 从 LQTS 患者中产生人类心肌细胞，并对这些人类心肌细胞进行了表征。我们之前的研究表明，功能获得性突变（G406R）影响人类心肌细胞的钙通道失活、动作电位、钙处理和收缩。我们发现 roscovitine (Ros) 是一种细胞周期蛋白依赖性激酶 (cdk) 抑制剂，可以挽救 LQTS 心肌细胞的表型。然而，Ros 如何恢复患者心肌细胞的生理功能仍然难以捉摸。我使用Ros类似物和cdk抑制剂的初步结果表明，cdk5/p35途径可能参与由心脏钙通道α1c亚基G406R突变引起的心脏钙通道病变的分子基础。基因表达分析表明，患者心肌细胞中该激酶途径分子的 mRNA 表达显着增加。然而，目前尚无关于cdk5/p35通路在心脏生理和心律失常发病机制中的作用的研究。本研究计划的目标是利用患者特异性 iPSC 来研究 cdk5/p35 通路在心脏钙通道调节和心律失常分子机制中的作用。该目标包括以下目标：目标 1 是验证 cdk5/p35 通路作为 LQTS 的新治疗靶点，并检验 cdk5/p35 抑制可挽救患者特异性心肌细胞表型的假设。将使用膜片钳和光学成像的生理记录在患者心肌细胞中测试抑制靶激酶分子的显性失活突变体和短发夹 RNA (shRNA)。目的 2 是检查 cdk5/p35 通路在钙通道功能调节中的作用。首先，我将使用蛋白质印迹检查 LQTS 心肌细胞中参与激酶途径的分子的蛋白质表达是否增加。其次，我将使用生化测定和定点诱变检查激酶是否直接与 alpha1c 亚基结合。第三，我将使用纯化的 alpha1c 亚基开发体外激酶活性测定和激酶蛋白来检查激酶是否磷酸化钙通道。使用该测定，我还将测试 Ros、Ros 类似物和 cdk 抑制剂，以检查这些小化合物是否抑制激酶对心脏钙通道的磷酸化。拟议的研究将为心脏钙通道调节的分子基础提供新的见解。该研究还将回答抑制激酶是否可以成为钙离子通道病引起的 LQTS 患者的新疗法的问题。