-Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The "NACHT, LRR and PYD domain containing protein 3" (NLRP3)-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. -NLRP3-inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal (pAF) or long-standing persistent (chronic) AF (cAF). To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knock-in 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, electrical activation pattern, Ca spark frequency (CaSF), atrial effective refractory period (AERP), and morphology of atria were evaluated in CM-KI mice and WT littermates. -NLRP3-inflammasome activity was increased in atrial CMs of pAF and cAF patients. 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-release, AERP shortening and atrial hypertrophy. Adeno-associated virus subtype-9 mediated CM-specific knockdown of suppressed AF development in CM-KI mice. Finally, genetic inhibition of prevented AF development in CREM transgenic mice, a well-characterized mouse model of spontaneous AF. -Our study establishes a novel pathophysiological role for CM NLRP3-inflammasome signaling with a mechanistic link to the pathogenesis of AF, and establishes inhibition of NLRP3 as a potential novel AF-therapy approach.

Diastolic calcium (Ca) leak via cardiac ryanodine receptors (RyR2) can cause arrhythmias and heart failure (HF). Ca/calmodulin (CaM)-dependent kinase II (CaMKII) is upregulated and more active in HF, promoting RyR2-mediated Ca leak by RyR2-Ser2814 phosphorylation. Here, we tested a mechanistic hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by promoting a pathological RyR2 conformation with reduced CaM affinity. Acute CaMKII activation in wild-type RyR2, and phosphomimetic RyR2-S2814D (vs. non-phosphorylatable RyR2-S2814A) knock-in mouse myocytes increased SR Ca leak, reduced CaM-RyR2 affinity, and caused a pathological shift in RyR2 conformation (detected via increased access of the RyR2 structural peptide DPc10). This same trio of effects was seen in myocytes from rabbits with pressure/ volume-overload induced HF. Excess CaM quieted leak and restored control conformation, consistent with negative allosteric coupling between CaM affinity and DPc10 accessible conformation. Dantrolene (DAN) also restored CaM affinity, reduced DPc10 access, and suppressed RyR2-mediated Ca leak and ventricular tachycardia in RyR2-S2814D mice. We propose that a common pathological RyR2 conformational state (low CaM affinity, high DPc10 access, and elevated leak) may be caused by CaMKII-dependent phosphorylation, oxidation, and HF. Moreover, DAN (or excess CaM) can shift this pathological gating state back to the normal physiological conformation, a potentially important therapeutic approach.

Background Enhanced sarcoplasmic reticulum (SR) Ca2+-leak via ryanodine receptor type-2 (RyR2) contributes to the pathogenesis of atrial fibrillation (AF). Recent studies have shown that the level of RyR2 protein is elevated in atria of paroxysmal AF (pAF) patients, suggesting that microRNA-mediated post-transcriptional regulation of RyR2 might be an underlying mechanism. Bioinformatic analysis suggests that miR-106b and miR-93, members of the miR-106b-25 cluster, could bind to RYR2-3′UTR and suppress its translation. Thus, we tested the hypothesis that loss of the miR-106b-25 cluster promotes AF via enhanced RyR2-mediated SR Ca2+-leak. Methods and Results Quantitative real-time PCR showed that the levels of mature miR-106b, miR-93 and miR-25 were lower in atria of pAF patients compared with patients with sinus rhythm. In vitro assay showed that miR-93 reduced RYR2-3′UTR luciferase activity. Total RyR2 protein in atrial tissue of miR-106b-25−/− mice was increased by 42% compared to wild-type (WT) littermates, but still maintained a normal subcellular distribution. Ca2+-spark frequency and total SR Ca2+-leak were increased in atrial myocytes of miR-106b-25−/− mice. Telemetry ECG recordings revealed that miR-106b-25−/− mice exhibited more frequent atrial ectopy and were also more susceptible to pacing-induced AF than WT littermates. Increased SR Ca2+-release and AF susceptibility in miR-106b-25−/− mice were abolished by the RyR2-blocker K201. Conclusions These results suggest that miR-106b-25 cluster mediated post-transcriptional regulation of RyR2 is a potential molecular mechanism involved in pAF pathogenesis. As such, the miR-106b-25 cluster could be a novel gene-therapy target in AF associated with enhanced RyR2 expression.

PP1 regulates RyR2 locally by counteracting CaMKII phosphorylation of RyR2. Decreased local PP1 regulation of RyR2 contributes to RyR2 hyperactivity and promotes AF susceptibility. This represents a novel mechanism for subcellular modulation of calcium channels and may represent a potential drug target of AF.

Background: Abnormal calcium (Ca 2+ ) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca 2+ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN. Methods: Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln -knockout mice. Ppp1r3a -knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a -knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca 2+ imaging. Results: Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca 2+ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF. Conclusions: PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca 2+ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.