Enhanced Sarcoplasmic Reticulum Ca2+ Leak and Increased Na+-Ca2+ Exchanger Function Underlie Delayed Afterdepolarizations in Patients With Chronic Atrial Fibrillation

Background-Delayed afterdepolarizations (DADs) carried by Na+-Ca2+-exchange current (I-NCX) in response to sarcoplasmic reticulum (SR) Ca2+ leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined. Methods and Results-Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca2+](i) (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca2+](i) and SR Ca2+ content (integrated I-NCX during caffeine-induced Ca2+ transient) were unchanged, whereas diastolic SR Ca2+ leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was approximate to 50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca2+ leak. Calmodulin expression (60%), Ca2+/calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca2+ leak, the frequency of spontaneous Ca2+ release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca2+ sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca2+](i) and I-NCX density revealed I-NCX upregulation in cAF. Spontaneous Ca2+ release events accompanied by inward INCX currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca2+](i) (diastolic [Ca2+](i)-voltage coupling gain) was higher in cAF compared with control. Conclusions-Enhanced SR Ca2+ leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I-NCX for a given SR Ca2+ release and increased diastolic [Ca2+](i)-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients. (Circulation. 2012;125:2059-2070.)