-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.

Rationale: Post-operative atrial fibrillation (POAF) is a common and troublesome complication of cardiac surgery. POAF is generally believed to occur when post-operative triggers act on a pre-existing 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 while spontaneous sarcoplasmic-reticulum (SR) Ca 2+ -release events and L-type Ca 2+ -current alternans occurred more frequently. Ca 2+ /calmodulin-dependent protein kinase-II (CaMKII) protein-expression, CaMKII-dependent phosphorylation of the cardiac ryanodine-receptor channel type-2 (RyR2) 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. POAF patients also showed stronger expression of activated components of the NLRP3-inflammasome system in atrial whole-tissue homogenates and cardiomyocytes. Acute application of interleukin-1beta caused NLRP3-signaling activation and CaMKII-dependent RyR2/phospholamban hyperphosphorylation in 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-1beta. Conclusions: Pre-existing 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.

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Background: Inflammation is a known risk factor of atrial fibrillation (AF), the most common arrhythmia; however, the mechanistic link between the inflammatory signaling and the pathophysiology of AF has not been established. ‘NACHT, LRR and PYD domains-containing protein 3’ (NLRP3) inflammasome is a signaling platform that is responsible for the activation of caspase-1 and interleukin (IL)-1b release. The activity of NLRP3 inflammasome is enhanced in atrial tissues of paroxysmal or long-lasting persistent AF patients. Thus, we tested the hypothesis that activation of NLRP3 inflammasome promotes the development of AF. Results: To elucidate the role of NLRP3 inflammasome in cardiomyocytes (CMs) and AF development, a CM-specific knockin murine model expressing a constitutive active NLRP3 (aMHC:NLRP3 A350V/+ , CKI) was developed. At 3-month old, telemetry recordings showed that 100% of CKΙ mice (n=5) developed premature atrial contractions (PACs), whereas only 25% of control mice (n=4, P<0.05) had PACs. Rapid atrial pacing induced AF in 89% of CKI mice (n=9), a much higher incidence than control mice (20%, n=5, P<0.05). Level of the active caspase-1 was increased in atrial tissues of 3-month old CKI mice, prior to a detectable increase in the level of macrophage marker at 7-month old, suggesting that the onset of PACs and AF vulnerability is not associated with the activated macrophages. Compared to the control mice, 3-month old CKI mice exhibited atrial hypertrophy, abnormal Ca 2+ release via RyR2, and shortening of atrial effective refractory period, which were associated with the upregulation of Mef2c , Ryr2 , Kcna5 (encoding Kv1.5), Kcnj 3 (encoding Kir3.1) and Kcnj5 (encoding Kir3.4) mRNA. Lastly, inflammasome inhibitor MCC950 (i.p., 10mg/kg, 10 days) reduced the AF inducibility in CKI mice (0%, n=3, P<0.05 vs vehicle-treated CKI). Conclusion: Activation of NLRP3 inflammasome promotes structural and electrical remodeling, permissive to the AF development. In addition to its canonical function, NLRP3 inflammasome may exhibit alternative function in regulating gene transcription. Our study establishes a mechanistic link between the inflammatory signaling and the pathogenesis of AF, and the inhibition of NLRP3 may become a novel anti-AF therapy.

Post-operative atrial fibrillation (poAF) is the most common perioperative arrhythmia associated with increased hospital morbidity and mortality. It is known that atrial myocyte Ca 2+ handling changes can play a crucial role in AF pathogenesis, although the specific mechanisms involved in poAF have not been elucidated. Our lab has previously identified Striated Muscle Preferentially Expressed Protein Kinase (SPEG) as a binding partner of the major cardiac Ca 2+ release channel RyR2 and its negative regulator JPH2, which are critical for cardiac Ca 2+ homeostasis. We hypothesized that decreased SPEG levels may create an arrhythmogenic substrate promoting poAF development. SPEG protein and mRNA levels were assessed using Western blotting and qRT-PCR, respectively. To establish a mouse model of poAF, we performed thoracotomy in C57Bl/6J mice followed by rapid atrial pacing three days post-operatively to assess for poAF susceptibility. To test for a cause-effect relationship between decreased SPEG and AF susceptibility, we used a cardiac specific tamoxifen-inducible SPEG conditional knockout (cKO) mouse. Rapid atrial pacing was performed in SPEG cKO mice two weeks post-tamoxifen injection prior to onset of heart failure as assessed by echocardiography. In human atrial samples from patients with poAF we found 41±9% lower SPEGβ mRNA expression (n=7, p=0.04) compared to patients with normal sinus rhythm (NSR, n=8), while SPEGα expression was unchanged. Similarly, SPEGβ protein was 45±7% lower in poAF vs NSR patients (n=6, p=0.04). In contrast, SPEGβ mRNA expression was not decreased in patients with persistent or paroxysmal AF. Similar to poAF patients, our novel mouse model exhibited 90±4% lower SPEGβ protein levels (n=4, p=0.03) compared to mice with NSR (n=4) after rapid atrial pacing three days post-thoracotomy. Furthermore, SPEG cKO mice had increased AF duration after rapid atrial pacing (19.43 ± 15.16 s, n=3) compared to controls (0.27 ± 0.27 s, n=6, p=0.01), suggesting that loss of SPEG is sufficient to drive AF. In conclusion, we discovered reduced SPEGβ mRNA and protein in both humans and mice with poAF and established that a loss of SPEG might causally contribute to poAF pathogenesis.

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