Abstract 13223: Calcium/Calmodulin-Dependent Protein Kinase II Decreases KCNH2-Related Current During Sustained Adrenergic Receptor Stimulation

Abstract

Introduction: KCNH2 codes for a potassium channel that mediates the primary cardiac repolarizing current, I Kr . Calcium/calmodulin-dependent protein kinase II (CaMKII) modulates several ion channels, is enhanced during sustained β-adrenergic receptor (βAR) stimulation, and is proarrhythmic. Furthermore, states of enhanced βAR stimulation/CaMKII activity, such as heart failure, alter microRNA (miR) expression profiles that suppress protein expression. Hypothesis: CaMKII decreases KCNH2-related current during sustained βAR stimulation through miR regulation. Methods: Putative miR binding sites (n=327) on KCNH2 3′-UTR were identified with bioinformatic tools. Binding sites were then constructed into a pIS0 plasmid, amplified, and transfected into hiPSC-cardiomyocytes. Cells were subjected to sustained βAR stimulation (Isoproterenol: 100 nM, 1 μM), constitutively active δCaMKII (Thr287Asp), or control conditions. Next-generation sequencing was used to identify altered miR binding sites expression and endogenous miR alterations. Whole-cell, voltage-clamp experiments were performed in HEK293 cells expressing KCNH2 and δCaMKII T287D . Cells were treated with isoproterenol, and miR mimics were used for validation of the bioassay. Results: Nine and eight predicted miR binding sites were downregulated in the CaMKII group (Δlog fold: -0.287 to -0.59, p<0.05) and sustained βAR group (Δlog fold: -0.29 to -0.72, p<0.05), respectively. Thirty miRs were predicted to bind to these sites on KCNH2 3′-UTR, of which seven were selected for validation based on a Δlog fold < -0.6 in one group (n=4) or < -0.5 in both treatment groups (n=3). Mimics of all seven miRs reduced KCNH2 protein expression, and three decreased KCNH2 currents compared to control (p<0.05). Sustained βAR stimulation decreased peak activation current in cells expressing both KCNH2 and δCaMKII T287D by 25% (17±7 pA/pF, p = 0.03) and tail current density by 27% (21±9 pA/pF, p = 0.03), but had no effect on currents in cells expressing only KCNH2. Conclusions: CaMKII hyperactivity alters miR expression profiles and decreases KCNH2 function during sustained βAR activation. The effect of the identified miRs on arrhythmia susceptibility with enhanced CaMKII activity warrants further study.