INTRACELLULAR FGFS:NOVEL REGULATIONS OF CARDIAC NAV CHANNELS

描述(由申请人提供)：电压门控Na+(Nav)通道负责心肌细胞动作电位的快速上升，并在控制动作电位的持续时间和传播方面发挥关键作用。心肌中主要的NAV孔形成(1)亚单位是由SCN5A编码的NaV1.5，SCN5A的突变与许多心律紊乱有关，包括长QT3综合征、Brugada综合征、心脏传导疾病、病态窦房结综合征和心房颤动。越来越多的证据表明，心肌NAV通道在多聚体蛋白复合体中发挥作用，包括一个NAV1亚基、辅助(2)亚基和许多其他辅助/调节蛋白，尽管辅助蛋白和调节蛋白在控制通道表达、性质和亚细胞分布中的作用尚不清楚。这项R21提案将检验细胞内成纤维细胞生长因子(IFGFs)作为心肌NaV1.5编码通道的新调节器的假设。这一假说源于最近的初步研究表明，iFGF13在成年和新生(小鼠)脑室表达，并且iFGF13靶向RNA干扰显著减弱(新生小鼠)心肌细胞的Nav电流密度。这项提议有两个相互关联的目标，这两个目标将同时实现。具体地说，这里概述的研究将检验iFGF13选择性地调节心室NAV电流并在心室动作电位的产生中发挥生理作用的假设(目标#1)。平行研究将探索iFGF13功能调节NaV1.5编码的心室NAV通道的稳定性、运输和/或亚细胞定位的假设(目标2)。为了达到这些目的，我们将利用小干扰RNA(SiRNAs)的靶向基因“敲除”策略在体外培养的小鼠心室肌细胞中操纵iFGF13的表达，并将确定这些操纵对Nav(和其他)通道的性质和细胞表面表达的功能后果。平行实验将在从含有Fgf13基因定点干扰的小鼠(Fgf13-/-)分离的心肌细胞上完成。预计本文提出的研究将为研究血管紧张性生长因子在心肌NAV通道的动态调节中的作用(S)提供新的和根本上重要的见解。此外，这些研究的结果将指导未来的研究，重点是描绘与心肌膜兴奋性的动态调节有关的分子、细胞和系统机制，以及与SCN5A突变相关的心脏兴奋性的紊乱。从长远来看，预计这些研究将为iFGFs作为治疗靶点调节遗传性和获得性心律失常的NAV通道功能的潜力提供重要的新见解。 与公共健康相关：在心脏，电压门控钠离子(Nav)通道负责动作电位的快速上升，并在控制动作电位的持续时间和传播方面发挥作用。因此，这些通道对于正常心律的产生至关重要。在许多遗传性和获得性心脏病中观察到NAV通道表达和/或特性的变化，这些变化可以产生深刻的生理后果，包括增加潜在威胁生命的心律失常的风险。越来越多的证据表明，心肌NAV通道是大分子蛋白复合体的组成部分，包括成孔(1)亚基和各种辅助(2)亚基，尽管目前对这些辅助亚基在调节心肌NAV通道稳定性、转运和/或性质中的作用知之甚少。这一新的研究计划将体内和体外分子遗传学策略与电生理和生化方法相结合，重点研究新的NAV通道调节蛋白家族-细胞内成纤维细胞生长因子(IFGFs)在调控心肌NAV通道表达和功能中的生理作用(S)。这些研究将对iFGFs在心肌NAV通道和心肌膜兴奋性的动态调节中的生理作用提供新的和根本的重要见解。从长远来看，这些研究也有望为iFGFs作为治疗靶点调节遗传性和获得性心律失常的NAV通道功能的潜力提供重要的新见解。

DESCRIPTION (provided by applicant): Voltage-gated Na+ (Nav) channels are responsible for the rapid upstroke of the action potential in cardiac cells and play critical roles in controlling action potential durations and propagation. The primary Nav pore- forming (1) subunit in the myocardium is Nav1.5, encoded by SCN5A, and mutations in SCN5A have been linked to a number of cardiac rhythm disorders, including Long QT3 syndrome, Brugada syndrome, cardiac conduction disease, sick sinus syndrome, and atrial fibrillation. Accumulating evidence suggests that myocardial Nav channels function in multimeric protein complexes, comprising one Nav1 subunit, accessory (2) subunits and a number other accessory/regulatory proteins, although the roles of accessory and regulatory proteins in controlling channel expression, properties and subcellular distributions are not well understood. This R21 proposal will test the hypothesis that intracellular fibroblast growth factors (iFGFs) function as novel regulators of myocardial Nav1.5-encoded channels. This hypothesis is motivated by recent preliminary studies demonstrating that iFGF13 is expressed in adult and neonatal (mouse) ventricles and that iFGF13-targeted RNA interference markedly attenuates Nav current densities in (neonatal mouse ventricular) myocytes. There are two related aims in this proposal, and these will be pursued in parallel. Specifically, the studies outlined here will test the hypothesis that iFGF13 selectively regulates ventricular Nav currents and plays a physiological role in the generation of ventricular action potentials (aim #1). Parallel studies will explore the hypothesis that iFGF13 functions to regulate the stability, the trafficking and/or the subcellular localization of Nav1.5-encoded ventricular Nav channels (aim #2). To achieve these aims, the expression of iFGF13 will be manipulated in (mouse) ventricular myocytes in vitro using targeted gene "knockdown" strategies with small interfering RNAs (siRNAs), and the functional consequences of these manipulations on the properties and the cell surface expression of Nav (and other) channels will be determined. Parallel experiments will be completed on myocytes isolated from mice (Fgf13-/-) harboring a targeted disruption of the Fgf13 locus. It is anticipated that the studies proposed here will provide new and fundamentally important insights into the role(s) of the iFGFs in the dynamic regulation of myocardial Nav channels. In addition, the results of these studies will guide future investigations focused on delineating the molecular, cellular and systemic mechanisms involved in the dynamic regulation of myocardial membrane excitability and in the derangements in cardiac excitability linked to mutations in SCN5A. In the long term, it is anticipated that these studies will provide important new insights into the potential of the iFGFs as therapeutic targets to modulate Nav channel functioning in inherited and acquired cardiac rhythm disorders. PUBLIC HEALTH RELEVANCE: In the heart, voltage-gated sodium Na+ (Nav) channels are responsible for the rapid upstroke of the action potential and play roles in controlling action potential durations and propagation. These channels, therefore, are critical for the generation of normal cardiac rhythms. Changes in Nav channel expression and/or properties are observed in a number of inherited and acquired cardiac diseases, and these changes can have profound physiological consequences, including increasing the risk of potentially life-threatening cardiac arrhythmias. Accumulating evidence suggests that myocardial Nav channels function as components of macromolecular protein complexes, comprising pore-forming (1) subunits and a variety of accessory (2) subunits, although very little is presently known about the roles of these accessory subunits in the regulation of myocardial Nav channel stability, trafficking and/or properties. Combining in vivo and in vitro molecular genetic strategies with electrophysiological and biochemical approaches, this new research program is focused on defining the physiological role(s) of the novel family of Nav channel regulatory proteins, the intracellular fibroblast growth factors (iFGFs), in the regulation of myocardial Nav channel expression and functioning. These studies will provide new and fundamentally important insights into the physiological roles of the iFGFs in the dynamic regulation of myocardial Nav channels and myocardial membrane excitability. In the long term, these studies are also expected to provide important new insights into the potential of the iFGFs as therapeutic targets to modulate Nav channel functioning in inherited and acquired cardiac rhythm disorders.