Model of Timothy Syndrome to Screen Drugs with Induced Pluripotent Stem Cells

描述（由申请方提供）：QT间期延长是心室肌细胞复极的电表现，是心律失常和猝死的主要原因。长QT综合征（LQTS）可能有遗传基础，也可能由药物暴露或生理应激引起。药物诱导的LQTS是许多已批准药物的副作用，也是临床试验中药物失败的常见原因。虽然许多基因被报道导致LQTS，但人类疾病的潜在机制尚未完全了解。 我的职业目标是开发新的系统，以揭示人类心律失常的分子和细胞机制，并找到用于治疗心律失常的药物应用的先导化合物。我进行这项研究的个人动机是，我的祖母患有严重心律失常，去年去世了。作为一名专业的科学家，我想为心血管领域做出贡献，帮助尽可能多的心律失常患者。我的职业目标的关键要素是：1）开发心律失常的人类模型，以研究心律失常如何在人类心脏中发生; 2）开发使用人类细胞的筛选方法，以发现新的先导化合物，这些化合物具有比现有化合物更好的效果，但副作用更小。 为了实现这一目标，我从本科开始就专注于心脏功能和发育中的钙信号。这是因为在大多数情况下，小鼠中钙相关分子的耗尽诱导致死性心脏功能障碍，并且据报道，分子中的许多突变与人类心脏疾病（包括LQTS）相关。在这里，我建议研究错义突变的L型钙离子通道，CaV1.2，导致LQTS和致命性心律失常患者蒂莫西综合征（TS），以探讨TS突变对人类心肌细胞（CMs）的电活动和收缩的影响。虽然TS是一种罕见的疾病，但CaV1.2通道在心肌动作电位的产生和兴奋-收缩偶联中起重要作用。因此，TS的人类模型将是一个有用的平台，研究心律失常的机制和测试药物，为未来的治疗心律失常。 在初步研究中，为了开发TS的人类模型，我重新编程了两名TS患者的人类皮肤细胞，以产生诱导多能干细胞（iPSC），并将这些细胞分化为CM。这些细胞的电生理记录和Ca2+成像研究显示，不规则的收缩，过量的Ca2+内流，延长动作电位，延迟后除极和不规则的Ca2+信号。使用这些细胞，我发现roscovitine恢复了TS CM的电和Ca2+信号传导特性。 使用iPSC衍生的CM的方法为研究人类心律失常的分子和细胞机制以及开发治疗这些疾病的新药提供了新的机会。然而，仍然难以使用人iPSC衍生的CM筛选化合物库以治疗致命性心律失常，因为电生理学记录不容易用于开发中等通量筛选以找到治疗心脏病的先导化合物。因此，本项目的目标是开发和验证一种基于iPSC的筛选方法，可用于识别心律失常的治疗方法。1）进一步研究TS心肌细胞的表型特征：通过多种方法探讨TS突变如何诱导致死性室性心动过速，以及TS突变是否改变了人心肌细胞的增殖、分化、基因表达、收缩力和超微结构，以进一步揭示TS心律失常的分子和细胞机制。 2)直接筛选药物以挽救TS表型：临床上使用几种离子通道阻滞剂家族以及2-受体阻滞剂来预防致死性心律失常。然而，尚不清楚这些阻断剂是否可以挽救在TS CM中观察到的心脏表型。我将测试这些阻断剂恢复正常Ca2+反应和减少TS CM不规则收缩的能力。此外，我还将测试roscovitine的衍生物，这些衍生物被测试用于拯救TS的细胞表型。 3)筛选方法的发展，以找到先导化合物：开发中通量筛选系统的化合物库，以挽救心脏表型的TS，我将测试两种不同的方法的基础上相对运动和钙反应的TS CM使用自动荧光显微镜。为了验证系统，我将使用2-激动剂和roscovitine，这已经在TS CM上测试，以优化方法的实验条件，以评估由Z '值确定的重现性。最后，我将使用LOPAC 1280化合物在TS CM中进行中试筛选，该化合物已用于人体，可通过斯坦福大学高通量筛选设施获得。 这些方法使用人类心脏模型的TS将是非常独特和创新的，以了解人类心律失常的机制。所提出的筛选化合物库以拯救TS表型的系统将提供一个平台，以发现不仅对TS而且对其他心律失常的治疗在临床上有用的新型先导化合物。

DESCRIPTION (provided by applicant): Prolonged QT interval, the electrical manifestation of repolarization in ventricular myocytes, is a major cause of cardiac arrhythmia and sudden death. Long QT syndrome (LQTS) can have a genetic basis or be induced by drug exposure or physiological stress. Drug-induced LQTS is a side effect of many drugs that have approved and is a common cause of drug failure in clinical trials. Though many of the genes are reported to cause LQTS, the mechanisms underlying the disease in humans are incompletely understood. My career goal is to develop novel systems to uncover molecular and cellular mechanisms underlying human cardiac arrhythmia and to find lead compounds for pharmaceutical applications to treat arrhythmia. My personal motivation for this study is that I have a grandmother who had suffered severe arrhythmia and then died last year. As a professional scientist I'd like to contribute to cardiovascular fields to help as many patients suffering arrhythmia as possible. Key elements of my career goal are 1) to develop human models of cardiac arrhythmia to examine how cardiac arrhythmia occurs in human hearts; 2) to develop screen methods using human cells to find new lead compounds that have better effects but less side effects than present ones. To accomplish this goal, I have focused calcium signaling in heart function and development since undergraduate studies. This is because depletion of calcium related molecules in mice induced lethal cardiac dysfunction in most cases and many mutations in the molecules are reported to be associated with human cardiac diseases including LQTS. Here I propose to study a missense mutation in the L-type Ca2+ channel, CaV1.2, which causes LQTS and lethal arrhythmia in patients with Timothy syndrome (TS) in order to explore the effect of the TS mutation on the electrical