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.
The risk of sudden death due to genetic and drug-induced cardiac arrhythmias is a major concern in the United States. This project will investigate molecular mechanisms of a genetic cardiac arrhythmia, long QT syndrome, using novel biological techniques to generate human cardiac cells from patient skin cells. This approach will provide an innovative platform to test potential therapeutics on human cardiac cells derived from patients for future treatment of cardiac arrhythmias caused by genetic bases and side effects of approved drugs.
|Song, LouJin; Park, Seon-Hye E; Isseroff, Yehuda et al. (2017) Inhibition of CDK5 Alleviates the Cardiac Phenotypes in Timothy Syndrome. Stem Cell Reports 9:50-57|