INTRACELLULAR FGFS:NOVEL REGULATIONS OF CARDIAC NAV CHANNELS

细胞内 FGFS：心脏 NAV 通道的新颖调节

• 批准号: 8031777
• 负责人: JEANNE M. NERBONNE
• 金额: $ 22.8万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8031777
• 关键词: Action Potentials; Adult; Affect; Arrhythmia; Atrial Fibrillation; Attenuated; Binding; Biochemical; C-terminal; Cardiac; Cell surface; Cells; Cytoplasmic Granules; Disease; Family; Fibroblast Growth Factor; Future; Gene Targeting; Generations; Goals; Heart; Heart Diseases; In Vitro; Inherited; Investigation; Ion Channel; Life; Link; Longitudinal Studies; Mediating; Membrane; Molecular; Molecular Genetics; Multiprotein Complexes; Mus; Muscle Cells; Mutation; Myocardial; Myocardium; Neonatal; Neurons; Pathway interactions; Physiological; Play; Property; Proteins; RNA Interference; Receptor Protein-Tyrosine Kinases; Regulation; Reporting; Research; Research Proposals; Risk; Role; Sick Sinus Syndrome; Small Interfering RNA; Sodium; Specificity; Syndrome; System; Testing; Ventricular; density; fibroblast growth factor 13; genetic regulatory protein; heart rhythm; hippocampal pyramidal neuron; in vivo; insight; neuronal excitability; novel; novel strategies; programs; protein complex; research study; small hairpin RNA; therapeutic target; trafficking; voltage

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.

描述（由申请人提供）：电压门控的NA+（NAV）通道负责心脏细胞中动作电位的快速上行，并在控制动作电位持续时间和传播中起关键作用。心肌中的主要NAV孔（1）亚基是NAV1.5，由SCN5A编码，SCN5A中的突变与许多心脏节律疾病有关，包括长QT3综合征，Brugada Syldrome，心脏传导疾病，患病的Sinus sinus综合征和肌纤维化。积累的证据表明，心肌NAV通道在多聚蛋白复合物中的功能，包括一个NAV1亚基，附件（2）亚基和其他附件/调节蛋白，尽管辅助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未很好地理解。该R21提案将检验以下假设：细胞内成纤维细胞生长因子（IFGFS）起着心肌NAV1.5编码通道的新调节剂的作用。该假设是由最近的初步研究激发的，表明IFGF13在成年和新生儿（小鼠）心室中表达，IFGF13靶向的RNA干扰显着衰减（新生儿小鼠心室）心肌细胞（新生儿小鼠）肌细胞。该提案中有两个相关的目标，这些目标将同时实现。具体而言，此处概述的研究将检验以下假设：IFGF13选择性调节心室NAV电流并在心室作用电位的产生中起生理作用（AIM＃1）。平行研究将探讨以下假设：IFGF13的功能可以调节NAV1.5编码的心室NAV通道的稳定性，贩运和/或亚细胞定位（AIM＃2）。为了实现这些目的，使用针对小型干扰RNA（siRNAS）的靶向基因“敲低”策略在体外（小鼠）心室心肌细胞（小鼠）心肌细胞（siRNA）中的表达以及这些操纵对Nave（和其他）通道的细胞表面表达的功能后果的表达。并行实验将在含有FGF13基因座的靶向破坏的小鼠（FGF13 - / - ）中分离的肌细胞上完成。预计此处提出的研究将为IFGF在心肌NAV通道的动态调节中的作用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，重点是描述与心肌膜兴奋性动态调节有关的分子，细胞和全身机制以及与SCN5A突变有关的心脏兴奋性的危险。从长远来看，预计这些研究将为IFGF作为治疗目标的潜力提供重要的新见解，以调节在遗传和获得的心律疾病中调节NAV通道的功能。 公共卫生相关性：在心脏中，电压门控钠Na+（NAV）渠道负责行动潜力的快速上行，并在控制动作潜在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。在许多遗传和获得的心脏病中观察到NAV通道表达和/或特性的变化，这些变化可能会带来深远的生理后果，包括增加潜在的威胁生命的心律不齐的风险。积累的证据表明，心肌NAV通道充当大分子蛋白复合物的组成部分，包括孔形成（1）亚基和各种附件（2）亚基，尽管目前对这些附件亚基在Myocardial海导通道稳定性的调节中的作用很少，但很少知道。将体内和体外分子遗传策略与电生理学和生化方法相结合，该新的研究计划着重于定义NAV通道调节蛋白的新型家族的生理作用，即胞内成纤维细胞成纤维细胞生长因子（IFGFS），在肌关节型海导通道表达和功能性的调节中。这些研究将为IFGF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重要见解。从长远来看，这些研究还有望提供有关IFGF作为治疗靶标的潜在的重要新见解，以调节遗传和获得性心律疾病中的NAV通道功能。