Novel Role of a Nucleoporin Gene in Atrial Fibrillation, the Most Common Cardiac

核孔蛋白基因在心房颤动（最常见的心脏疾病）中的新作用

• 批准号: 8242821
• 负责人: QING Kenneth WANG
• 金额: $ 38.86万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-16 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242821
• 关键词: 5p13; Action Potentials; Affect; Alleles; American; Antibodies; Area; Arrhythmia; Atrial Fibrillation; Binding; Biochemical; Blood Circulation; Cardiac; Catheters; Cell Membrane Permeability; Cell Nucleus; Cell membrane; Cell surface; Cells; Chromosomes; Complex; Computer Simulation; Confocal Microscopy; Cytoplasm; Data; Development; Digitonin; Disease; Docking; Down-Regulation; Drug Delivery Systems; Early Diagnosis; Eukaryotic Cell; Evaluation; Family; Frequencies; Gene Expression; Genes; Genetic; Goals; Heart Atrium; Human; Image Analysis; In Situ; In Vitro; Ion Channel; Knock-out; Knockout Mice; Lead; Link; Macromolecular Complexes; Maps; Measures; Membrane; Messenger RNA; Methods; Mitosis; Modeling; Molecular; Molecular Weight; Morbidity - disease rate; Mus; Muscle Cells; Mutation; Nuclear; Nuclear Envelope; Nuclear Export; Nuclear Import; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nucleoporin Gene; Organ; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Physiology; Play; Potassium; Property; Proteins; Refractory; Regulation; Regulator Genes; Reporting; Role; Signal Transduction; Sinus; Small Interfering RNA; Structure; Surface; Testing; Therapeutic Intervention; atrioventricular node; base; density; gambogic acid; improved; in vivo; mRNA Export; macromolecule; member; models and simulation; mortality; mutant; novel; protein expression; public health relevance; trafficking

DESCRIPTION (provided by applicant): Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia with more than 2 million Americans affected, and is growing exponentially. The major goal of this project is to identify new molecular determinants and novel molecular mechanisms of AF by molecular characterization of the newly-discovered AF gene NUP155. The NUP155 gene encodes a 155 kDa nucleoporin, which is required for the formation of the nuclear pore complex (NPC) and the assembly of the nuclear envelope during mitosis. The NPC is a large macromolecular complex of about 30 nucleoporins, and plays a key role in bi-directional transport of macromolecules with a molecular weight of >40 kDa across the nucleus membrane. Export of mRNA from the nucleus to the cytoplasm plays an important role in gene expression in eukaryotic cells. The NUP155 protein contains a binding domain that interacts directly with mRNA export factor Gle1, which may anchor GLe1 onto the NPC. NUP155 also interacts directly with a NUP53 which forms further complex with other structural nucleoporins. Thus, NUP155 may play an important role in the assembly of the NPC and regulated control of nuclear export of mRNAs. Mutations in NUP155 cause AF. Two NUP155 mutations have been identified, including mutation R391H reported previously by our group (Zhang et al 2008 Cell) and a newly identified mutation H1104P located within the Gle1 binding domain. Homozygous NUP155-/- knockout (KO) mice die before E8.5, but heterozygous NUP155 mice faithfully recapitulate the human AF phenotype. We have demonstrated that atrial myocytes from NUP155 KO mice show significant shortening of action potential duration (APD). However, the molecular mechanisms by which NUP155 mutations cause APD shortening and consequently AF remain unknown. Based on our new preliminary data that IK1 current densities are increased in NUP155 KO atrial myocytes compared to wild type control myocytes, here we propose that the NPC incorporating a mutant NUP155 subunit or less NUP155, or lacking NUP155 becomes defective structurally and/or functionally. The defective NPC may mis-regulate nuclear export of mRNAs for important atrial ion channel genes and/or their regulatory genes (e.g. genes for IK1 subunits Kir2.1, Kir2.2, Kir2.3 or Kir2.x trafficking factors), which leads to abnormal electrical remodeling of ionic currents in the atria (e.g. IK1). Enhanced IK1 and/or other electrical remodeling cause the shortening of APD and shortening of atrial effective refractory period (ERP), and triggers reentry arrhythmias and AF. To test this hypothesis, we will combine cellular and biochemical approaches, electrophysiological studies, computer modeling, and in vivo KO mouse studies to identify new molecular mechanisms of AF. We will first characterize the AF mutations in NUP155 (R391H, H1104P, NUP155 siRNA mimicking KO allele) for their structural effects on the NPC (interaction with Gle1 and NUP53, and complex formation with other nucleoporins, and nuclear envelope localization) as well as for their functional effects on the NPC (nuclear membrane permeability, nuclear export of mRNAs, nuclear import of proteins using Hsp70 as a marker). Secondly, we will use in vivo intracardiac electrophysiological studies to characterize NUP155 KO mice to assess whether the APD shortening at the cellular level is associated with a shortened atrial ERP and increased inducibility of AF at the organ level. The effects of an IK1 specific blocker, gambogic acid, will be evaluated as potential therapy for AF. Finally, we will evaluate the roles of NUP155 in the nuclear export of mRNAs for IK1 subunits, regulation of cell surface trafficking of IK1 subunits, remodeling of IK1 currents, and effects of IK1 blockers on IK1 currents and atrial APD in NUP155 KO mice. In combination with computer modeling, these studies will investigate the functional impact of down-regulation of NUP155 expression on atrial arrhythmias and identify the substrates and important mechanisms for AF cause by the NUP155 mutations. Results obtained from this study will serve our long-term goal of understanding the cardiac-specific signaling by NUP155 in cardiac physiology and disease. PUBLIC HEALTH RELEVANCE: AF is the most common sustained cardiac arrhythmia and causes substantial morbidity and mortality. The proposed studies should find a new disease-causing gene for AF and identify a novel NUP155-linked molecular mechanism for development of AF. These studies may lead to early diagnosis of AF patients with NUP155 mutations, and new and improved interventions and therapy for AF by targeting NUP155.

