INTRACELLULAR FGFS:NOVEL REGULATIONS OF CARDIAC NAV CHANNELS

描述（申请人提供）：电压门控Na+ (Nav)通道负责心脏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孔形成亚基是由SCN5A编码的Nav1.5， SCN5A的突变与许多心律失常有关，包括Long QT3综合征、Brugada综合征、心传导疾病、病窦综合征和房颤。越来越多的证据表明，心肌Nav通道在多聚体蛋白复合物中起作用，包括一个Nav1亚基、辅助(2)亚基和许多其他辅助/调节蛋白，尽管辅助和调节蛋白在控制通道表达、特性和亚细胞分布中的作用尚不清楚。这项R21提案将验证细胞内成纤维细胞生长因子（iFGFs）作为心肌nav1.5编码通道的新调节剂的假设。最近的初步研究表明，iFGF13在成年和新生儿（小鼠）心室中表达，并且iFGF13靶向的RNA干扰显著降低了新生儿小鼠心室肌细胞中的Nav电流密度。这项建议有两个相关的目标，这两个目标将并行进行。具体来说，这里概述的研究将验证iFGF13选择性调节心室Nav电流并在心室动作电位的产生中起生理作用的假设（目的1）。平行研究将探索iFGF13功能调节nav1.5编码心室Nav通道的稳定性、运输和/或亚细胞定位的假设（目标#2）。为了实现这些目标，将在体外（小鼠）心室肌细胞中使用小干扰rna （sirna）靶向基因“敲低”策略来操纵iFGF13的表达，并确定这些操作对Nav（和其他）通道的特性和细胞表面表达的功能后果。平行实验将在从小鼠（Fgf13-/-）分离的肌细胞上完成，这些肌细胞含有Fgf13位点的靶向破坏。预计本文提出的研究将为iFGFs在心肌Nav通道动态调节中的作用提供新的和根本性的重要见解。此外，这些研究的结果将指导未来的研究，重点是描述心肌膜兴奋性动态调节的分子、细胞和系统机制，以及与SCN5A突变相关的心脏兴奋性紊乱。从长远来看，预计这些研究将为iFGFs作为调节遗传和获得性心律失常的Nav通道功能的治疗靶点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.