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.

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 ≈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 I_NCX 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.

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.