Biogenesis of hERG1a/1b ion channels in health and disease model cardiomyocytes

健康和疾病模型心肌细胞中 hERG1a/1b 离子通道的生物发生

• 批准号: 10723869
• 负责人: Lisandra Flores Aldama
• 金额: $ 13.22万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723869
• 关键词: Action Potentials; Affect; Affinity; Arrhythmia; Behavior; Binding; Biogenesis; Bioinformatics; Biophysics; Calorimetry; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular system; Cause of Death; Cells; Complex; Data; Defect; Disease; Disease model; Electrophysiology (science); Ethers; Fluorescent in Situ Hybridization; General Population; Genes; Genetic Transcription; Goals; Grant; Half-Life; Health; Heart Abnormalities; Heart Diseases; Hela Cells; Human; Immunoprecipitation; Impairment; In Vitro; Individual; Inherited; Ion Channel; Link; Long QT Syndrome; Mediating; Mediator; Messenger RNA; Microscopy; Molecular; Mutagenesis; Mutation; Myocardium; Outcome; Patients; Phase; Post-Transcriptional Regulation; Potassium; Prevalence; Probability; Process; Proteins; RNA; RNA-Binding Proteins; Research; Role; Shapes; Specificity; Structure; Sudden Death; System; Testing; Time; Transcript; Transfection; Translations; Type 2 Long QT syndrome; United States; Ventricular; Ventricular Arrhythmia; Voltage-Gated Potassium Channel; Western Blotting; Work; behavior in vitro; career; design; experimental study; heart rhythm; helicase; human disease; human model; hybrid protein; induced pluripotent stem cell; insight; knock-down; live cell imaging; mRNA Stability; mortality; mutant; novel; posttranscriptional; reconstitution; ribosome profiling; single molecule; skills; structural determinants; sudden cardiac death

PROJECT SUMMARY/ABSTRACT Cardiac IKr is a critical repolarizing potassium current shaping the human ventricular action potential. It is conducted by heteromeric assemblies of the human ether-à-go-go-related gene (hERG1) 1a and 1b subunits. These subunits are encoded by alternate transcripts of the hERG/KCNH2 gene and differ only in their amino- terminal regions. hERG1a/1b heteromerization is vital for normal CM function, as the imbalance of subunit expression and/or function results in cellular pro-arrhythmic behaviors. hERG1a/1b assembly is mediated by the co-translational association of the encoding mRNAs in HEK293 cells, cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), and human myocardium. Evidence suggests that interaction between the nascent proteins is not required for the co-translational complex assembly. This grant's preliminary findings indicate that this complex assembly occurs post-transcriptionally and is promoted by direct interactions between hERG1a and 1b mRNAs governed by their secondary structures. In preliminary studies, RNA binding proteins DDX3X and DDX5 were identified as part of the complex, and purified DDX3X promoted hERG1a/1b mRNAs' association in vitro. In the K99 phase, I will define the mRNA structural features promoting the co-translational association and determine the affinity and energies of the RNA/RNA interaction using in vitro systems, isothermal calorimetry (ITC), mutagenesis, hybrid protein-RNA immunoprecipitation (RIP), and live-cell imaging. I will also determine whether DDX3X and DDX5 affect hERG1a and 1b mRNAs stability, translation, and association in hiPSC-CMs using qPCR, electrophysiology, Western Blot, ribosome profiling, RIP, and single molecule fluorescent in situ hybridization (smFISH). I will use quantitative ITC and in vitro reconstitution approaches to determine the specificity, affinity, and energies of the interaction between purified DDX3X and DDX5 with hERG1a and 1b mRNAs. I will also evaluate if DDX3X and DDX5 promote the association of the mRNAs in in vitro systems. In the R00 phase, I will determine whether the stability, translation, and association of hERG1a and 1b mRNAs are impaired in arrhythmias associated with type 2 long QT syndrome (LQT2). I will use hiPSC-CM disease models to evaluate half-life, translation rate, and association of the mRNAs with qPCR, ribosome profiling, RIP, and smFISH. These experiments will contribute to understanding ion channel biogenesis and elucidate molecular mechanisms underlying LQT2 related arrhythmias. This proposal is designed to fulfill my short-term goals of expanding my skills in cardiovascular research and biophysics and transitioning into the independent phase of my career. This will ultimately allow me to obtain my long-term purpose of linking RNA and ion channel biophysics to translational cardiovascular research.

项目摘要/摘要 心脏IKR是一个关键的复极化钾电流，塑造了人心室动作电位。这是 由人类醚-go相关基因（HERG1）1A和1B亚基的异源组件进行。 这些亚基是由HERG/KCNH2基因的替代转录物编码的，并且仅在其氨基中不同 终端区域。 HERG1A/1B异构化对于正常CM功能至关重要，因为亚基的​​不平衡 表达和/或功能导致细胞促性心律失常行为。 HERG1A/1B组件由 HEK293细胞中编码的mRNA的共译，源自人的心肌细胞 诱导多能干细胞（HIPSC-CM）和人心肌。证据表明相互作用 共同翻译配合物组装不需要新生蛋白之间。这笔赠款是 初步发现表明，这种复杂的组装在转录后发生，并通过直接促进 HERG1A和1B mRNA之间的相互作用受二级结构支配。在初步研究中， RNA结合蛋白DDX3X和DDX5被鉴定为复合物的一部分，并促进了纯化的DDX3X HERG1A/1B mRNA的体外协会。在K99阶段，我将定义mRNA结构特征 促进共译结合并确定RNA/RNA相互作用的亲和力和能量 使用体外系统，等温量热法（ITC），诱变，杂化蛋白RNA免疫沉淀 （RIP）和活细胞成像。我还将确定DDX3X和DDX5是否影响HERG1A和1B mRNA 使用QPCR，电生理学，蛋白质印迹，核糖体的HIPSC-CMS稳定性，翻译和关联 分析，RIP和单分子荧光原位杂交（Smfish）。我将使用定量ITC和 体外重构方法来确定相互作用之间相互作用的特异性，亲和力和能量 带有HERG1A和1B mRNA的纯化DDX3X和DDX5。我还将评估DDX3X和DDX5是否促进 体外系统中mRNA的关联。在R00阶段，我将确定稳定性是否 在与2型长的心律不齐相关的心律不齐中，HERG1A和1B mRNA的翻译和关联受损 QT综合征（LQT2）。我将使用HIPSC-CM疾病模型评估半衰期，翻译率和 mRNA与QPCR，核糖体分析，RIP和Smfish的关联。这些实验将有助于 了解离子通道生物发生和阐明LQT2相关的分子机制 心律不齐。该建议旨在实现我扩大心血管技能的短期目标 研究和生物物理学，并过渡到我职业生涯的独立阶段。这最终将允许 我要获得将RNA和离子通道生物物理学与翻译的心血管链接的长期目的 研究。