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.

项目摘要/摘要 遗传性心律失常是导致心脏性猝死(SCD)的重要和破坏性原因，在 美国和全球都是如此。一个突出的例子是Brugada综合征(BRS)，它是#年SCD的一个重要原因 年轻患者，典型的心脏结构正常。第一个BRS相关基因SCN5A，它编码 心脏钠通道NaV1.5是在1998年被报道的，从那时起，其他几个离子通道基因和 他们的互动者被牵连了进来。尽管取得了这些进展，但只有30%的BRS病例有已知的变异 在这些基因中的一个，剩下的~70%的基因没有被诊断出来。最近，我们的合作 进行了迄今为止规模最大的BRS全基因组关联研究，确定了9个新的遗传基因座。 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人。我的初步数据同时使用了mapre2空(KO)和N- 末端截短突变体(ΔN-EB2)支持MAPRE2作为一个新的致病基因。 具体地说，Mapre2功能丧失会导致胚胎和成年脑室的NAV功能降低 肌细胞，BRS的标志，以及普遍的肌节紊乱和微管网络紊乱。 此外，MAPRE2可能与已知的心血管发育基因HEY2存在遗传上的相互作用 这与BRS有很大的牵连。最后，RNA测序表明Wnt通路与mapre2丢失有关。 GSK3β抑制剂和WNT激活剂SB216763对心电信号异常的挽救作用 在成年Mapre2突变鱼中。这些证据和其他证据让我假设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，更广泛地说 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作为一个与BRS相关的新基因，并定义其 发病机制，特别注意其独特的43个氨基酸N-末端，这在其他家族中是不存在的 成员(EB1和EB3)。在R00阶段，我将研究HEY2的S基因与MAPRE2和MAPRE2的基因相互作用 在BRS和NaV1.5功能障碍的背景下的SCN5A。我还将更广泛地定义EB2和 微管网络在心脏Wnt信号和心律失常发生中的作用 利用斑马鱼胚胎体内筛选Wnt/β-Catenin活性，探索抑制GSK3β的新途径 BRS及相关心律失常的治疗途径，并研究MAPRE2与AN的遗传相互作用 已确定为致心律失常性心肌病突变体。总之，这项提议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，以及建立新的工具和 K99阶段的遗传模型。这将使我能够在R00期间实现我的长期目标 阶段及以后：定义我们对遗传性心律失常理解的范式转变并发现 可用于治疗BRS和相关NAV心律失常的新疗法。