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: 9754865
• 负责人: David K. Jones
• 金额: $ 24.52万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9754865
• 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; cell behavior; 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.

项目总结/摘要 人类ether-a-go-go基因（hERG）和KCNQ 1基因编码的蛋白质， 复极化电流IKr和IKs。复极受损会增加潜在致命的风险 室性心律失常，是长QT综合征（LQTS）和心力衰竭的标志。治疗 受损的复极化主要由于其脱靶效应的高发生率而受到限制。hERG胞浆 结构域相互作用以负调节IKr，使其成为治疗复极疾病的潜在靶点。 这项资助的初步数据表明，针对不同区域的小抗体肽片段 细胞质hERG Per-Arnt-Sim（PAS）结构域选择性增加IKr并缩短动作电位 持续时间（APD）。作为一种治疗方法，这种片段可能会缩短APD并恢复受损的 LQTS和心力衰竭中的复极。此外，针对PAS和其他免疫原的抗体片段也可用于免疫治疗。 结构域将是表征特定门控过程如何调节细胞行为的有用工具， 整体心脏生理学在这个项目的指导阶段，我将描述的机制， 这些抗体片段使用电生理学和光谱学技术修饰hERG门控。我会 还表征了抗体片段hERG在健康人干细胞衍生的心肌细胞中的调节 （iPSC-CM）和使用经由记录细胞内递送的scFv从LQTS患者获得的iPSC-CM。 移液管。在这个项目的独立阶段，我将联合收割机将scFv抗体与两个细胞内 递送技术：（1）细胞穿透心脏靶向肽CTP，和（2）腺相关病毒载体。 病毒血清型9（AAV 9）。我将在iPSC-CM中引入抗体-CTP融合蛋白，并监测所产生的 生物物理和生理效应与膜片钳和微电极技术。开发一个 为了实现更长期的治疗方法，我将使用病毒AAV 9来产生稳定的转移。 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， 探索自然生理学中不同通道过程的机械作用。此外，这些实验 将作为未来实验的概念验证，旨在开发抗hERG抗体片段， 复极受损的治疗。这个建议是为了实现我的短期目标， 心脏电生理学的技能，并过渡到我职业生涯的独立阶段。这将 最终让我实现了我的长期目标，研究翻译心脏电生理学研究， 使用患者来源的iPSC-CM来探索心律失常的触发因素和治疗方法。