Cardiomyocyte NLRP3 signaling in right heart failure is sexually dimorphic via estrogen receptor α

Figures

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  Fig 1:. NLRP3 in RVs from pulmonary artery hypertension (PAH) patients with RV failure (RVF) is activated in a sexually dimorphic manner. (A) Experimental design. (B, C) Heat maps demonstrating RV expression of NLRP3 inflammasome pathway components in human control samples and samples from PAH patients with RVF by RNA-Seq (B) and Proteomics (C). N=13 for control; n=6 for RVF. Data were analyzed from publicly available data in references (35) and (36). (D, E) RNA and proteomic profiles of RV NLPR3 signaling components expression in control and RVF subjects stratified by sex. N=8 for male control, n=1 for male RVF, n=5 for female control, and n=5 for female RVF. (F-H) Correlations of protein abundance of NLRP3 inflammasome downstream mediators caspase-1, IL-1β, and IL-18 with cardiac index (CI) in male and female human PAH RVF samples. (I) Representative images (left) and quantification (right) of colocalization of NLRP3 and its binding partner ASC in RVs from male or female human control samples and samples from PAH patients with RVF. Magnification is 20x. Human RV samples were stained for NLRP3 (green), ASC (red) and DAPI (blue). Co-localization was analyzed in the whole RV sample;…

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  Fig 2:. RV NLRP3 inflammasome activation in rats with monocrotaline (MCT)-induced PH exhibits a male bias that is due to inhibitory effects of 17β-estradiol (E2). (A) Experimental design. RVs were isolated from male, intact or ovariectomized (OVX) female control or MCT rats. Subgroups of MCT-PH males were treated with E2 (75 μg/kg/day by subcutaneous [s.c.] pellets) or NLRP3 inhibitor MCC950 (10 mg/kg by s.c. injection) for 4 weeks (starting at MCT injection). Subgroups of OVX MCT-PH females were treated with E2 (75 μg/kg/day by s.c. pellets) for 4 weeks (starting at MCT injection). (B) Representative immunofluorescence images and quantification of fluorescence intensity of RV NLRP3-ASC colocalization. RVs were stained for NLRP3 (green), ASC (red), and DAPI (blue). Co-localization images were analyzed in ten different fields, and values were then averaged. Images were obtained at 100x magnification. Representative images were digitally zoomed 5x. NLRP3 and ASC co-localization (yellow; arrows) were quantified by determining Pearson correlation coefficient. (C, D) Representative Western blot images and densitometric quantification of caspase-1/pro-caspase-1 ratio (C) and IL-1β/pro-IL-1β…

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  Fig 3:. NLRP3 activation decreases RV isovolumic contractility in a sex-specific and male-biased manner, and this decrease is prevented by E2 in male RVs. (A) Experimental design. (B) Representative immunofluorescence images and quantification of fluorescence intensity of NLRP3-ASC co-localization in RVs from male or female control rat hearts perfused with lipopolysaccharide (LPS; 1 μg/mL, 130 min prior to start of dP/dt max measurement and continued throughout measurement) and ATP (2 mM, 10 min prior to start of dP/dt max measurement and continued throughout measurement) or vehicle, in presence or absence of MCC950 (1 μM, 30 min prior to LPS and continued throughout LPS exposure) or E2 (1 nM, 30 min prior to LPS and continued throughout LPS exposure). Rat RVs were stained for NLRP3 (green), ASC (red), and DAPI (blue). Images were obtained at 100x magnification. Co-localization images were analyzed in ten different fields per animal, and averaged values for each animal are shown. NLRP3 and ASC co-localization (yellow; indicated by arrows) was quantified by determining Pearson correlation coefficient. Representative images were digitally zoomed 5x. (C, D) Maximum rate of rise in RV…

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  Fig 4:. RV cardiomyocyte (RVCM) NLRP3 inflammasome activation and NLRP3-induced RVCM contractile dysfunction in MCT-PH rats are sexually dimorphic, male-biased, and attenuated by 17β-estradiol (E2). (A) Experimental design. (B) Representative immunofluorescence images and quantification of fluorescence intensity of NLRP3-ASC co-localization in RVCMs from male, intact or ovariectomized (OVX) female control or MCT rats. Subgroups of MCT-PH males were treated with NLRP3 inhibitor MCC950 (10 mg/kg by subcutaneous [s.c.] injection) or E2 (75 μg/kg/day by s.c. pellets) for 4 weeks (starting at MCT injection). Subgroups of MCT-PH females were ovariectomized and treated with E2 (75 μg/kg/day by s.c. pellets) for 4 weeks (starting at MCT injection). Rat RVCMs were stained for NLRP3 (green), ASC (red), and DAPI (blue). Images were obtained at 100x magnification. Co-localization images were analyzed in ten different RVCMs per animal, and averaged values for each animal are shown. NLRP3 and ASC co-localization (yellow; indicated by arrows) was quantified by determining Pearson correlation coefficient. Representative RVCM images were digitally zoomed 5x. (C, D) Representative Western blot images and…

