The Role of End-Binding Protein 2 and Microtubule Network in Inherited Cardiac Arrhythmias

末端结合蛋白 2 和微管网络在遗传性心律失常中的作用

• 批准号: 10351800
• 负责人: David Yi-Eng Chiang
• 金额: $ 16.82万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10351800
• 关键词: APC gene; Action Potentials; Acute; Adaptor Signaling Protein; Adult; Antisense Oligonucleotides; Arrhythmia; Attention; Binding; Binding Proteins; Biochemical; Bioinformatics; Brugada syndrome; CRISPR/Cas technology; Cardiac; Cardiac Electrophysiologic Techniques; Cardiac Myocytes; Cardiovascular system; Cell Size; Cells; Chemicals; Collaborations; Cytoskeleton; Dangerousness; Data; Defect; Developmental Gene; Disease; Electrocardiogram; Electron Microscopy; Embryo; Family member; Fishes; Functional disorder; Genes; Genetic; Genetic Models; Genetic study; Goals; Heart; Heterozygote; Human; Immunoprecipitation; Inherited; Ion Channel; Knock-out; Lead; Link; Manuscripts; Mass Spectrum Analysis; Mechanics; Microtubules; Molecular; Muscle Cells; Mutate; N-terminal; Nature; Pathogenesis; Pathway interactions; Patients; Pharmacology; Phase; Phenotype; Physiology; Plus End of the Microtubule; Predisposition; Promoter Regions; Proteins; Reporting; Research; Role; Shapes; Signal Transduction; Sodium Channel; Syndrome; Testing; Therapeutic; Time; Transcription Repressor; Variant; Ventricular; WNT Signaling Pathway; Zebrafish; arrhythmogenic cardiomyopathy; base; beta catenin; cell motility; design; drug discovery; gene discovery; gene interaction; genome wide association study; genomic locus; glycogen synthase kinase 3 beta inhibitor; heart function; in vivo; loss of function; mutant; novel; novel therapeutics; overexpression; skills; sudden cardiac death; tool; trafficking; transcriptome sequencing

PROJECT SUMMARY / ABSTRACT Inherited cardiac arrhythmias are a significant and devastating cause of sudden cardiac death (SCD) both in the US and globally. One prominent example is Brugada syndrome (BrS), which is a significant cause of SCD in young patients, typically with structurally normal hearts. The first BrS-associated gene, SCN5A, which encodes the cardiac sodium channel NaV1.5, was reported in 1998 and since then several other ion channel genes and their interactors have been implicated. Despite these advances, only ~30% of BrS cases have a known variant in one of these genes, leaving the remaining ~70% genetically undiagnosed. Recently, our collaboration conducted the largest BrS genome-wide association study (GWAS) to date, which identified 9 novel genetic loci. At one locus, MAPRE2, which encodes the microtubule plus end-binding protein 2 (EB2), emerged as one of the top candidates based on bioinformatic analyses. My preliminary data using both a mapre2 null (KO) and N- terminus truncated mutant (ΔN-EB2) support the role of MAPRE2 as a novel gene contributing to BrS. Specifically, mapre2 loss-of-function leads to decreased NaV function both in the embryonic and adult ventricular myocytes, a hallmark of BrS, as well as general sarcomeric disarray and microtubule network disorganization. Furthermore, MAPRE2 may interact genetically with HEY2, a well-known cardiovascular developmental gene which has been strongly implicated in BrS. Finally, RNA-sequencing implicates the Wnt pathway in mapre2 loss- of-function and treatment with SB216763, a GSK3β inhibitor and activator of Wnt, rescues ECG abnormalities in adult mapre2 mutant fish. These and other evidence led me to hypothesize that MAPRE2 loss-of-function leads to trafficking and subcellular localization defects of NaV1.5 and associated proteins, and more generally disrupts the microtubule network and cytoskeleton, contributing to cardiac arrhythmogenesis. During the K99 phase, I will explore MAPRE2 as a novel gene contributing to BrS and define its pathogenesis, paying special attention to its unique 43 aa N-terminus which is absent in the other family members (EB1 and EB3). During the R00 phase, I will study HEY2’s gene-gene interaction with MAPRE2 and SCN5A in the context of BrS and NaV1.5 dysfunction. I will also define more broadly the role of EB2 and microtubule network in cardiac Wnt signaling and arrhythmogenesis including carrying out a phenotypic chemical screen using zebrafish embryos based on in vivo Wnt/β-catenin activity, explore GSK3β inhibition as a novel therapeutic avenue for BrS and related arrhythmias, and study genetic interaction between MAPRE2 with an established arrhythmogenic cardiomyopathy mutant. Together, this proposal will allow me to fulfill my short-term goal of gaining skills and expertise in cardiac genetics and zebrafish research, as well as build novel tools and genetic models during the K99 phase. This will enable me to pursue my long-term objective during the R00 phase and beyond: to define a paradigm shift in our understanding of inherited cardiac arrhythmias and discover novel therapeutics useful in treating BrS and related NaV arrhythmias.

