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 ²⁺ leak. Calmodulin expression (60%), Ca ²⁺ /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.

Background: Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The NLRP3 (NACHT, LRR, and PYD domain containing protein 3) inflammasome mediates caspase-1 activation and interleukin-1β release in immune cells but is not known to play a role in cardiomyocytes (CMs). Here, we assessed the role of CM NLRP3 inflammasome in AF. Methods: NLRP3 inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal AF or long-standing persistent (chronic) AF. To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knockin mouse model expressing constitutively active NLRP3 (CM-KI) was established. In vivo electrophysiology was used to assess atrial arrhythmia vulnerability. To evaluate the mechanism of AF, electric activation pattern, Ca ²⁺ spark frequency, atrial effective refractory period, and morphology of atria were evaluated in CM-KI mice and wild-type littermates. Results: NLRP3 inflammasome activity was increased in the atrial CMs of patients with paroxysmal AF and chronic AF. CM-KI mice developed spontaneous premature atrial contractions and inducible AF, which was attenuated by a specific NLRP3 inflammasome inhibitor, MCC950. CM-KI mice exhibited ectopic activity, abnormal sarcoplasmic reticulum Ca ²⁺ release, atrial effective refractory period shortening, and atrial hypertrophy. Adeno-associated virus subtype-9–mediated CM-specific knockdown of Nlrp3 suppressed AF development in CM-KI mice. Finally, genetic inhibition of Nlrp3 prevented AF development in CREM transgenic mice, a well-characterized mouse model of spontaneous AF. Conclusions: Our study establishes a novel pathophysiological role for CM NLRP3 inflammasome signaling, with a mechanistic link to the pathogenesis of AF, and establishes the inhibition of NLRP3 as a potential novel AF therapy approach.

Rationale: Postoperative atrial fibrillation (POAF) is a common and troublesome complication of cardiac surgery. POAF is generally believed to occur when postoperative triggers act on a preexisting vulnerable substrate, but the underlying cellular and molecular mechanisms are largely unknown. Objective: To identify cellular POAF mechanisms in right atrial samples from patients without a history of atrial fibrillation undergoing open-heart surgery. Methods and Results: Multicellular action potentials, membrane ion-currents (perforated patch-clamp), or simultaneous membrane-current (ruptured patch-clamp) and [Ca ²⁺ ] _i -recordings in atrial cardiomyocytes, along with protein-expression levels in tissue homogenates or cardiomyocytes, were assessed in 265 atrial samples from patients without or with POAF. No indices of electrical, profibrotic, or connexin remodeling were noted in POAF, but Ca ²⁺ -transient amplitude was smaller, although spontaneous sarcoplasmic reticulum (SR) Ca ²⁺ -release events and L-type Ca ²⁺ -current alternans occurred more frequently. CaMKII (Ca ²⁺ /calmodulin-dependent protein kinase-II) protein-expression, CaMKII-dependent phosphorylation of the cardiac RyR2 (ryanodine-receptor channel type-2), and RyR2 single-channel open-probability were significantly increased in POAF. SR Ca ²⁺ -content was unchanged in POAF despite greater SR Ca ²⁺ -leak, with a trend towards increased SR Ca ²⁺ -ATPase activity. Patients with POAF also showed stronger expression of activated components of the NLRP3 (NACHT, LRR, and PYD domains-containing protein-3)-inflammasome system in atrial whole-tissue homogenates and cardiomyocytes. Acute application of interleukin-1β caused NLRP3-signaling activation and CaMKII-dependent RyR2/phospholamban hyperphosphorylation in an immortalized mouse atrial cardiomyocyte cell-line (HL-1-cardiomyocytes) and enhanced spontaneous SR Ca ²⁺ -release events in both POAF cardiomyocytes and HL-1-cardiomyocytes. Computational modeling showed that RyR2 dysfunction and increased SR Ca ²⁺ -uptake are sufficient to reproduce the Ca ²⁺ -handling phenotype and indicated an increased risk of proarrhythmic delayed afterdepolarizations in POAF subjects in response to interleukin-1β. Conclusions: Preexisting Ca ²⁺ -handling abnormalities and activation of NLRP3-inflammasome/CaMKII signaling are evident in atrial cardiomyocytes from patients who subsequently develop POAF. These molecular substrates sensitize cardiomyocytes to spontaneous Ca ²⁺ -releases and arrhythmogenic afterdepolarizations, particularly upon exposure to inflammatory mediators. Our data reveal a potential cellular and molecular substrate for this important clinical problem.