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  Fig 5:. Lipopolysaccharide (LPS)- and adenosine triphosphate (ATP)-induced NLRP3 activation in RVCMs is sexually dimorphic and prevented by E2 in male RVCMs. (A) Experimental design. (B) Representative immunofluorescence images and quantification of fluorescence intensity of NLRP3-ASC co-localization in RVCMs from male or female control rats treated in vitro with LPS+ATP in absence or presence of NLRP3 inhibitor MCC950 or E2. RVCMs were treated with LPS (1 μg/mL) for 4 hours and ATP (2 mM) for 10 min prior to cell collection. MCC950 (1 μM) was given 30 min prior to LPS and continued throughout LPS and ATP exposure. E2 (1 nM) was given for 24 hours prior to LPS and continued throughout LPS and ATP exposure. Rat RVCMs were stained for NLRP3 (green), ASC (red), and DAPI (blue). Images were obtained at 100x magnification. Co-localization images were analyzed in ten different RVCMs per animal, and averaged values for each animal are shown. NLRP3 and ASC co-localization (yellow; indicated by arrows) was quantified by determining Pearson correlation coefficient. Representative RVCM images were digitally zoomed 5x. (C, D) Representative Western blot images and densitometric quantification of…

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  Fig 6:. Endothelin 1-induced NLRP3 activation in human induced pluripotent stem cell derived cardiac myocytes (hiPSC-CMs) is sexually dimorphic, male biased, and prevented by E2 in male cells. (A) Experimental design. (B) Representative immunofluorescence images and quantification of fluorescence intensity of NLRP3-ASC co-localization in hiPSC-CMs treated with endothelin-1 (ET-1; 1 nM) given 4 hours prior to cell collection +/− MCC950 (1 μM; administered 30 min prior to ET-1 and continued throughout ET-1 exposure) or E2 (1 nM, administered 24 hours prior to ET-1 and continued throughout ET-1 exposure). Cells were stained for NLRP3 (green), ASC (red), and DAPI (blue). Co-localization was quantified in ten different hiPSC-CMs per patient line by determining Pearson correlation between NLRP3 and ASC, and values were then averaged. Each data point = one human iPSC-CM clone obtained from 3 male and 3 female iPSC-CM cell line (means ± SEM). *p<0.05, **p<0.01 by one-way ANOVA with Holm-Šídák post-test (male cells) or Student t-test (female cells).

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  Fig 7:. Loss of ERα prevents E2 from attenuating RV contractile dysfunction in male RVs and renders female RVs susceptible to contractile dysfunction after NLRP3 activation. (A) Experimental design. (B) Representative immunofluorescence images and quantification of fluorescence intensity of NLRP3-ASC co-localization in RVs from male or female control rat hearts perfused with LPS (1 μg/mL, 130 min prior to start of dP/dt max measurement and continued throughout measurement) + ATP (2 mM, 10 min prior to start of dP/dt max measurement and continued throughout measurement), in the presence or absence of MCC950 (1 μM, 30 min prior to LPS and continued throughout LPS exposure) or E2 (1 nM, 30 min prior to LPS and continued throughout LPS exposure). Rat RVs were stained for NLRP3 (green), ASC (red), and DAPI (blue). Images were obtained at 100x magnification. Co-localization images were analyzed in ten different fields per animal, and averaged values for each animal are shown. NLRP3 and ASC co-localization (yellow; indicated by arrows) was quantified by determining Pearson correlation coefficient. Representative RVCM images were digitally zoomed 5x. Maximum rate of rise in RV pressure during…

