Mechanistic re-evaluation of fibroblast growth factor homologous factors in the heart

Abstract Cardiac ion channels are tightly regulated—in abundance, subcellular location, turnover, and activity—to control the electrical signaling that drives heart muscle contraction. Any perturbation of this regulation can lead to arrhythmogenic sudden death. Most studies of how ion channel dysfunction contributes to pathophysiology have focused on defects of the channel pore-forming subunits. Contributions of channel auxiliary subunits are understudied and complicated by alternative or additional roles for this set of proteins. Here, we focus on fibroblast growth factor homologous factors (FHFs), proteins that bind directly to voltage-gated sodium channels (VGSCs) including the cardiac NaV1.5 channel and that are implicated in life-threatening arrhythmias. Studies on FHFs in heart have focused almost exclusively on how they regulate NaV1.5 via direct binding the channel's cytoplasmic C-terminus. The goal of this proposal is to expand knowledge of how FHFs influence cardiac physiology and how variants contribute to arrhythmias, thus providing a platform for targeted therapies. Our preliminary data uncover three unexpected aspects of FHF function in heart: 1) FHFs regulates VGSCs independent of channel interaction; 2) FHFs regulate trafficking and membrane targeting of multiple cardiac channels via control of microtubule stability (we focus on connexin43 [Cx43]); 3) FHFs affect adrenergic signaling to channels independent of ion channel interaction. We propose to define the underlying mechanisms using a novel structurally guided approach that generates an FHF incapable of binding NaV1.5 and by exploiting unbiased proteomic analyses using protein proximity labeling in cardiomyocytes in the following Aims: Aim 1: Test the hypothesis that FGF13 confers VGSC regulation by affecting local membrane cholesterol. FGF13 is the main FHF in rodent hearts. Our preliminary data offer insight into how FGF13 affects NaV1.5 via regulation of local accessible membrane cholesterol, a mechanism independent of channel binding. Among other implications this aim may explain the vulnerability of subjects with Brugada syndrome to arrhythmias during febrile illnesses. Aim 2: Test the hypothesis that FGF13 stabilizes cardiomyocyte microtubules and thus regulates ion channel trafficking through regulation of MAP4. Focusing on Cx43 trafficking, we show that ablation of FGF13 in mice destabilizes microtubules and mislocalizes Cx43 in ventricular cardiomyocytes via effects on MAP4, a key regulator of microtubules in cardiomyocytes. We will investigate the underlying mechanisms of this additional NaV1.5-binding independent effect. Aim 3: Test the hypothesis that FGF13 regulates adrenergic responses in cardiomyocytes. Exploiting an unbiased proteomic screen, we uncover that FGF13 affects adrenergic regulation of NaV1.5 and CaV1.2 Ca2+ channels. Building on our unbiased discovery platforms, we propose to determine the mechanisms by which FGF13 regulates this critical cardiomyocyte signaling pathway. Together, these Aims propose to expand an understanding of FHFs in cardiomyocyte biology and more generally uncover mechanisms for how ion channel auxiliary subunits regulate their targets.