Understanding Molecular Basis of Cardiac Calcium Channelopathy

 说明(申请人提供)：心脏钙通道在心脏发育和功能中起重要作用。钙通道α1c亚基错义突变与多种类型的心律失常有关，包括长QT综合征(LQTS)和致死性心动过速。在此之前，我们已经使用人诱导多能干细胞(IPSCs)从LQTS患者中产生人心肌细胞，并对这些人心肌细胞进行了鉴定。我们先前的研究表明，功能获得突变(G406R)影响人心肌细胞的钙通道失活、动作电位、钙处理和收缩。我们发现，细胞周期蛋白依赖性激酶(CDK)抑制剂罗斯科维汀(ROS)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不清楚。我使用ROS类似物和CDK抑制剂的初步结果表明，CDK5/p35通路可能参与了由心肌钙通道α1c亚单位G406R突变引起的心肌钙通道病的分子基础。基因表达分析表明，在患者心肌细胞中，该激酶通路中分子的mRNA表达显著增加。然而，目前还没有关于CDK5/p35通路在心脏生理学和心律失常发病机制中作用的研究。本研究的目的是研究CDK5/p35通路在心脏钙通道调节中的作用，以及使用患者特有的IPSCs在心律失常的分子机制中的作用。目的1验证CDK5/p35通路作为治疗LQTS的新靶点，并验证抑制CDK5/p35可挽救患者特异性心肌细胞表型的假说。利用膜片钳生理记录和光学成像技术，在患者心肌细胞中检测抑制靶蛋白激酶分子的显性负性突变体和短发夹状RNA(ShRNAs)。目的2研究CDK5/p35通路在钙通道功能调节中的作用。首先，我将用蛋白质印迹法检测LQTS心肌细胞中是否有参与激酶通路的分子的蛋白表达增加。其次，我将使用生化分析和定点突变来检查该激酶是否直接与α1c亚基结合。第三，我将开发一种使用纯化的alpha1c亚基的体外激酶活性分析方法。 以及检测该激酶是否使钙通道磷酸化的蛋白。利用这项测试，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了心肌钙通道的磷酸化。这项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。这项研究还将回答抑制该激酶是否可能成为钙通道病引起的LQTS患者的新疗法的问题。

 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.