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: 8399063
• 负责人: Masayuki Yazawa
• 金额: $ 8.63万
• 依托单位: STANFORD UNIVERSITY
• 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 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8399063
• 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; 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

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.

描述（申请人提供）：QT间期延长（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诱发。药物引起的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。尽管据报道许多基因会导致 LQTS，但人类疾病的机制尚不完全清楚。 我的职业目标是开发新的系统来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。我个人进行这项研究的动机是我的一位祖母患有严重的心律失常，并于去年去世。作为一名专业科学家，我希望为心血管领域做出贡献，帮助尽可能多的心律失常患者。我职业目标的关键要素是 1) 开发心律失常的人体模型，以研究心律失常是如何在人类心脏中发生的； 2）开发利用人体细胞的筛选方法来寻找比现有化合物具有更好效果但副作用更少的新先导化合物。 为了实现这一目标，自本科学习以来，我一直专注于心脏功能和发育中的钙信号传导。这是因为在大多数情况下，小鼠体内钙相关分子的消耗会引起致命的心脏功能障碍，并且据报道，分子中的许多突变与包括 LQTS 在内的人类心脏病有关。在此，我提出研究L型Ca2+通道CaV1.2的错义突变，该突变会导致蒂莫西综合征（TS）患者出现LQTS和致死性心律失常，以探讨TS突变对电活动和收缩的影响人类心肌细胞（CM）。虽然 TS 是一种罕见疾病，但 CaV1.2 通道在动作电位的产生和心肌的兴奋-收缩耦合中发挥着重要作用。因此，TS人体模型将成为研究心律失常机制和测试未来治疗心律失常药物的有用平台。 在初步研究中，为了开发 TS 人类模型，我对两名 TS 患者的人类皮肤细胞进行了重新编程，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+ 成像研究揭示了不规则的收缩、过量的 Ca2+ 流入、延长的动作电位、延迟的后除极和不规则的 Ca2+ 信号传导。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，使用人 iPSC 衍生的 CM 筛选用于治疗致命性心律失常的化合物库仍然很困难，因为电生理记录不易用于开发中等通量筛选以寻找治疗心脏病的先导化合物。因此，该项目的目标是开发和验证一种基于 iPSC 的筛查方法，可用于确定心律失常的治疗方法。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 突变如何诱导致命性室性心动过速，以及 TS 突变是否改变增殖、分化、基因表达、收缩性和超微结构在人类 CM 中揭示 TS 心律失常背后的进一步分子和细胞机制。 2）直接筛选挽救TS表型的药物：临床上使用多个离子通道阻滞剂家族以及2-阻滞剂来预防致命性心律失常。然而，尚不清楚这些阻滞剂是否可以挽救 TS CM 中观察到的心脏表型。我将测试这些阻滞剂恢复正常 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，这些衍生物被测试可以挽救TS的细胞表型。 3) 开发寻找先导化合物的筛选方法：为了开发用于挽救 TS 心脏表型的化合物库的中通量筛选系统，我将使用自动荧光测试基于 TS CM 中的相对运动和钙反应的两种不同方法显微镜。为了验证系统，我将使用 2-激动剂和 roscovitine（已在 TS CM 上进行过测试）来优化方法的实验条件，以评估由 Z' 值确定的再现性。最后，我将使用已用于人体的 LOPAC 1280 化合物对 TS CM 进行试点筛选，该化合物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了解人类心律失常的机制将是非常独特和创新的。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来寻找新型先导化合物，这些化合物在临床上不仅可用于治疗 TS，而且还可用于治疗其他心律失常。