Fibroblast growth factor homologous factors:modulation of L-type calcium channel

描述（由申请人提供）：背景：越来越多的心血管疾病如心律失常和某些心肌病可归因于离子通道功能障碍，其由通道内的突变引起。这些“通道病”有助于理解离子通道的扰动如何导致疾病。最近的工作表明，离子通道的辅助和调节亚基是有效的通道调节剂，往往赋予多种离子电流的影响。成纤维细胞生长因子同源因子（FHF，FGF 11-FGF 14）是电压门控钠通道的调节剂，并且Pitt实验室最近表征了FGF 13在调节心脏中的电压门控钠通道中的作用。初步数据显示，FHF，特别是FGF 13可以影响Cav1.2，心脏中的L型Ca 2+通道（LTCC），这表明FHF在兴奋-收缩偶联和心肌发生中起重要作用。本研究将建立在这些观察与以下两个目标：目标1：定义FGF 13调节CaV1.2的机制。待检验的假设是：FGF 13通过直接相互作用调节心脏钙通道CaV 1.2。目的2：探讨内源性FGF 13对心室肌细胞CaV 1.2的调节作用。待检验的假设是：FGF 13调节CaV 1.2电流和心肌细胞中的定位，从而影响心脏动作电位和兴奋-收缩偶联。研究方法：目的1将利用一种异源表达系统，通过全细胞膜片钳技术来确定各种FGF 13亚型在调节Cav1.2电流中的作用。这些研究将随后进行生化分析，以确定FGF 13如何影响CaV 1.2电流，利用免疫共沉淀，表面生物素化，和体外结合测定。目的2利用全细胞膜片钳技术研究FGF 13基因敲低对小鼠心室肌细胞钙电流的影响。作用机制将通过免疫细胞化学分析确定敲低后Cav1.2分布的变化。单个FGF 13同种型在影响电流和Cav1.2靶向中的作用将使用内源性FGF 13的敲低与单个标记同种型的过表达的组合来定义。目的：目的1的结果将定义突出的心脏FHF的特定亚型FGF 13如何影响LTCC功能。将进一步确定FGF 13和CaV1.2亚基之间的相互作用，并将显示FGF 13是否在将Cav1.2靶向细胞表面中起作用。这些结果将为目标2提供信息，其中 内源性FGF 13对LTCC的作用将通过敲低研究和伴随的特定同种型的敲低和过表达来阐明。这将证明FGF 13通过其对LTCC的调节在心脏动作电位中是突出的参与者，并将FGF 13功能障碍定义为获得性和遗传性心律失常的介导者。 公共卫生相关性：在美国，目前约有500万患者患有心力衰竭，估计有400，000例死亡归因于心脏性猝死（SCD）。与心力衰竭和伴随的危及生命的心律失常相关的钙处理不当可能由多种病因引起，包括Ca 2+通道本身的成孔亚基或其相互作用蛋白的功能障碍。本研究的目的是表征一个新的钙通道相互作用蛋白家族，成纤维细胞生长因子同源因子，从而为SCD风险增加提供新的遗传标记，并为抗血小板药物提供治疗靶点。

DESCRIPTION (provided by applicant): Background: A growing number of cardiovascular disorders such as arrhythmias and certain cardiomyopathies are attributable to ion channel dysfunction that results from mutations within channels. These "channelopathies" have been instrumental in providing an understanding about how perturbation of ion channels can lead to disease. Recent work has shown that ion channel auxiliary and modulatory subunits are potent regulators of channels and often impart effects upon multiple ionic currents. Fibroblast growth factor homologous factors (FHFs, FGF11-FGF14) are modulators of voltage-gated sodium channels and the Pitt lab has recently characterized the role of FGF13 in modulating voltage-gated sodium channels in the heart. Preliminary data show that FHFs, particularly FGF13 can affect Cav1.2, the L-type Ca2+ channel (LTCC) in the heart, as well, which suggests an important role for FHFs in excitation-contraction coupling and arrhythmogenesis. This study will build upon these observations with the following two aims: Aim 1: Define the mechanisms by which FGF13 regulates CaV1.2. The hypothesis to be tested is: FGF13 regulates the cardiac calcium channel, CaV1.2, through a direct interaction. Aim 2: Determine the role of endogenous FGF13 in regulating the CaV1.2 in ventricular myocytes. The hypothesis to be tested is: FGF13 modulates CaV1.2 currents and localization in cardiac myocytes, thereby affecting the cardiac action potential and excitation-contraction coupling. Methods: Aim 1 will employ a heterologous expression system to define the roles of various FGF13 isoforms in modulating Cav1.2 curent via whole-cell patch clamp. These studies will be followed with biochemical analyses to define how FGF13 affects the CaV1.2 current, utilizing co-immunoprecipitation, surface biotinylation, and in vitro binding asays. Aim 2 wil utilize whole-cell patch clamp of mouse ventricular myocytes to define the effects on the Ca2+ current with FGF13 knocked down. The mechanism of action will be defined with immunocytochemical analysis for changes in Cav1.2 distribution after knockdown. The roles of individual FGF13 isoforms in affecting current and Cav1.2 targeting wil be defined using a knockdown of endogenous FGF13 combined with overexpression of a single tagged isoform. Objectives: The results of Aim 1 will define how specific isoforms of the prominent heart FHF, FGF13, affect LTCC function. The interactions between FGF13 and CaV1.2 subunits will be further determined and it will be shown whether FGF13 plays a role in targeting Cav1.2 to the cell surface. The results will inform Aim 2, in which the effects of endogenous FGF13 on the LTCC will be elucidated through knockdown studies and concomitant knockdown and overexpression of specific isoforms. This will demonstrate that FGF13 is a prominent player in the cardiac action potential through its modulation of the LTCC and define FGF13 dysfunction as a mediator of acquired and inherited arrhythmias. PUBLIC HEALTH RELEVANCE: In the United States, about 5 million patients are currently affected with heart failure with an estimated 400,000 deaths attributed to sudden cardiac death (SCD). Mishandling of calcium associated with heart failure and the accompanying life-threatening arrhythmias can result from multiple etiologies, including dysfunction of the pore-forming subunit of the Ca2+ channel itself or its interacting proteins. This study aims to characterize a new family of Ca2+ channel-interacting proteins, fibroblast growth factor homologous factors, thus providing new genetic markers for increased SCD risk and therapeutic targets for anti-arrhythmic medications.