Inhibition of the NLRP3/IL‐1β axis protects against sepsis‐induced cardiomyopathy

Figures

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  Figure 1. Nlrp3 KO mice show less cardiac atrophy and a preserved cardiac function during sepsis. 12‐ to 16‐week‐old male Nlrp3 KO and WT mice were subjected to CLP or sham surgery. (A) Heart weight normalized to tibia length and expressed as percent‐wise change compared with the respective sham group 96 h after CLP or sham surgery. CLP‐treated Nlrp3 KO (n = 16); sham Nlrp3 KO (n = 8), CLP Nlrp3 WT (n = 12), sham Nlrp3 WT (n = 13). (B) Mean myocyte cross‐sectional area (MCSA) determined in haematoxylin and eosin‐stained histological cross sections from hearts of CLP‐treated Nlrp3 KO (n = 6), sham Nlrp3 KO (n = 6), CLP Nlrp3 WT (n = 6) and sham Nlrp3 WT (n = 6) mice (n = 100 myocytes per mouse). Data are presented as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001. (C) Results of transthoracic echocardiography of Nlrp3 WT sham (n = 13), Nlrp3 WT CLP (n = 13), Nlrp3 KO sham (n = 18) and Nlrp3 KO CLP (n = 18) mice 24 h after CLP or sham operation. (D) Serum IL‐1β concentration in sham and CLP mice. Nlrp3 WT sham (n = 15), CLP Nlrp3 WT (n = 12), Nlrp3 KO sham (n = 14), Nlrp3 KO CLP (n = 14). Data are presented as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01. qRT‐PCR analysis of Il1b (E) and Il6 (F)…

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  Figure 2. IL‐1β via the IL‐1 receptor reduces contractility, relaxation and deformation of cardiomyocytes. (A–D) Adult rat ventricular cardiomyocytes were treated with vehicle (PBS), recombinant IL‐1β (10 ng/mL) or the IL‐1 receptor antagonist (IL‐1RA) (10 μg/mL), as indicated, and IonOptix measurements were performed. Quantitation of effects on cell shortening (A) and Ca2+ transients (B) are shown. The threshold is defined as a reduction of cell shortening by −7.70% and is displayed by the red line. It equates to the mean effect on cell shortening induced by vehicle minus twice the standard deviation of this effect. (C) Time‐to‐50% relengthening. (D) Change of relaxation speed. Values are expressed as mean percentage change from baseline ± SEM. *P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001. (E–I) Mechanical high‐throughput characterization of HL‐1 cardiomyocytes by RT‐DC was used to investigate the effects of IL‐1β to cell size and deformation. (E) RT‐DC scatter plots of cell size (area, μm2) and deformation of vehicle (PBS, control, n = 2948 cells) and IL‐1β (n = 2554 cells) treated HL‐1 cells. Isoelasticity lines in grey highlight areas of equal elastic Young's modulus. Colour code indicates red…

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  Figure 3. Principal component analysis and hierarchical clustering of RNA sequencing data. (A) Principal component analysis (PCA) performed using DESeq2 rlog‐normalized RNA‐seq data. Loadings for principal components 1 (PC1), PC2 and PC3 are shown in graph on x‐, y‐ and z‐axis. Genotypes and treatments are indicated. (B) Hierarchical clustering analyses performed using DESeq2 rlog‐normalized RNA‐seq data. Colour code (from white to dark blue) refers to the distance metric used for clustering (dark blue corresponds to the maximum of correlation values). Genotypes and treatments are indicated. (C) Venn diagrams showing the number of twofold upregulated (top panel) and twofold downregulated (bottom panel) genes and their overlap in hearts of septic Nlrp3 WT and Nlrp3 KO mice. (D) Selected enrichment scores of GO term analysis of genes, which were upregulated (left panel) or downregulated (right panel) in hearts of septic Nlrp3 KO compared with Nlrp3 WT mice. (E) Heatmaps of normalized expression values of genes involved in the NF‐κB signalling pathway (left panel) and the autophagy and lysosomal pathway of protein degradation (right panel). Genotypes and treatments as well as the z‐score are…

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  Figure 4. Sepsis‐induced expression of autophagy genes is attenuated in hearts of Nlrp3 KO mice. (A) qRT‐PCR analysis of Sqstm1, Map 1LC3b, Ctsl, Atg13 and Bnip3 in hearts of Nlrp3 WT sham (n = 13), Nlrp3 WT CLP (n = 12), Nlrp3 KO sham (n = 8) and Nlrp3 KO CLP (n = 15) mice. mRNA expression was normalized to Gapdh. (B) Western blot analysis with anti‐p62 and anti‐LC3B antibody. Actin was used as loading control. (C) qRT‐PCR analysis of Trim63 and Fbxo32 in hearts of Nlrp3 WT sham, Nlrp3 WT CLP, Nlrp3 KO sham and Nlrp3 KO CLP mice. mRNA expression was normalized to Gapdh. Data are presented as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001.

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  Figure 5. The IL‐1β signalling pathway is contained and active in H9c2 cardiomyocytes. (A) Seven days differentiated H9c2 myotubes were treated with recombinant vehicle, IL‐1β (50 ng/mL) and TNF (5 ng/mL), as indicated, for 25 min. Western blot analysis was performed with anti‐phospho‐NF‐κB p65 and anti‐NF‐κB p65 antibody. GAPDH was used as loading control. (B) Differentiated H9c2 cells were treated with recombinant vehicle, IL‐1β (50 ng/mL) and TNF (5 ng/mL), as indicated, for 15 and 35 min, and NF‐κB‐DNA‐response element complex formation was analysed by EMSA. (C) Differentiated H9c2 cells were treated with recombinant IL‐1β (50 ng/mL), vehicle, the IL‐1 receptor antagonist (IL‐1RA) (10 μg/mL) and the NF‐κB inhibitor BMS‐345541 (25 μM), as indicated. qRT‐PCR analysis of Il6 (left panel) and Nlrp3 (right panel) is shown. mRNA expression was normalized to Gapdh. (D) Differentiated H9c2 cells were treated with solvent or increasing amounts of recombinant IL‐1β (10, 20 and 50 ng/mL) for 72 h. Western blot analysis of isolated proteins with anti‐myosin heavy chain (MyHC)‐slow antibody is shown. GAPDH was used as loading control. (E) Frequency distribution histograms of cell width of vehicle,…

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  Figure 6. Proposed mechanism of septic cardiomyopathy. (Left panel) Sepsis, caused by polymicrobial infection leads to an activation of the NLRP3 inflammasome, resulting in increased conversion of inactive pro‐IL‐1ß in active IL‐1ß, which acts on cardiomyocytes via the IL‐1 receptor complex (IL‐1R1/IL‐1RAcP). Binding of IL‐1 to its receptor causes an activation of NF‐κB, which leads to increased protein degradation by the autophagy–lysosomal pathway (ALP), a decrease in myosin heavy chain content (MyHC), a reduced deformability and a decreased cardiomyocyte contraction and relaxation. This in turn causes septic cardiomyopathy with cardiac atrophy, systolic and diastolic cardiac dysfunction and increased expression of pro‐inflammatory cytokines in the heart. (Right panel) In Nlrp3 knockout mice, sepsis can no longer activate the NLRP3 inflammasome, generation of IL‐1ß is much reduced, and activation of NF‐κB as well as NF‐κB signalling is attenuated, which maintains cardiomyocyte size, deformability, contraction and relaxation. As a result, Nlrp3 knockout mice show a normal cardiac function in sepsis without cardiac atrophy and preserved expression of pro‐inflammatory cytokines. Inhibitors…