Enhanced sarcoplasmic reticulum Ca 2+ Leak and increased Na +-Ca 2+ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation

摘要

Background-Delayed afterdepolarizations (DADs) carried by Na +-Ca 2+-exchange current (I NCX) in response to sarcoplasmic reticulum (SR) Ca 2+ 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 [Ca 2+] i (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca 2+] i and SR Ca 2+ content (integrated I NCX during caffeine-induced Ca 2+ transient) were unchanged, whereas diastolic SR Ca 2+ leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was 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 Ca leak. Calmodulin expression (60%), Ca/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 Ca 2+ leak, the frequency of spontaneous Ca 2+ 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 Ca 2+ sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca 2+] i and I NCX density revealed I NCX upregulation in cAF. Spontaneous Ca 2+ release events accompanied by inward I NCX currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca 2+]i (diastolic [Ca 2+] i-voltage coupling gain) was higher in cAF compared with control. Conclusions-Enhanced SR Ca 2+ leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I NCX for a given SR Ca 2+ release and increased diastolic [Ca 2+] i-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.