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

摘要：QT间期延长是心肌细胞复极的电学表现。 心律失常和猝死的主要原因。QT间期延长综合征(LQTS)可能有遗传基础或 由药物暴露或生理压力引起。药物诱导的LQTS是许多药物的副作用， 已经被批准，并且是临床试验中药物失败的常见原因。尽管已有许多基因被报道 导致LQTS的原因是人类潜在的疾病机制还不完全清楚。 我的职业目标是开发新的系统来揭示潜在的分子和细胞机制 人类心律失常，并寻找用于治疗心律失常的先导化合物。我的 这项研究的个人动机是，我有一位祖母患有严重的心律失常，然后 去年去世了。作为一名专业科学家，我愿意为心血管领域做出贡献，帮助更多的患者 尽可能地患上心律失常。我的职业目标的关键要素是：1)开发心脏的人体模型 心律失常以检查心律失常是如何在人体心脏中发生的；2)开发使用 人类细胞寻找新的先导化合物，比目前的化合物有更好的效果和更少的副作用。 为了实现这一目标，我一直关注钙信号在心脏功能和发育中的作用 本科生学习。这是因为小鼠体内钙相关分子的耗尽导致了致命的心脏 据报道，大多数情况下的功能障碍和许多分子突变与人类 心脏疾病，包括LQTS。在这里，我建议研究L型钙离子通道的一个错义突变， CaV1.2，其中导致LQTS和致命性心律失常的Timothy综合征(TS)患者以探讨 TS突变对人心肌细胞电活动和收缩的影响而当 TS是一种罕见的疾病，CaV1.2通道在动作电位的产生和兴奋中起重要作用。 用于心脏肌肉的收缩偶联。因此，TS的人体模型将是一个有用的研究平台 研究心律失常的机制，并测试未来治疗心律失常的药物。 在初步研究中，为了建立人类TS模型，我从两个TS中重新编程了人类皮肤细胞 患者产生诱导多能干细胞(IPSCs)，并将这些细胞分化为CMS。 电生理记录和钙成像研究显示这些细胞不规则收缩、过度收缩、过度收缩。 Ca~(2+)内流、动作电位延长、后除极延迟和钙信号不规则。使用这些 细胞我发现，罗斯科维汀恢复了TS CMS的电和钙信号特性。 利用IPSC衍生CMS的方法为研究分子和分子生物学提供了新的机会 人类心律失常的细胞机制和开发治疗这些疾病的新药。 然而，仍然很难筛选出使用人类来治疗致命心律失常的化合物库。 IPSC衍生的CMS，因为电生理记录不容易用于显影介质- 通过筛选寻找治疗心脏病的先导化合物。因此，这个项目的目标是 开发并验证一种基于IPSC的筛查方法，该方法可用于识别 心律失常。这一目标包括以下方法： 1)进一步鉴定TS心肌细胞的表型：使用各种分析方法我将 问TS突变是如何导致致死性室性心动过速的，TS突变是否改变了细胞增殖， 人类CMS的分化、基因表达、收缩能力和超微结构进一步揭示分子 以及TS心律失常的细胞机制。 2)直接筛选挽救TS表型的药物：几种离子通道阻滞剂家族 在临床上用作阻滞剂，以防止致命的心律失常。然而，目前还不清楚这些 阻滞剂可以挽救TS CMS患者的心脏表型。我会测试这些阻滞剂的能力 使TS CMS恢复正常的钙反应，减少不规则收缩。此外，我还将测试 罗斯科维汀的衍生物，经测试可挽救TS的细胞表型。 3)开发寻找先导化合物的筛选方法：开发中等产量 为了挽救TS的心脏表型，我将测试两个化合物库中的筛选系统 利用自动荧光检测TS CMS的相对运动和钙反应的不同方法 显微镜。为了验证这些系统，我将使用已经在TS上测试的激动剂和罗斯科维汀 CMS，以优化Z‘测定重现性评价方法的实验条件 价值。最后，我将在TS CMS中使用Lopac 1280化合物进行试点筛选，这些化合物已经在 人类，这是通过斯坦福大学的高通量筛选设施获得的。 这些使用人类心脏模型的TS的方法将是非常独特和创新的 人类心律失常的潜在机制。拟议的筛选化合物库的系统 挽救TS表型将提供一个平台，寻找将在临床上有用的新的先导化合物 不仅是TS的治疗，还包括其他心律失常的治疗。

Abstract: 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 ¿-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 ¿-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.