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.
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.
|Hennessey, Jessica A; Boczek, Nicole J; Jiang, Yong-Hui et al. (2014) A CACNA1C variant associated with reduced voltage-dependent inactivation, increased CaV1.2 channel window current, and arrhythmogenesis. PLoS One 9:e106982|
|Hennessey, Jessica A; Wei, Eric Q; Pitt, Geoffrey S (2013) Fibroblast growth factor homologous factors modulate cardiac calcium channels. Circ Res 113:381-8|
|Hennessey, Jessica A; Marcou, Cherisse A; Wang, Chuan et al. (2013) FGF12 is a candidate Brugada syndrome locus. Heart Rhythm 10:1886-94|
|Wei, Eric Q; Barnett, Adam S; Pitt, Geoffrey S et al. (2011) Fibroblast growth factor homologous factors in the heart: a potential locus for cardiac arrhythmias. Trends Cardiovasc Med 21:199-203|