Enhanced Cardiomyocyte NLRP3 Inflammasome Signaling Promotes Atrial Fibrillation

Abstract

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, Ca²⁺ 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 Ca²⁺ 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.

Keywords: AAV9; NLRP3 inflammasome; atrial fibrillation; electrical remodeling.

Figure 1. Enhanced activation of NLRP3-inflammatory signaling pathway in AF patients, dogs subjected to atrial tachycardia pacing (ATP), and CREM-TG mice with spontaneous AF

Representative Western blots (WBs) and quantification of NLRP3, ASC, and Casp1-p20 in atrial tissue of patients with paroxysmal AF (pAF, A–B) and chronic AF (cAF, C–D). Representative WBs (E) and quantification (F) of NLRP3, ASC, and p20 in atrial tissue of ATP and control (Ctl) dogs. Representative WBs (G) and quantification (H) of NLRP3, ASC, and Casp1-p20 in atrial tissues of CREM-TG mice and WT littermates. *P<0.05, **P<0.01.

Figure 2. Enhanced activation of NLRP3-inflammatory signaling pathway in cardiomyocytes (CMs) of AF patients

(A) Validation of atrial CM-enriched cell pellets isolated from human atrial biopsies by detecting a CM marker - calsequestrin (CSQ) - using Western blotting (top). Tissue lysate (Lys) was used as positive control and cardiac fibroblasts (CFs) as negative control. Validation of absence of immune cells including macrophages (MFs) in human CMs and CFs preparations by detecting the specific MF/eosinophilic granulocyte marker F4/80 (bottom). Differentiated bone marrow-derived mouse MFs served as a positive control. (B–E) Representative WBs and quantification of NLRP3, ASC, and Casp1-p20 in atrial CMs of patients with paroxysmal AF (pAF, B–C) or chronic AF (cAF, D–E). Representative WBs and quantification of NLRP3, ASC, and Casp1-p20 in atrial CMs of ATP dogs (F–G). *P<0.05, **P<0.01.

Figure 3. Constitutive activation of NLRP3 in cardiomyocytes (CMs) predisposes mice to atrial arrhythmias

(A–B) Representative WBs and quantification of NLRP3, ASC, and Caspase-1 in atrial tissue of CM-KI and Ctl mice. (C) Representative telemetry ECG recording demonstrated sinus rhythm in Ctl mice and premature atrial contractions (PACs, indicated by arrows) in CM-KI mice. (D) Box plot summarizing the average events of PACs with whiskers indicating the minimum and maximum values in Ctl and CM-KI mice. (E) Representative simultaneous recordings of surface ECG (lead 1) and intracardiac electrograms in CM-KI and Ctl mice following programmed intracardiac stimulation (indicated by arrows). (F) Incidence of pacing-induced AF in Ctl mice (combining WT and Myh6^Cre) and CM-KI mice. (G) Representative WBs and quantification of CD68 (macrophage marker) and IL-1β in atrial tissue of CM-KI and Ctl mice. *P<0.05.

Figure 4. Constitutive activation of NLRP3 in cardiomyocytes (CMs) promotes electrical remodeling associated with AF development

(A–B) Representative activation maps revealed an early ectopic activation (indicated by asterisk) corresponding to premature atrial contraction (P′-wave on ECG, B) in the right atrium of a CM-KI mouse, at a location different from sinus rhythm origin corresponding to the normal P-wave in a Ctl mouse (A). (C) Incidence of ectopic activity. (D) Representative recordings of Ca²⁺ transients. (E–F) Quantification of fractional release and decay (time constant, Tau) in atrial CMs of Ctl and CM-KI mice. (G) Representative line-scan confocal images and the quantification of Ca²⁺ sparks (CaSF) normalized to SR Ca²⁺ load in CMs of Ctl and CM-KI mice. (H) Representative WBs and quantification of total whole-tissue RyR2 and the phosphorylated RyR2-pS2808 or RyR2-pS2814 relative to RyR2. (I) Quantification of AERP in Ctl and CM-KI mice. (J) Representative recordings of I_Kur in atrial CMs of Ctl and CM-KI mice (in the presence and absence of the selective I_Kur inhibitor DPO-1, 0.5 μmol/L), respectively. (K) Quantification of I_Kur density. *P<0.05, **P<0.01.

Figure 5. Constitutive activation of NLRP3 in cardiomyocytes (CMs) promotes structural remodeling associated with AF development

(A) Whole mount images of hearts and (B) quantification of atrial weight to tibia length (AW/TL) ratio in Ctl and CM-KI mice. (C) qPCR revealed increased levels of hypertrophic marker Mef2c, but not Rcan1 in CM-KI mice compared to Ctl mice. (D) Masson’s trichrome staining and (E) quantification of atrial fibrosis (blue) in CM-KI and Ctl mice. (F) qPCR revealed elevated levels of Col1a (encoding collagen-1a) and Lgals3 (encoding galectin-3) mRNA in the atria of CM-KI compared to Ctl mice. *P<0.05, **P<0.01.

Figure 6. Cardiomyocyte (CM)-specific knockdown of Nlrp3 reduces AF-inducibility

(A) Design of AAV9 vector to achieve the CM-specific shRNA-mediated Nlrp3 knockdown after injecting CM-KI mice. (B) qPCR confirmed the Nlrp3 knockdown efficacy by AAV9-shNlrp3 in CM-KI atria. (C) qPCR demonstrated that knockdown of Nlrp3 by AAV9-shNlrp3 reduced the levels of Ryr2, Kcna5, Girk1, and Girk4 mRNA in CM-KI mice. (D) Representative simultaneous recordings of surface ECG (lead 1) and intracardiac electrograms in CM-KI treated with either AAV9-scramble or AAV9-shNlrp3. (E) Incidence of pacing-induced AF in CM-KI mice treated with either AAV9-scramble or AAV9-shNlrp3. *P<0.05, **P<0.01.

Figure 7. Genetic ablation of Nlrp3 prevents the development of spontaneous AF

Representative WBs (A) and quantification (B) of caspase-1 in atrial tissues of WT, CREM-TG and CREM-TG:NLRP3^−/− (DM) mice at the age of 7 months. Representative telemetry ECG recordings (C) and incidence of spontaneous AF (D) in WT, CREM-TG and DM mice (# indicated breathing artifact). Representative B-mode echocardiography recordings (E) and quantification (F) of left-atrial (LA) area in WT, CREM-TG and DM mice. *P<0.05, **P<0.01. (G) Working model of AF-promoting mechanisms resulting from CM-restricted NLRP3-inflammasome activation. Solid lines indicate proven mechanisms, and dash lines indicate putative mechanisms that need further investigation. DADs, delayed afterdepolarizations; DAMPs, danger-associated molecular patterns; ERP, effective refractory period; MΦ1, M1 macrophage; TNFα, tumor necrosis factor alpha.