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: 10580832
• 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/10580832
• 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; Link; Manuscripts; Mass Spectrum Analysis; Mechanics; Microtubules; Molecular; Muscle Cells; Mutate; N-terminal; Nature; Pathogenesis; Pathway interactions; Patients; Phase; Phenotype; Physiology; Plus End of the Microtubule; Predisposition; Promoter Regions; Proteins; Reporting; Research; Role; Shapes; Signal Transduction; Sodium Channel; Syndrome; Testing; Therapeutic; Transcription Repressor; Variant; Ventricular; WNT Signaling Pathway; Zebrafish; arrhythmogenic cardiomyopathy; beta catenin; cell motility; design; drug discovery; gene discovery; gene interaction; genome wide association study; genomic locus; glycogen synthase kinase 3 beta; glycogen synthase kinase 3 beta inhibitor; heart function; in vivo; loss of function; mutant; novel; novel therapeutics; overexpression; pharmacologic; 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 遗传性心律失常是猝死（SCD）的重大且毁灭性的原因 我们和全球。一个突出的例子是布鲁加达综合征（BRS），这是SCD中的重要原因 年轻患者，通常具有结构正常的心脏。第一个与BRS相关的基因SCN5A，编码 1998年报告了心脏钠通道NAV1.5，此后，其他几个离子通道基因和 他们的互动者被暗示。尽管有这些进展，但只有约30％的BRS案例具有已知变体 在这些基因之一中，剩余的约70％通常未诊断。最近，我们的合作 迄今为止，进行了最大的BRS基因组关联研究（GWAS），该研究确定了9个新的遗传基因座。 在一个基因座，MapRe2编码微管加上终端结合蛋白2（EB2），出现为其中之一 基于生物信息学分析的顶级候选人。我使用MAPRE2 NULL（KO）和N-的初步数据 末端截断突变体（ΔN-EB2）支持MAPRE2作为对BRS的新基因的作用。 具体而言，MAPRE2功能丧失会导致胚胎和成人心室的NAV功能降低 心肌细胞，BRS的标志，以及一般的肌肉混乱和微管网络混乱。 此外，MAPRE2可能会与Hey2相互作用，Hey2是一个众所周知的心血管发育基因 这与BRS密切相关。最后，RNA测序在MAPRE2损耗中实现了Wnt途径 - 使用SB216763（GSK3β抑制剂和WNT激活剂）的功能和处理，反应ECG异常 在成人Mapre2突变鱼中。这些和其他证据使我假设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，更普遍地 破坏微管网络和细胞骨架，导致心律失常发生。 在K99阶段，我将探索MAPRE2作为一个新的基因，并定义其 发病机理，特别注意其独特的43 AA N末端，另一个家庭不存在 成员（EB1和EB3）。在R00阶段，我将研究Hey2与MapRe2和 SCN5A在BRS和NAV1.5功能障碍的背景下。我还将更广泛地定义EB2和 心脏Wnt信号传导和心律失常的微管网络，包括进行表型化学 筛选基于体内Wnt/β-catenin活性的斑马鱼胚胎，探索GSK3β作为一种新型 用于BRS和相关心律失常的治疗途径，并研究MAPRE2与A的遗传相互作用 已建立的心律失常性心肌病突变体。这项提议在一起将使我能够履行我的短期 在心脏遗传学和斑马鱼研究方面获得技能和专业知识的目标，以及建立新颖的工具和 K99阶段的遗传模型。这将使我能够在R00期间追求自己的长期目标 阶段及以后：在我们对遗传心律不齐的理解中定义范式转变并发现 新型疗法可用于治疗BR和相关的NAV心律失常​​。