Impaired local regulation of ryanodine receptor type-2 by protein phosphatase 1 promotes atrial fibrillation

Abstract

Purpose: Atrial fibrillation (AF) is the most common cardiac arrhythmia with a growing prevalence yet current pharmaceutical treatments are ineffective. Previous studies suggest that protein phosphatase 1 (PP1) is dysregulated globally in AF patients but its local regulation on key Ca2+-handling proteins such as ryanodine receptor type-2 (RyR2) is not known. In this study we hypothesize that decreased PP1 regulation of RyR2 promotes AF through increasing Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2. Methods: To study the local regulation of RyR2 by PP1, we used spinophilin knockout mice (KO), since PP1 targets RyR2 via spinophilin. The interaction between RyR2 and PP1 was measured using co-immunoprecipitation while RyR2 phosphorylation level was quantified using Western blot. RyR2 activity was determined by measuring 1) single RyR2 channel activity in a lipid bilayer system and 2) Ca2+ sparks frequency in atrial myocytes loaded with Fluo4-AM. Baseline cardiac characteristics were determined using echocardiography and histological staining while AF inducibility was assessed by intracardiac electrophysiology study. Results: The interaction of PP1 with RyR2 was reduced by 55% in the KO hearts compared to WT (P<0.001). RyR2 phosphorylation at Serine 2814 was increased by 43% in the KO atria (P<0.05 vs. WT) while unchanged at Serine 2808. Single RyR2 channels isolated from KO hearts had a higher open probability (0.10±0.02) compared to that from WT (0.02±0.01; P<0.01). Ca2+ spark frequency normalized to sarcoplasmic reticulum (SR) Ca2+ content was increased in atrial myocytes isolated from KO mice (1.72±0.26 a.u.) compared to WT (0.77±0.20 a.u.; P<0.05). Furthermore, this increase was rescued by CaMKII-inhibitor KN93 but not by protein kinase A (PKA) inhibitor H89. End diastolic diameter of hearts from KO mice was 10% smaller than that from WT (P<0.01) but both the heart weight to body weight ratio and ejection fraction were unchanged. Also, there was no difference in fibrosis between KO and WT heart based on histological sections stained with Masson's trichome. However, when subjected to intracardiac electrical stimulation, KO mice had a higher incidence of pacing-induced AF (100%) compared to WT (33%; P<0.01). Conclusions: PP1 regulates RyR2 by counteracting CaMKII but not PKA phosphorylation of RyR2. Spinophilin deficiency leads to decreased PP1 regulation of RyR2 which may promote AF. This regulation may represent a novel therapeutic target for treating AF.