Facilitation of SK Channel Activity via Inhibition OF PYK2-Dependent Tyrosine Phosphorylation Alleviates Ventricular Tachyarrhythmia in Cardiac Hypertrophy

Abstract

Small conductance calcium (Ca2+)-activated K channels (SK) expressed in ventricular myocytes (VMs) are thought to play an antiarrhythmic role in cardiac disease by mitigating reduced repolarization reserve. Using the rat model of hypertrophy induced by thoracic aortic banding (TAB), we thought to test the antiarrhythmic potential of enhanced SK channel activity by inhibiting a putative negative regulator of SK activity, tyrosine kinase Pyk2. Immunoprecipitated SK channels (SK2) from TAB VMs demonstrated increased levels of Tyr phosphorylation reversible by incubation with PF431396 (10 µM, n=3), a specific Pyk2 inhibitor. Western blot analysis showed ∼2 fold increase in expression levels of Tyr kinase Pyk2 but not Src/Fyn in TAB VMs. In ex vivo optically mapped TAB hearts stained with voltage sensitive dye di-4 ANNEPS, VT/VF was observed in 8/8 hearts challenged with β-adrenergic agonist isoproterenol (50 nM). PF431496 (2 μM, 30 min.) alleviated VT/VF and shortened APD under isoproterenol (n=3). To unravel the mechanism of Pyk2-dependent modulation of SK activity, we used cultured adult rat VMs overexpressing SK2. Simultaneous whole-cell patch clamp and confocal Ca2+ imaging revealed that Ca2+-voltage(Vm) dependence of SK channels is biphasic with inhibition at higher [Ca2+]/Vm and this can be enhanced by stimulation of Pyk2 (phenylephrine, 100 µM and propranolol, 1 µM). Furthermore, N-terminus de-phosphomimetic SK2 mutant Y138F was completely insensitive to phenylephrine, suggesting phosphorylation of this Tyr residue confers Pyk2-mediated enhancement of Ca2+-Vm dependent inhibition of SK2. Coexpression of specific Pyk2 inhibiting peptide, CRNK, fully restored SK2 responsiveness to Ca2+ in phenylephrine-treated cells. In conclusion, SK channel activity in TABs can be further increased by inhibiting Pyk2. Additional facilitation of SK channels via Pyk2 inhibition may be a rational antiarrhythmic target, increasing repolarization reserve that is diminished in cardiac disease.