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

项目总结/摘要 遗传性心律失常是心脏性猝死（SCD）的重要和毁灭性原因， 美国和全球。一个突出的例子是Brugada综合征（BrS），这是SCD的一个重要原因， 年轻患者，通常心脏结构正常。第一个BrS相关基因SCN 5A，编码 心脏钠离子通道NaV1.5，在1998年被报道，从那时起，其他几个离子通道基因， 他们的联系人被牵连了尽管取得了这些进展，但只有约30%的BrS病例具有已知的变体 在这些基因之一，剩下的约70%的基因未被诊断。最近，我们的合作 进行了迄今为止最大的BrS全基因组关联研究（GWAS），发现了9个新的遗传基因座。 在一个位点，编码微管加末端结合蛋白2（EB 2）的MAPRE 2作为一种蛋白质出现， 根据生物信息学分析，我的初步数据同时使用mapre 2空值（KO）和N- 末端截短突变体（Δ N-EB 2）支持MAPRE 2作为促进BrS的新基因的作用。 具体来说，mapre 2功能丧失导致胚胎和成人心室肌细胞NaV功能降低， 肌细胞，BrS的标志，以及一般的肌节紊乱和微管网络紊乱。 此外，MAPRE 2可能与HEY 2（一个众所周知的心血管发育基因）发生遗传相互作用 这与BrS有很大关系最后，RNA测序表明Wnt途径与mapre 2丢失有关。 SB 216763（一种GSK 3 β抑制剂和Wnt激活剂）治疗可挽救ECG异常 在成年mapre 2突变鱼中。这些和其他证据使我假设MAPRE 2功能丧失 导致NaV1.5和相关蛋白的运输和亚细胞定位缺陷，更普遍地说， 破坏微管网络和细胞骨架，促进心脏血管生成。 在K99阶段，我将探索MAPRE 2作为一个新的基因有助于BrS，并定义其在BrS中的作用。 致病机制，特别注意其独特的43 aa的N-末端，这是在其他家族中缺失的 成员（EB 1和EB 3）。在R 00阶段，我将研究HEY 2与MAPRE 2的基因-基因相互作用， 在BrS和NaV1.5功能障碍的背景下的SCN 5A。我还将更广泛地定义EB 2的作用， 微管网络在心脏Wnt信号传导和血管生成中的作用，包括进行表型化学 利用斑马鱼胚胎筛选体内Wnt/β-catenin活性，探索GSK 3 β抑制作为一种新的 BrS和相关心律失常的治疗途径，并研究MAPRE 2与 建立了致心肌病突变体。总之，这个提议将使我能够履行我的短期 目标是获得心脏遗传学和斑马鱼研究的技能和专业知识，以及建立新的工具和 K99阶段的遗传模型。这将使我能够在R 00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的理解的范式转变，并发现 用于治疗BrS和相关NaV心律失常的新疗法。

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.