Influenza a Blockers with Reduced Resistance Formation

Abstract

arrhythmogenic early afterdepolarizations (EADs) characteristic of LQT2 myocytes under b-adrenergic stimulation. Parameters of Ca handling were measured in ventricular myocytes isolated from normal and LQT2 hearts using confocal Ca imaging and whole-cell patch clamp. Ca imaging revealed no LQT2-mediated changes in amplitude of Ca transients and SR Ca content under basal conditions. Experiments in saponin-permeabilized cells using SRentrapped low-affinity Ca indicator revealed enhanced RyR-mediated SR Ca leak and SERCA-mediated Ca uptake in LQT2 myocytes. Correspondingly, western blot analyses using phospho-specific antibodies showed that phosphorylation of both RyR and phospholamban is significantly higher in LQT2 CMs vs. controls at both PKA and CaMKII sites. In the presence of isoproterenol (50 nM) LQT2 CMs exhibited diminished Ca transient amplitudes and SR Ca content compared to controls. Stimulation of LQT2 CMs with isoproterenol resulted in prolongation of plateau of action potentials accompanied by aberrant Ca releases and phase 3 EADs, in contrast to controls. Importantly, preincubation of LQT2 CMs with CaMKII inhibitor KN93 (10 min, 500 nM) prevented ISO-mediated changes in AP duration, disturbances in Ca handling and EADs. Analysis of CaMKII activity revealed no differences between LQT2 and control heart tissues, while Western-blot analysis demonstrated 30% decrease in the abundance of catalytic subunit of protein phosphatase type 1 (PP1). These data suggest that hyperactive RyRs due to adaptive reduction in PP1 activity and thereby RyR hyperphosphorylation is a key contributor to enhanced triggered activity in hereditary LQT2 syndrome.