Domain-specific gating modulation to restore action potential duration in long QT patient-derived cardiomyocytes

域特异性门控调节可恢复长 QT 患者来源的心肌细胞的动作电位持续时间

• 批准号: 9463521
• 负责人: David K. Jones
• 金额: $ 9.86万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9463521
• 关键词: Action Potentials; Alleles; Antibodies; Arrhythmia; Behavior; Binding; Biophysics; C-terminal; Cardiac; Cardiac Electrophysiologic Techniques; Cardiac Myocytes; Cause of Death; Cells; Chimeric Proteins; Cyclic Nucleotides; Cytoplasmic Tail; Dangerousness; Data; Dependovirus; Disease; Electrophysiology (science); Epitopes; Ethers; Fluorescence Resonance Energy Transfer; Future; Genes; Goals; Grant; Heart; Heart Diseases; Heart failure; Human; Immunoglobulin Fragments; Impairment; Incidence; Individual; Kinetics; Long QT Syndrome; Mentors; Microelectrodes; Modeling; Monitor; Patch-Clamp Techniques; Patients; Peptide Fragments; Peptide antibodies; Peptides; Phase; Physiological; Physiology; Process; Property; Proteins; Research; Risk; Role; Serotyping; Spectrum Analysis; Techniques; Testing; Therapeutic; Tissues; United States; Ventricular Arrhythmia; Viral; Viral Vector; base; career; design; experimental study; human stem cells; in vivo; induced pluripotent stem cell; monolayer; novel; patch clamp; protein aminoacid sequence; restoration; sensor; skills; tool; voltage

PROJECT SUMMARY/ABSTRACT The human ether-a-go-go gene (hERG) and KCNQ1 genes encode proteins that conduct the cardiac repolarizing currents IKr and IKs, respectively. Impaired repolarization increases the risk of potentially lethal ventricular arrhythmias and is a hallmark of long QT syndrome (LQTS) and heart failure. Treatments for impaired repolarization are limited largely due to their high incidence of off-target effects. hERG cytoplasmic domains interact to negatively modulate IKr, making them potential targets to treat diseases of repolarization. The preliminary data for this grant demonstrate that small antibody peptide fragments targeting distinct regions of the cytoplasmic hERG Per-Arnt-Sim (PAS) domain selectively increase IKr and shorten action potential duration (APD). As a therapeutic, such fragments would potentially shorten APD and restore impaired repolarization in LQTS and heart failure. Additionally, antibody fragments directed toward the PAS and other domains would be useful tools to characterize how specific gating processes regulate cellular behavior and overall cardiac physiology. In the mentored phase of this project, I will characterize the mechanisms by which these antibody fragments modify hERG gating using electrophysiological and spectroscopy techniques. I will also characterize antibody fragment hERG modulation in healthy human stem cell-derived cardiomyocytes (iPSC-CMs) and iPSC-CMs obtained from LQTS patients using scFvs delivered intracellularly via the recording pipette. In the independent phase of this project, I will combine the scFv antibodies with two intracellular delivery techniques: (1) the cell-penetrating Cardiac Targeting Peptide, CTP, and (2) the adeno-associated virus serotype 9 (AAV9). I will introduce antibody-CTP fusion proteins into iPSC-CMs and monitor the resulting biophysical and physiological effects with patch clamp and micro-electrode techniques. To develop an approach leading to a longer-term treatment, I will use the viral AAV9 to produce stable transfer of the antibody-encoding sequence into mammalian cardiomyocytes. These experiments will generate novel tools to probe the mechanistic role of distinct channel processes in native physiology. Additionally, these experiments will act as a proof-of-concept for future experiments designed to develop anti-hERG antibody fragments into treatments for impaired repolarization. This proposal is designed to fulfill my short-term goals of expanding my skills in cardiac electrophysiology and transitioning into the independent phase of my career. This will ultimately allow me to obtain my long-term goal of studying translational cardiac electrophysiology research by using patient-derived iPSC-CMs to explore triggers and treatments for cardiac arrhythmia.

项目摘要/摘要 人类的乙醚-a-go-go基因(Herg)和KCNQ1基因编码的蛋白质传导心脏 再极化电流分别为Ikr和Iks。复极受损增加了潜在致命的风险 室性心律失常，是长QT综合征(LQTS)和心力衰竭的标志。治疗 受损的复极化在很大程度上是有限的，这在很大程度上是因为它们的偏离目标效应的发生率很高。HERG细胞质 结构域相互作用，负向调节IKR，使其成为治疗复极化疾病的潜在靶点。 这项资助的初步数据表明，针对不同区域的小抗体多肽片段 胞质Herg Per-Arnt-Sim(PAS)结构域选择性地增加IKR和缩短动作电位 持续时间(时长)。作为一种治疗方法，这样的碎片可能会缩短apd并恢复受损的 LQTS的复极与心力衰竭。此外，针对PAS和其他抗体片段 结构域将是有用的工具来表征特定的门控过程如何调节细胞行为和 整体心脏生理学。在这个项目的指导阶段，我将描述 这些抗体片段使用电生理和光谱学技术修改HERG门控。这就做 健康人干细胞来源的心肌细胞中抗体片段Herg的调节特性 (IPSC-CMS)和IPSC-CMS通过记录从LQTS患者细胞内传递的scFv获得 移液管。在这个项目的独立阶段，我将把scFv抗体与两个细胞内的 给药技术：(1)细胞穿透性心脏靶向肽，CTP；(2)腺相关肽 病毒血清9型(AAV9)。我将把抗体-CTP融合蛋白导入IPSC-CMS并监测其结果。 膜片钳和微电极技术的生物物理和生理效应。要开发一个 方法导致更长期的治疗，我会用AAV9病毒产生稳定的转移 将抗体编码序列导入哺乳动物心肌细胞。这些实验将产生新的工具来 探索不同的经络过程在自然生理学中的机制作用。此外，这些实验 将作为未来实验的概念验证，旨在将抗HERG抗体片段开发成 复极受损的治疗。这项提议旨在实现我的短期目标，即扩大我的 心脏电生理学方面的技能，并过渡到我职业生涯的独立阶段。这将是 最终让我实现我的长期目标，研究翻译心脏电生理学研究，通过 使用患者衍生的IPSC-CMS探索心律失常的触发因素和治疗方法。