Abstract 12860: Enhanced Expression and PKCδ-mediated Hyperphosphorylation Underlie the Proarrhythmic Increase in Na + -Ca 2+ Exchanger Activity in Patients With Chronic Atrial Fibrillation

Abstract

Background: Enhanced Na + -Ca 2+ exchanger type-1 (NCX1) function is implicated in triggering and maintaining atrial fibrillation (AF), but the molecular mechanisms of dysfunctional NCX in AF patients are unknown and were the main subject of this study. Methods: We used perforated patch-clamp technique to measure transient inward currents (I ti ) in atrial cardiomyocytes from sinus rhythm (Ctl) and chronic AF (cAF) patients. Co-immunoprecipitation (co-IP) and Western blot in atrial tissue lysates and membrane fractions and in-cell co-IP (DuoLink ® ) in human atrial cardiomyocytes were employed to identify regulatory partners of NCX1 and potential differences in their expression in Ctl versus cAF patients. Results: Frequency of spontaneous NCX-mediated I ti and amplitude of caffeine-induced peak I ti were higher in cAF than Ctl patients, with no change in SR Ca 2+ content (Fig A), validating the increased NCX function in cAF. Expression of the functional 160 kDa full-length NCX1 protein was strongly increased in cAF membrane fractions and was responsible for more than 90% of total membranous NCX1 (Fig B). Co-IPs followed by immunoblotting identified two protein kinase-C isoforms (PKCα and PKCδ), protein phosphatase-type-1α (PP1α), phospholemman (PLM) and AMP-activated protein kinase-α (AMPKα) as parts of the NCX1 complex, whereas calcineurin-Aβ (CnAβ) and protein phosphatase type-2A (PP2A) were absent (Fig C). Although expression levels of PKCδ were 50% lower in cAF lysates, attachment of PKCδ to NCX1 was 875% higher in cAF vs. Ctl (Fig D). The positive interaction between NCX1 and PKCδ in the plasma membrane of human atrial cardiomyocytes was shown using Duolink (red dots in Fig E), but was absent in fibroblasts. Conclusions: We identified PKCα, PKCδ, PP1α, PLM and AMPKα as part of the human atrial NCX1 protein complex. Increased membranous NCX1 protein and a stronger PKCδ-mediated NCX1 phosphorylation (Fig F) may underlie the proarrhythmic NCX dysfunction in cAF.