描述（由申请人提供）：房颤（AF）是最常见的持续性心律失常，超过200万美国人受到影响，并且呈指数级增长。本项目的主要目标是通过对新发现的AF基因NUP155的分子表征，确定AF的新分子决定因素和新的分子机制。NUP155基因编码一个155 kDa的核孔蛋白，这是核孔复合物（NPC）的形成和有丝分裂过程中核膜组装所必需的。NPC是一个由大约30个核孔蛋白组成的大分子复合物，在分子量约为40 kDa的大分子在核膜上的双向运输中起着关键作用。mRNA从细胞核向细胞质的输出在真核细胞的基因表达中起着重要作用。NUP155蛋白含有一个直接与mRNA输出因子Gle1相互作用的结合域，该结合域可能将Gle1锚定在NPC上。NUP155也直接与NUP53相互作用，NUP53与其他结构核孔蛋白形成复合物。因此，NUP155可能在NPC的组装和mrna核输出的调控中发挥重要作用。NUP155突变导致AF。已经鉴定出两种NUP155突变，包括我们小组先前报道的突变R391H （Zhang et al . 2008 Cell）和位于Gle1结合域的新发现突变H1104P。纯合子NUP155-/-敲除（KO）小鼠在E8.5之前死亡，但杂合子NUP155小鼠忠实地再现了人类AF表型。我们已经证明，来自NUP155 KO小鼠的心房肌细胞显示出动作电位持续时间（APD）的显著缩短。然而，NUP155突变导致APD缩短和AF的分子机制尚不清楚。根据我们新的初步数据，与野生型对照肌细胞相比，NUP155 KO心房肌细胞的IK1电流密度增加，这里我们提出含有突变型NUP155亚基或较少NUP155或缺乏NUP155的NPC在结构和/或功能上存在缺陷。有缺陷的NPC可能会错误地调控重要心房离子通道基因和/或其调控基因（例如IK1亚基Kir2.1、Kir2.2、Kir2.3或Kir2的基因）mrna的核输出。x运输因子)，导致心房离子电流的异常电重构（如IK1）。IK1增强和/或其他电重构导致APD缩短和心房有效不应期（ERP）缩短，并引发再入性心律失常和房颤。为了验证这一假设，我们将结合细胞和生化方法、电生理研究、计算机建模和体内KO小鼠研究来确定房颤的新的分子机制。我们将首先表征NUP155 (R391H, H1104P，NUP155 siRNA模拟KO等位基因)对鼻咽癌的结构影响（与Gle1和NUP53的相互作用，与其他核孔蛋白的复合物形成，以及核膜定位）以及它们对鼻咽癌的功能影响（核膜通透性，mrna的核输出，以Hsp70为标记的蛋白质的核输入）。其次，我们将使用体内心内电生理研究来表征NUP155 KO小鼠，以评估细胞水平APD缩短是否与心房ERP缩短和器官水平AF诱导性增加有关。IK1特异性阻滞剂甘草酸的作用将被评估为AF的潜在治疗方法。最后，我们将评估NUP155在IK1亚基mrna的核输出、IK1亚基细胞表面运输的调节、IK1电流的重塑以及IK1阻滞剂对NUP155 KO小鼠IK1电流和心房APD的影响。结合计算机建模，这些研究将探讨NUP155表达下调对心房心律失常的功能影响，并确定NUP155突变引起心房心律失常的底物和重要机制。这项研究的结果将有助于我们了解NUP155在心脏生理和疾病中的心脏特异性信号传导。