activity and contraction of human cardiomyocytes (CMs). While TS is a rare disorder, CaV1.2 channels play important roles in generation of action potential and in excitation- contraction coupling for heart muscles. Therefore, human model of TS would be a useful platform to study mechanisms of arrhythmia and to test drugs for future treatment of cardiac arrhythmia. In preliminary studies, to develop human models of TS, I reprogrammed human skin cells from two TS patients to generate induced pluripotent stem cells (iPSCs) and differentiated these cells into CMs. Electrophysiological recording and Ca2+ imaging studies of these cells revealed irregular contraction, excess Ca2+ influx, prolonged action potentials, delayed afterdepolarizations and irregular Ca2+ signaling. Using these cells I found that roscovitine restored the electrical and Ca2+ signaling properties of TS CMs. The approach using iPSC-derived CMs provides new opportunities for studying the molecular and cellular mechanisms of cardiac arrhythmias in humans and for developing new drugs to treat these diseases. However, it is still difficult to screen a library of chemical compounds to treat lethal arrhythmia using human iPSC-derived CMs because electrophysiological recordings are not easily used for developing medium- throughput screen to find lead compounds to treat cardiac disease. Therefore, the goal of this project is to develop and validate an iPSC-based screening method that can be used to identify therapies for cardiac arrhythmia. This goal encompasses the approaches as follow: 1) Further characterization of phenotypes in TS cardiomyocytes: Using a variety of assays I will ask how TS mutation induce lethal ventricular tachycardia and whether TS mutation alters proliferation, differentiation, gene expression, contractility and ultra-structures in human CMs to uncover further molecular and cellular mechanisms that underlie cardiac arrhythmia of TS. 2) Direct screen of drugs to rescue TS phenotypes: Several families of ion channel blockers are used clinically as well as 2-blockers to prevent lethal cardiac arrhythmia. However, it is not clear that these blockers can rescue the cardiac phenotypes observed in TS CMs. I will test these blockers for their ability to restore normal Ca2+ responses and reduce irregular contraction in TS CMs. In addition, I will also test derivates of roscovitine, which are tested to rescue the cellular phenotypes of TS. 3) Development of screen methods to find lead compounds: To develop medium throughput screen systems for a library of chemical compounds to rescue the cardiac phenotypes of TS, I will test two different methods based on relative motion and calcium response in TS CMs using automated fluorescent microscopes. To validate the systems, I will used 2-agonists and roscovitine, which have been tested on TS CMs, to optimize experimental conditions for the methods to assess the reproducibility as determined by Z' value. Finally, I will conduct a pilot screen in TS CMs using LOPAC 1280 compounds that have been used in human, which is available through Stanford high-throughput screening facility. These approaches using human cardiac model of TS would be very unique and innovative to understand the mechanisms underlying human cardiac arrhythmia. The proposed systems to screen a library of compounds to rescue TS phenotypes will provide a platform to find novel lead compounds that would be clinically useful for the treatment of not only TS but also other cardiac arrhythmias.