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: 8063582
• 负责人: QING Kenneth WANG
• 金额: $ 39.25万
• 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/8063582
• 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）：心房颤动（AF）是最常见的持续性心律失常，超过200万美国人受到影响，并且呈指数增长。本项目的主要目的是通过对新发现的AF基因NUP 155的分子特征分析，确定新的AF分子决定因素和新的AF分子机制。NUP 155基因编码一个155 kDa的核孔蛋白，它是有丝分裂过程中核孔复合体（NPC）形成和核膜组装所必需的。NPC是约30个核孔蛋白的大分子复合物，并且在分子量>40 kDa的大分子跨核膜的双向转运中起关键作用。mRNA从细胞核到细胞质的输出在真核细胞中的基因表达中起重要作用。NUP 155蛋白含有与mRNA输出因子Gle 1直接相互作用的结合结构域，其可将Gle 1锚到NPC上。NUP 155还与NUP 53直接相互作用，NUP 53与其他结构核孔蛋白形成进一步的复合物。因此，NUP 155可能在NPC的组装和mRNA的核输出的调节控制中起重要作用。 NUP 155中的突变导致AF。已经鉴定了两个NUP 155突变，包括我们小组先前报道的突变R391 H（Zhang et al 2008 Cell）和位于Gle 1结合结构域内的新鉴定的突变H1104 P。纯合NUP 155-/-敲除（KO）小鼠在E8.5之前死亡，但杂合NUP 155小鼠忠实地再现了人AF表型。我们已经证明，NUP 155 KO小鼠的心房肌细胞表现出明显的动作电位时程（APD）缩短。然而，NUP 155突变导致APD缩短并因此导致AF的分子机制仍然未知。基于我们的新的初步数据，即与野生型对照肌细胞相比，NUP 155 KO心房肌细胞中IK 1电流密度增加，在此我们提出，掺入突变NUP 155亚基或更少NUP 155或缺乏NUP 155的NPC在结构上和/或功能上变得有缺陷。有缺陷的NPC可能错误调节重要心房离子通道基因和/或其调节基因（例如IK 1亚基Kir2.1、Kir2.2、Kir2.3或Kir2.x运输因子的基因）的mRNA的核输出，这导致心房中离子电流（例如IK 1）的异常电重构。增强的IK 1和/或其他电重构导致APD缩短和心房有效不应期（ERP）缩短，并触发折返性心律失常和AF。为了验证这一假设，我们将结合联合收割机细胞和生化方法，电生理研究，计算机模拟，和体内KO小鼠研究以鉴定AF的新分子机制。我们将首先表征NUP 155中的AF突变，（R391 H、H1104 P、NUP 155 siRNA模拟KO等位基因）对NPC的结构影响（与Gle 1和NUP 53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NPC的功能作用（核膜渗透性、mRNA的核输出、使用Hsp 70作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学研究来表征NUP 155 KO小鼠，以评估细胞水平上的APD缩短是否与心房ERP缩短和器官水平上的AF诱导增加相关。最后，我们将评估NUP 155在IK 1亚基mRNA核输出、IK 1亚基细胞表面运输调节、IK 1电流重塑以及IK 1阻滞剂对NUP 155 KO小鼠IK 1电流和心房APD的影响。结合计算机建模，这些研究将研究NUP 155表达下调对房性心律失常的功能影响，并确定NUP 155突变导致AF的底物和重要机制。从这项研究中获得的结果将有助于我们理解NUP 155在心脏生理学和疾病中的心脏特异性信号传导的长期目标。 公共卫生相关性：AF是最常见的持续性心律失常，可导致大量发病率和死亡率。这些研究可能会导致对NUP 155突变的AF患者的早期诊断，以及通过靶向NUP 155对AF进行新的和改进的干预和治疗。