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

蒂莫西综合征模型用诱导多能干细胞筛选药物

• 批准号: 8598272
• 负责人: Masayuki Yazawa
• 金额: $ 24.9万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598272
• 关键词: Action Potentials; Adverse effects; Affect; Agonist; Arrhythmia; Biological; Biological Assay; Calcium; Calcium Signaling; Cardiac; Cardiac Myocytes; Cardiovascular system; Cell Proliferation; Cell Separation; Cells; Clinical Trials; Coculture Techniques; Contracts; Coupling; Defect; Development; Disease; Drug Exposure; Elements; Failure; Family; Functional disorder; Future; Gene Expression; Generations; Genes; Genetic; Goals; Heart; Heart Atrium; Heart Diseases; Human; Image; In Vitro; Induced Mutation; Isoproterenol; L-type calcium channel alpha(1C); Lead; Libraries; Long QT Syndrome; Methods; Missense Mutation; Modeling; Molecular; Motion; Motivation; Mus; Muscle Cells; Muscle Contraction; Mutation; Myocardium; Nodal; Patent Ductus Arteriosus; Patent Foramen Ovale; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Physiological; Play; Preclinical Drug Evaluation; Property; Rare Diseases; Relative (related person); Reporter; Reporting; Reproducibility; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; Scientist; Signal Transduction; Skin; Stimulus; Stress; Structure; Sudden Death; System; Techniques; Testing; Tetralogy of Fallot; Timothy syndrome; United States; Ventricular; Ventricular Fibrillation; Ventricular Septal Defects; Ventricular Tachycardia; abstracting; base; cardiogenesis; career; design; drug testing; fluorescence microscope; heart function; high throughput screening; immunocytochemistry; induced pluripotent stem cell; innovation; ion channel blocker; novel; patch clamp; prevent; research study; response; roscovitine; screening; small molecule libraries; voltage

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.

【摘要】：QT间期延长是心室肌细胞复极的电学表现。 心律失常和猝死的主要原因。长 QT 综合征 (LQTS) 可能有遗传基础或 由药物暴露或生理应激诱发。药物引起的 LQTS 是许多药物的副作用， 已获批准，是临床试验中药物失败的常见原因。尽管许多基因已被报道 导致 LQTS 的人类疾病机制尚不完全清楚。 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药物应用的先导化合物。我的 这项研究的个人动机是我的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，帮助尽可能多的患者 尽量避免心律失常。我职业目标的关键要素是 1) 开发心脏的人体模型 心律失常检查人类心脏中心律失常是如何发生的； 2) 开发筛选方法 人类细胞寻找新的先导化合物，其效果比现有化合物更好，但副作用更少。 为了实现这一目标，自那时以来，我一直专注于心脏功能和发育中的钙信号传导 本科学习。这是因为小鼠体内钙相关分子的消耗会导致致命的心脏损伤。 据报道，大多数情况下功能障碍和分子中的许多突变与人类有关 心脏病，包括 LQTS。在这里我建议研究L型Ca2+通道的错义突变， CaV1.2，引起蒂莫西综合征（TS）患者的 LQTS 和致死性心律失常，以探索 TS 突变对人类心肌细胞 (CM) 电活动和收缩的影响。尽管 TS 是一种罕见疾病，CaV1.2 通道在动作电位的产生和兴奋中发挥重要作用 心肌收缩耦合。因此，TS的人体模型将是一个有用的研究平台 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，为了开发 TS 的人类模型，我对两个 TS 的人类皮肤细胞进行了重新编程 患者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+ 成像研究揭示了不规则收缩、过度收缩 Ca2+ 流入、动作电位延长、后除极延迟和不规则 Ca2+ 信号传导。使用这些 我发现 roscovitine 恢复了 TS CM 的电学和 Ca2+ 信号传导特性。 使用 iPSC 衍生的 CM 的方法为研究分子和 人类心律失常的细胞机制以及开发治疗这些疾病的新药。 然而，筛选用于人类治疗致命性心律失常的化合物库仍然很困难。 iPSC 衍生的 CM 因为电生理记录不易用于开发培养基 通过通量筛选来寻找治疗心脏病的先导化合物。因此，该项目的目标是 开发并验证基于 iPSC 的筛选方法，可用于识别治疗方法 心律失常。这一目标包括以下方法： 1) TS 心肌细胞表型的进一步表征：使用各种测定法，我将 询问 TS 突变如何诱发致命性室性心动过速以及 TS 突变是否会改变增殖， 人类 CM 的分化、基因表达、收缩性和超微结构，以揭示进一步的分子 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：离子通道阻滞剂的几个家族 临床上与β受体阻滞剂一样用于预防致命性心律失常。然而，尚不清楚这些 阻滞剂可以挽救 TS CM 中观察到的心脏表型。我将测试这些拦截器的能力 恢复正常的 Ca2+ 反应并减少 TS CM 的不规则收缩。另外我也会测试一下 roscovitine 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：开发介质通量 为了挽救 TS 心脏表型的化合物库的筛选系统，我将测试两个 使用自动荧光技术基于 TS CM 中的相对运动和钙反应的不同方法 显微镜。为了验证系统，我将使用 ¿-激动剂和 roscovitine，它们已经在 TS 上进行了测试 CM，优化方法的实验条件，以评估由 Z' 确定的再现性 价值。最后，我将使用 LOPAC 1280 化合物在 TS CM 中进行试点筛选，这些化合物已用于 人类，可通过斯坦福大学高通量筛选设施获得。 这些使用 TS 人类心脏模型的方法对于理解 TS 来说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 拯救 TS 表型将提供一个平台来寻找新的先导化合物，这些化合物将在临床上有用 不仅可以治疗 TS，还可以治疗其他心律失常。