项目概要/摘要 遗传性心律失常是心源性猝死 (SCD) 的一个重要且具有破坏性的原因，无论是在 美国和全球。一个突出的例子是 Brugada 综合征 (BrS)，它是导致 SCD 的重要原因 年轻患者，通常心脏结构正常。第一个 BrS 相关基因 SCN5A，编码 心脏钠通道 NaV1.5 于 1998 年被报道，此后还有其他几个离子通道基因和 他们的互动者受到牵连。尽管取得了这些进展，但只有约 30% 的 BrS 病例具有已知的变异 在这些基因之一中，剩下约 70% 的基因未被诊断出来。最近，我们的合作 BrS 进行了迄今为止最大规模的全基因组关联研究 (GWAS)，确定了 9 个新的遗传位点。 在一个基因座上，编码微管加末端结合蛋白 2 (EB2) 的 MAPRE2 成为 基于生物信息学分析的最佳候选者。我使用mapre2 null（KO）和N-的初步数据 末端截短突变体 (ΔN-EB2) 支持 MAPRE2 作为促进 BrS 的新基因的作用。 具体来说，mapre2 功能丧失会导致胚胎和成人心室中 NaV 功能下降 肌细胞（BrS 的标志）以及一般肌节紊乱和微管网络紊乱。 此外，MAPRE2 可能与众所周知的心血管发育基因 HEY2 存在遗传相互作用。 这与 BrS 密切相关。最后，RNA 测序表明 Wnt 通路与 mapre2 丢失有关 功能丧失并用 SB216763（一种 GSK3β 抑制剂和 Wnt 激活剂）治疗可挽救心电图异常 在成年mapre2突变鱼中。这些和其他证据使我推测 MAPRE2 功能丧失 导致 NaV1.5 和相关蛋白的运输和亚细胞定位缺陷，更普遍的是 破坏微管网络和细胞骨架，导致心律失常。 在 K99 阶段，我将探索 MAPRE2 作为促进 BrS 的新基因，并定义其 发病机制，特别注意其独特的 43 个氨基酸 N 末端，这是其他家族中不存在的 成员（EB1 和 EB3）。在R00阶段，我将研究HEY2与MAPRE2的基因间相互作用以及 BrS 和 NaV1.5 功能障碍背景下的 SCN5A。我还将更广泛地定义 EB2 的作用和 心脏 Wnt 信号传导和心律失常发生中的微管网络，包括进行表型化学 基于体内 Wnt/β-catenin 活性筛选斑马鱼胚胎，探索 GSK3β 抑制作为一种新的 BrS 和相关心律失常的治疗途径，并研究 MAPRE2 与 建立了致心律失常性心肌病突变体。总之，这个提案将使我能够实现我的短期目标 目标是获得心脏遗传学和斑马鱼研究方面的技能和专业知识，以及构建新颖的工具和 K99阶段的遗传模型。这将使我能够在 R00 期间追求我的长期目标 阶段及以后：定义我们对遗传性心律失常的理解的范式转变并发现 可用于治疗 BrS 和相关 NaV 心律失常的新型疗法。