Atrial fibrillation (AF) and heart failure (HF) are common cardiovascular diseases that often co-exist. Animal models have suggested complex AF-promoting atrial structural, electrical, and Ca²⁺-handling remodeling in the setting of HF, but data in human samples are scarce, particularly regarding Ca²⁺-handling remodeling. Here, we evaluated atrial remodeling in patients with severe left ventricular (LV) dysfunction (HFrEF), long-standing persistent (‘chronic’) AF (cAF) or both (HFrEF-cAF), and sinus rhythm controls with normal LV function (Ctl) using western blot in right-atrial tissue, sharp-electrode action potential (AP) measurements in atrial trabeculae and voltage-clamp experiments in isolated right-atrial cardiomyocytes. Compared to Ctl, expression of profibrotic markers (collagen-1a, fibronectin, periostin) was higher in HFrEF and HFrEF-cAF patients, indicative of structural remodeling. Connexin-43 expression was reduced in HFrEF patients, but not HFrEF-cAF patients. AP characteristics were unchanged in HFrEF, but showed classical indices of electrical remodeling in cAF and HFrEF-cAF (prolonged AP duration at 20% and shorter AP duration at 50% and 90% repolarization). L-type Ca²⁺ current (I_Ca,L) was significantly reduced in HFrEF, cAF and HFrEF-cAF, without changes in voltage-dependence. Potentially proarrhythmic spontaneous transient-inward currents were significantly more frequent in HFrEF and HFrEF-cAF compared to Ctl, likely resulting from increased sarcoplasmic reticulum (SR) Ca²⁺ load (integrated caffeine-induced current) in HFrEF and increased ryanodine-receptor (RyR2) single-channel open probability in HFrEF and HFrEF-cAF. Although expression and phosphorylation of the SR Ca²⁺-ATPase type-2a (SERCA2a) regulator phospholamban were unchanged in HFrEF and HFrEF-cAF patients, protein levels of SERCA2a were increased in HFrEF-cAF and sarcolipin expression was decreased in both HFrEF and HFrEF-cAF, likely increasing SR Ca²⁺ uptake and load. RyR2 protein levels were decreased in HFrEF and HFrEF-cAF patients, but junctin levels were higher in HFrEF and relative Ser2814-RyR2 phosphorylation levels were increased in HFrEF-cAF, both potentially contributing to the greater RyR2 open probability. These novel insights into the molecular substrate for atrial arrhythmias in HF-patients position Ca²⁺-handling abnormalities as a likely trigger of AF in HF patients, which subsequently produces electrical remodeling that promotes the maintenance of the arrhythmia. Our new findings may have important implications for the development of novel treatment options for AF in the context of HF.

Background: Enhanced diastolic calcium (Ca ²⁺ ) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca ²⁺ leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca ²⁺ /calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. Methods: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca ²⁺ leak in atrial cardiomyocytes was assessed using confocal Ca ²⁺ imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9. Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca ²⁺ spark frequency in atrial cardiomyocytes with Ca ²⁺ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca ²⁺ leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca ²⁺ release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.

Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein βγ dimer (Gβγ) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in Gβ ₁ γ ₂ dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Gα _s and Gα _i subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish Gβ ₁ orthologs, Gβ ₁ and Gβ _1like , causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of Gβ ₁ /Gβ _1like is associated with a down-regulation in caveolins, AC-regulating Gα-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with Gβγ dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.