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.

项目概要/摘要 心脏 IKr 是塑造人类心室动作电位的关键复极钾电流。 由人类 ether-à-go-go 相关基因 (hERG1) 1a 和 1b 亚基的异聚体组装进行。 这些亚基由 hERG/KCNH2 基因的替代转录物编码，仅在氨基上有所不同。 hERG1a/1b 异聚化对于正常 CM 功能至关重要，因为亚基不平衡。 hERG1a/1b 组装的表达和/或功能导致细胞促心律失常行为。 HEK293 细胞（源自人类的心肌细胞）中编码 mRNA 的共翻译关联 诱导多能干细胞（hiPSC-CM）和人类心肌之间存在相互作用。 共翻译复合体组装不需要新生蛋白质之间的相互作用。 初步结果表明，这种复杂的组装发生在转录后，并由直接促进 hERG1a 和 1b mRNA 之间的相互作用受其二级结构控制。 RNA 结合蛋白 DDX3X 和 DDX5 被鉴定为复合物的一部分，纯化的 DDX3X 促进 hERG1a/1b mRNA 的体外关联 在 K99 阶段，我将定义 mRNA 的结构特征。 促进共翻译关联并确定 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、混合蛋白-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、核糖体研究 hiPSC-CM 的稳定性、翻译和关联 我将使用定量 ITC 和 in 分析、RIP 和单分子荧光原位杂交 (smFISH)。 体外重建方法来确定之间相互作用的特异性、亲和力和能量 纯化的 DDX3X 和 DDX5 与 hERG1a 和 1b mRNA 我还将评估 DDX3X 和 DDX5 是否促进 在R00阶段，我将确定mRNA在体外系统中的关联性。 hERG1a 和 1b mRNA 的翻译和关联在与 2 型长波相关的心律失常中受损 QT 综合征 (LQT2) 我将使用 hiPSC-CM 疾病模型来评估半衰期、转化率和 mRNA 与 qPCR、核糖体分析、RIP 和 smFISH 的关联这些实验将有所贡献。 了解通道生物发生并阐明 LQT2 相关的分子机制 该提案旨在实现我扩展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。 我的长期目标是将 RNA 和离子通道生物物理学与转化心血管联系起来 研究。