Calcium/Calmodulin-Dependent Protein Kinase II Regulation of the Slow Delayed Rectifier Potassium Current, I(ks), During Sustained Beta-Adrenergic Receptor Stimulation

Tyler Andrew Shugg, Purdue University

Abstract

Background: Sustained elevations in catecholaminergic signaling, mediated primarily through β-adrenergic receptor (β-AR) stimulation, are a hallmark neurohormonal alteration in heart failure (HF) that contribute to pathophysiologic cardiac remodeling. An important pathophysiological change during sustained β-AR stimulation is functional inhibition of the slow delayed rectifier potassium current, IKs, which has been demonstrated to prolong action potential duration (APD) and increase ventricular arrhythmogenesis in HF. Though functional inhibition of IKs has been consistently reproduced in cellular, animal, and limited human studies of HF, the mechanisms that mediate IKs inhibition during HF remain poorly understood. In addition, HF results in aberrant calcium handling that is known to contribute to the disease. HF has been demonstrated to increase the expression and function of calcium/calmodulin-dependent protein kinase II (CaMKII), a key regulator of calcium homeostasis and excitation-contraction coupling in cardiomyocytes. Enhanced CaMKII signaling has been consistently demonstrated to contribute to increased arrhythmogenesis in a number of cardiac diseases, including HF. CaMKII is a known pathological regulator of many cardiac ion channels resulting in APD prolongation and the development of arrhythmias. Objective: This investigation aims to assesses the potential for CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and to characterize the potential functional implications on IKs. Furthermore, this investigation seeks to elucidate the mechanism underlying CaMKII-mediated IKs inhibition during sustained β-AR stimulation. Methods: Phosphorylation of KCNQ1 was assessed using a tandem liquid chromatography- mass spectrometry/ mass spectrometry (LCMS/MS) approach during sustained β-AR stimulation via treatment with 100 nM isoproterenol (ISO) for 4-24 hours and during co-expression with KCNE1. Whole-cell, voltage-clamp patch clamp electrophysiology experiments were performed in HEK 293 cells transiently co-expressing wild-type (WT) or mutant KCNQ1 (mutations conferring mimics of dephosphorylation and phosphorylation were introduced at phosphorylation sites identified by LCMS/MS) and KCNE1 (auxiliary subunit) during ISO treatment, treatment with CaMKII or protein kinase A (PKA) inhibitors, or during lentiviral δCaMKII overexpression. A robotic peptide synthesizer was used to create fifteen residue peptide fragments on a nitrocellulose membrane corresponding to KCNQ1 intracellular domains and the KCNQ1 residues identified via LCMS/MS; membranes were incubated with activated CaMKII or PKA in the presence of radiolabeled ATP to identify potential sites of phosphorylation. Bimolecular fluorescence complementation (BiFC) experiments were performed in HEK 293 cells to assess the impact of CaMKII-mediated KCNQ1 phosphorylation on the interaction of KCNQ1 and KCNE1 subunits. Protein immunoblot experiments were performed to (1) assess CaMKII activation during ISO treatment and (2) to assess plasma membrane expression of KCNQ1 and KCNE1 subunits with mimics of differential KCNQ1 phosphorylation following a membrane protein biotinylation procedure. Results: In Aim 1, we investigated the regulation of the KCNQ1 carboxyl terminus during sustained β-AR stimulation and assessed the associated functional implications on IKs. An LCMS/MS approach identified five novel KCNQ1 carboxyl terminal sites that demonstrated basal phosphorylation, with T482 and S484 having enhanced phosphorylation during treatment with 100 nM ISO for 24 hours (p<0.01 at both sites). Using patch clamp electrophysiology, we demonstrated that treatment with 100 nM ISO for 12-24 hours reduced IKs current density (p=0.01) and produced a depolarizing shift in the voltage dependence of activation (p<0.01) relative to vehicle.

Degree

Ph.D.

Advisors

Overholser, Purdue University.

Subject Area

Analytical chemistry|Chemistry|Genetics|Medicine|Physiology

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