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

描述(申请人提供)：房颤(房颤)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房颤基因NUP155进行分子特征分析，以确定房颤的新分子决定因素和新的分子机制。NUP155基因编码155 kDa的核孔蛋白，在有丝分裂过程中核孔复合体(NPC)的形成和核膜的组装是必需的。NPC是一个由大约30个核孔蛋白组成的大分子复合体，在大分子在核膜上的双向运输中起着关键作用。在真核细胞中，mRNA从细胞核输出到细胞质在基因表达中起着重要的作用。NUP155蛋白含有一个结合域，它直接与mRNA输出因子Gle1相互作用，Gle1可能将Gle1锚定在NPC上。NUP155还与NUP53直接相互作用，与其他结构的核孔蛋白形成进一步的复合体。因此，NUP155可能在NPC的组装和对mRNAs核出口的调控中发挥重要作用。NUP155基因突变可引起房颤。已鉴定出两个NUP155突变，包括本课题组(Zhang Et Al 2008 Cell)先前报道的突变R391H和新发现的位于Gle1结合区的突变H1104P。纯合子NUP155基因敲除(KO)小鼠在E8.5之前死亡，但杂合NUP155小鼠忠实地概括了人类房颤的表型。我们已经证明了NUP155 KO小鼠的心房肌细胞动作电位时程显著缩短。然而，NUP155突变导致动作电位时程缩短从而导致房颤的分子机制仍不清楚。根据我们的新的初步数据，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，我们认为含有或少于NUP155突变亚基的NPC在结构和/或功能上存在缺陷。鼻咽癌可能错误调控重要的心房离子通道基因和/或其调控基因(如IK1亚单位Kir2.1、Kir2.2、Kir2.3或Kir2.x转运因子)的核输出，从而导致心房离子电流的异常电重构(如IK1)。增强的IK1和/或其他电重构导致时程缩短和心房有效不应期缩短，并触发折返性心律失常和房颤。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、计算机模拟和KO小鼠体内研究来确定房颤的新分子机制。我们将首先研究NUP155(R391H，H1104P，NUP155 siRNA模拟KO等位基因)的AF突变对NPC的结构影响(与Gle1和NUP53的相互作用，与其他核孔蛋白的复合体形成，以及核膜定位)以及它们对NPC的功能影响(核膜通透性，mRNAs核输出，以Hsp70为标记的蛋白质核输入)。其次，我们将使用体内心内电生理研究来描述NUP155 KO小鼠的特征，以评估细胞水平的时程缩短是否与器官水平的心房ERP缩短和房颤的诱发性增加有关。IK1特异性阻滞剂藤黄酸的效果将被评估为房颤的潜在治疗方法。最后，我们将评估NUP155在IK1亚基的核输出、IK1亚基细胞表面转运的调节、IK1电流的重构以及IK1阻滞剂对IK1电流和心房动作电位的影响中所起的作用。结合计算机模拟，这些研究将探讨NUP155表达下调对房性心律失常的功能影响，并确定NUP155突变导致房颤的底物和重要机制。这项研究的结果将为我们理解NUP155在心脏生理和疾病中的心脏特异信号的长期目标服务。 公共卫生相关性：房颤是最常见的持续性心律失常，可导致相当大的发病率和死亡率。建议的研究将发现一个新的房颤致病基因，并确定一个新的与NUP155连锁的房颤发生的分子机制。这些研究可能导致对具有NUP155突变的房颤患者的早期诊断，并通过靶向NUP155来改进对房颤的新的干预和治疗。

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.