Abstract 3501: Calmodulin Kinase II Activation of Mutant Ryanodine Channels Promotes Ectopic Activity and Atrial Fibrillation

Abstract

Some individuals with mutations in the cardiac ryanodine receptor gene (RyR2) develop stress-induced atrial fibrillation (AF), but the underlying mechanisms are unknown. This study explored the molecular basis of AF susceptibility in mice heterozygous for the human arrhythmia-linked RyR2 mutation R176Q (RQ mice). AF inducibility was assessed with atrial burst pacing in WT (n=34), RQ mice (n=43) and RQ mice crossed with transgenic mice expressing the CaMKII-inhibitory peptide AC3I (RQxAC3I, n=6) or the inactive analog AC3C (RQxAC3C, n=9). Dual voltage (RH237) and calcium (Rhod-2 AM) optical mapping was applied to monitor simultaneous Ca2+ and membrane-voltage changes. Ca2+ fluorescence (Fluo-4 AM) was used to study SR Ca2+ release. Expression/phosphorylation of CaMKII and RyR2 were determined by immunostaining in mice and human atrial biopsies from sinus rhythm (SR) (n=7) and chronic AF (n=7) patients. Atrial burst pacing-induced AF was more common in RQ (55%) than in WT mice (11%*; *P<0.05) and was associated with ~50% higher CaMKII-dependent RyR2 phopshorylation (Ser2814 site). Expression and phosphorylation (Thr287) of CaMKIId were increased in AF patients by 63%* and 40%*, respectively, and CaMKII-phopshorylation of RyR2 at Ser2814 was 80%* higher in AF than in SR patients. Genetic inhibition of CaMKII reduced AF incidence in RQxAC3I (16%*), but not in RQxAC3C mice (62%). Optical mapping revealed more atrial burst pacing-induced ectopic beats (and re-entry) in RQ than WT atria (296 vs.19* ectopic beats, 120 ms cycle length) that were depressed by CaMKII inhibiton with KN-93 (1 μM). [Ca2+]i release by 10 mM local caffeine puffs (an index of SR Ca2+ content) were similar in RQ and WT mice (4.59±0.42 vs 4.04±0.41 [a.u.], respectively), whereas SR Ca2+ leak defined as tetracaine (5 μM)-sensitive decrease in diastolic [Ca2+]i was higher in RQ than in WT mice (% of SR Ca2+ content: 10.2±0.9 vs 7.7±0.8*). CaMKII inhibition with KN-93 suppressed SR Ca2+ leak. High rate-associated CaMKII activation promotes AF inducibility in mice with a stress-induced arrhythmia-linked R176Q mutation in RyR2. Inhibition of CaMKII may be a viable drug target for effective AF therapy. This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).