NLRP3 inflammasome-mediated pyroptosis contributes to the pathogenesis of non-ischemic dilated cardiomyopathy

Abstract

Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure, and the underlying mechanism remains largely elusive. Here we investigated whether NLRP3 inflammasome-mediated pyroptosis contributes to non-ischemic DCM and dissected the underlying mechanism. We found that hyper activated NLRP3 inflammasome with pyroptotic cell death of cardiomyocytes were presented in the myocardial tissues of DCM patients, which were negatively correlated with cardiac function. Doxorubicin (Dox)-induced DCM characterization disclosed that NLRP3 inflammasome activation and pyroptosis occurred in Dox-treated heart tissues, but were very marginal in either NLRP3^-/- or caspase-1^-/- mice. Mechanistically, Dox enhanced expressions of NOX1 and NOX4 and induced mitochondrial fission through dynamin-related protein 1 (Drp1) activation, leading to NLRP3 inflammasome-mediated pyroptosis in cardiomyocytes via caspase-1-dependent manner. Conversely, both inhibitions of NOX1 and NOX4 and Drp1 suppressed Dox-induced NLPR3 inflammasome activation and pyroptosis. The alterations of NOX1 and NOX4 expression, Drp1 phosphorylation and mitochondrial fission were validated in DCM patients and mice. Importantly, Dox-induced Drp1-mediated mitochondrial fission and the consequent NLRP3 inflammasome activation and pyroptosis were reversed by NOX1 and NOX4 inhibition in mice. This study demonstrates for the first time that cardiomyocyte pyroptosis triggered by NLRP3 inflammasome activation via caspase-1 causally contributes to myocardial dysfunction progression and DCM pathogenesis.

Keywords: Dilated cardiomyopathy; Heart failure; Mitochondrial fission; NLRP3 inflammasome; NOX; Pyroptosis.

Graphical abstract

Fig. 1

NLRP3 inflammasomes are activated in heart tissues from non-ischemic DCM patients. (A) The enrichment results of inflammatory response, TLR and NLR signature genes in the myocardial tissues of DCM patients (data from GEO data-set GSE84796 and GSE111544) (n = 31). (B) Heatmap showing the expression of NLRP3 inflammasome components, IL-1β, IL-18, NOX1, NOX2 and NOX4 in myocardial tissues of cDCM patients (n = 10), iDCM patients (n = 14) and normal controls (n = 7) (data from GEO data-set GSE84796 and GSE111544). ^# indicated that control vs iDCM, p > 0.05. (C) HE and Masson's trichrome staining of heart tissues. (D) Immunofluorescent staining showing ASC specks formation in cardiomyocytes. (E) Representative immunoblots of the indicated proteins in myocardial tissues and the corresponding quantification (n = 9). (F) Plasma levels of IL-1β and IL-18 (n = 9). Casp-1: caspase-1. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs control group.

Fig. 2

Pyroptosis is occurred in heart tissues from non-ischemic DCM patients. (A) Representative immunoblots and the corresponding quantification of the cleavage status of GSDMD (n = 9). (B) Triple-immunostaining of active caspase-1, TUNEL and α-actinin. Red arrows: pyroptotic cardiomyocytes (active caspase-1⁺/TUNEL⁺/α-actinin⁺ cells), white arrow: pyroptotic non-cardiomyocytes (active caspase-1⁺/TUNEL⁺/α-actinin- cells), blue arrows: apoptotic cardiomyocytes (active caspase-1-/TUNEL⁺/α-actinin⁺ cells). (C) The ratio of pyroptotic cells to apoptotic cells in all types of cells or in cardiomyocytes (n = 9) and (D) the ratio of cardiomyocytes to non-cardiomyocytes in all pyroptotic cells or apoptotic cells in DCM heart tissues (n = 9). (E) Correlationship of EF with TUNEL positive, pyroptotic and apoptotic cardiomyocytes in DCM patients (n = 9). **p < 0.01 vs control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

NOX1 and NOX4 are associated with the cleavage of caspase-1 and GSDMD in non-ischemic DCM patients. (A) Representative immunoblots and the corresponding quantification of NOX1, NOX2 and NOX4 (n = 9). (B) Immunoblots of the indicated proteins in myocardial tissues in all nine DCM patients (n = 9). The relative expression level of each protein are quantified and shown under each blots. (C–D) Correlationship of the cleaved caspase-1 (C) or GSDMD-NT (D) with NOX1 and NOX4 in myocardial tissues of DCM patients (n = 9). Casp-1: caspase-1. *p < 0.05 and **p < 0.01 vs control group.

Fig. 4

NLRP3 inflammasome-mediated pyroptosis via caspase-1 contributes to Dox-induced DCM. (A) Representative echocardiographic (Echo), HE and Masson's trichrome staining images. (B) Cardiac function index (n = 6). (C) Representative immunoblots and the corresponding quantification of the indicated proteins in myocardial tissues (n = 6). Casp-1: caspase-1. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig. 5

Dual inhibition of NOX1 and NOX4 attenuates NLRP3 inflammasome-mediated pyroptosis in DCM. (A) Representative echocardiographic (Echo), HE and Masson's trichrome staining images. (B) Cardiac function index (n = 6–8). (C) Representative immunoblots and the corresponding quantification of the indicated proteins (n = 6–8). (D) Immunofluorescent staining showing ASC specks formation in cardiomyocytes. Casp-1: caspase-1. *p < 0.05, **p < 0.01, ***p < 0.001 and ****P < 0.0001.

Fig. 6

NOX1 and NOX4 contribute to NLRP3 inflammasome activation through Drp1-mediated mitochondrial fission. (A, C and G) Representative flow cytometric image and quantitative analysis of intracellular ROS levels (A) or mitochondrial ROS levels (C and G) (n = 3). (B, D, F and H) Representative immunoblots and the corresponding quantification of the indicated proteins in H9c2 cells (n = 3). (E) Representative confocal microscopic images of MitoTracker and Drp1. Casp-1: caspase-1. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig. 7

NOX1 and NOX4 facilitate Drp1 activation and mitochondrial fission in DCM. (A and C) Representative immunoblots and the corresponding quantification of the expression and phosphorylation of Drp1 in Dox-induced DCM mice (n = 6–8) (A) and human DCM patients (n = 9) (C). (B) Mouse myocardial TEM images and quantitative analysis of mitochondrial length, width, and the ratio of length to width (n = 6–8). (D) Myocardial TEM images showing mitochondrial morphology of DCM patients. Note: besides the morphology of small spheroid mitochondria (white arrows), disrupted myofibrils, dispersed and disorganized mitochondria with abnormal internal membrane whorls and cristae lysis were also observed. *p < 0.05, **p < 0.01 and ****p < 0.0001.

Fig. 8

Targeting NOX1 and NOX4 or Drp1 reduced cardiomyocyte pyroptosis in primary NRVCs. (A) Representative flow cytometric dot plots and (B) the corresponding quantification showing active caspase-1⁺/7-AAD⁺ cells as pyroptosis population (n = 3). (C) LDH levels in supernatant (n = 3). (D) Representative immunoblots and the corresponding quantification of the cleavage status of caspase-1, IL-18 and GSDMD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.