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: 8442341
• 负责人: QING Kenneth WANG
• 金额: $ 36.99万
• 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/8442341
• 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 Targeting; 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; 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 Targeting; 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.

描述（由申请人提供）：房颤（AF）是最常见的持续性心律失常，受影响超过200万美国人，并且正在成倍增长。该项目的主要目的是通过新发现的AF基因NUP155的分子表征来鉴定AF的新分子决定因素和新型分子机制。 NUP155基因编码155 kDa核孔蛋白，这是核孔复合物（NPC）和有丝分裂过程中核包膜组装所必需的。 NPC是大约30个核孔蛋白的大型大分子复合物，在整个核膜上的分子量> 40 kDa的大分子的双向转运中起着关键作用。将mRNA从细胞核出口到细胞质在真核细胞中的基因表达中起重要作用。 NUP155蛋白包含一个与mRNA输出因子GLE1直接相互作用的结合结构域，该因子可能会将GLE1锚定在NPC上。 NUP155还与NUP53直接相互作用，该NUP53与其他结构核孔形成进一步的复合物。因此，NUP155可能在NPC组装和对mRNA核出口的控制中起重要作用。 NUP155中的突变导致AF。已经确定了两个NUP155突变，包括我们组先前报道的突变R391H（Zhang等，2008 Cell）和位于GLE1结合域内的新鉴定的突变H1104p。纯合子NUP155 - / - 敲除（KO）小鼠在E8.5之前死亡，但杂合的NUP155小鼠忠实地概括了人AF表型。我们已经证明，来自NUP155 KO小鼠的心肌细胞表现出明显的动作电位持续时间缩短（APD）。但是，NUP155突变导致APD缩短的分子机制，因此AF仍然未知。基于我们的新初步数据，与野生型对照肌细胞相比，NUP155 KO心房心肌细胞中IK1电流密度增加，在这里我们建议NPC NPC NPC NUP155突变的NUP155或更少的NUP155，或缺乏NUP155的NPC在结构和/或功能上有缺陷。有缺陷的NPC可能会错误地调节对重要心房离子通道基因和/或其调节基因的mRNA的核出口（例如，IK1亚基Kir2.1，Kir2.2，Kir2.2，Kir2.3或Kir2.x运输因素的基因），导致电气重塑，导致a atriak in a atria intria。增强的IK1和/或其他电重塑会导致APD缩短并缩短心房有效期（ERP），以及触发重新进入心律不齐和AF。 为了检验这一假设，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，以鉴定AF的新分子机制。我们将首先表征NUP155（R391H，H1104P，NUP155模仿KOELERELE）对NPC的结构效应（与GLE1和NUP53的相互作用，以及与其他核苷蛋白的复杂形成以及对NPC的复杂形成，以及对NPC的复杂形成，以及对NPC的相互作用，以及对NPC的相互作用（以及核包含的NPC），MRNP（MRNP）效果（MRNP），MR效应了NPC，则效应，MR使用HSP70作为标记的蛋白质进口蛋白质）。其次，我们将使用体内的体内电生理研究来表征NUP155 KO小鼠的表征，以评估细胞水平的APD缩短是否与缩短的心房ERP相关，并增加了AF在器官水平上的诱导性。 IK1特异性阻断剂甘醇酸的影响将被评估为对AF的潜在治疗。最后，我们将评估NUP155在IK1亚基的核输出mRNA中的作用，IK1亚基的细胞表面运输，IK1电流的重塑以及IK1阻断剂对IK1电流的影响以及NUP155 KO小鼠中IK1电流的影响。结合计算机建模，这些研究将研究NUP155表达对心律失常的下调的功能影响，并通过NUP155突变确定AF原因的底物和重要机制。从这项研究中获得的结果将达到我们的长期目标，即了解NUP155在心脏生理和疾病中的心脏特异性信号传导。