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  Fig 8:. Lack of functioning estrogen receptor α (ERα) eliminates sexual dimorphisms in NLRP3 activation in RVCMs from MCT-PH rats and prevents E2 from attenuating NLRP3 activation in these cells. (A) Experimental design. (B) Representative immunofluorescence images and quantification of NLRP3-ASC colocalization in RVCMs from male or female ERα loss-of-function mutant (ERαmut) rats with MCT-PH. Subgroups of ERαmut MCT-PH rats were treated with E2 (75 μg/kg/day via s.c. pellets) for 4 weeks (starting at MCT injection). Images were obtained at 100x magnification. RVCMs were stained for NLRP3 (green), ASC (red), and DAPI (blue). Images were obtained at 100x magnification. Co-localization images were analyzed in ten different RVCMs per animal, and averaged values for each animal are shown. NLRP3 and ASC co-localization (yellow; arrows) was quantified by determining Pearson correlation coefficient. Representative images were digitally zoomed 5x. (C, D) Representative Western blot images and densitometric quantification of caspase-1/pro-caspase-1 ratio (C) and IL-1β/pro-IL-1β ratio (D) in RVCMs from male or female ERαmut MCT-PH rats with or without E2 treatment in vivo. (E, F) Representative…

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  Fig 9:. Lack of functioning estrogen receptor α (ERα) eliminates sexual dimorphisms in LPS + ATP-induced NLRP3 activation in rat RVCMs and prevents E2 from attenuating NLRP3 activation in these cells. (A) Experimental design. (B) Representative immunofluorescence images and quantification of NLRP3-ASC co-localization in RVCMs from male or female ERα loss-of-function mutant (ERαmut) rats after treatment with LPS and ATP in vitro. LPS (1 μg/mL) was given 4 hours prior to cell collection. ATP (2 mM) was given 10 min prior to cell collection. MCC950 (1 μM) or E2 (1 nM) were given 30 min or 24 hours prior LPS and ATP, respectively, and continued throughout the exposure. RVCMs were stained for NLRP3 (green), ASC (red), and DAPI (blue). Images were obtained at 100x magnification. Co-localization images were analyzed in ten different RVCMs per animal, and averaged values for each animal are shown. NLRP3 and ASC co-localization (yellow; arrows) was quantified by determining Pearson correlation coefficient. Representative images were digitally zoomed 5x. (C, D) Representative Western blot images and densitometric quantification of caspase-1/pro-caspase-1 ratio (C) and IL-1β/pro-IL-1β ratio (D) in…

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  Fig 10:. ERα co-localizes with NLRP3 in RVs from PAH patients with RV failure and in human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs). (A) Experimental design for PAH patient studies. (B, C) ERα-NLRP3 co-localization in RVs from PAH patients with RV failure. (B) Representative images and (C) quantification of co-localization of NLRP3 (green) with ERα (red). Magnification is 20x. Co-localization was analyzed in the whole RV sample; representative images are 40x. Arrows indicate ERα-NLRP3 co-localization (yellow). Co-localization was quantified by determining Pearson correlation coefficient between NLRP3 and ERα. Note sexual dimorphism and male bias with more pronounced co-localization in male PAH RVs. (D) ERα nuclear intensity in all experimental groups. (E, F) demonstrate correlations between ERα-NLRP3 co-localization intensity and (E) NLRP3 activation (NLRP3-ASC co-localization) and (F) nuclear ERα abundance. Note decrease in nuclear ERα abundance with increase in ERα-NLRP3 co-localization. (G) Experimental design for hiPSC-CM studies. (H, I) ERα-NLRP3 co-localization in hiPSC-CMs with and without endothelin 1 (ET-1; 1 nM, 4 hours prior to cell collection). (H)…

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  Fig 11:. E2 improves RV-pulmonary artery (PA) coupling and survival in male MCT-PH rats in an ERα-dependent manner. Male wild type (WT) or ERαmut MCT-PH rats were treated with MCC950 (WT only) or E2 (WT and ERαmut) as outlined in Fig. 2&8. End-systolic elastance (Ees) and arterial elastance (Ea) were measured using pressure-volume loops. Additional animals were utilized for survival studies. (A) Ees/Ea in WT MCT-PH groups. Individual values for Ees and Ea are listed in the table in (B). (C) Survival curves for WT MCT-PH groups. Animal numbers at risk are shown below x-axis. Note that curve for E2 group overlaps with curve of control group due to lack of mortality in either group. (D-F) Corresponding studies in ERαmut MCT-PH rats (note that ERαmut MCT-PH rats were not treated with MCC950). (A, D) *p<0.05, **p<0.01, ns = not significant by one-way ANOVA with Holm-Šídák post-test; (B, E) *p<0.05 vs. male WT or ERαmut control, #p<0.05 vs. male WT or ERαmut MCT by one-way ANOVA with Holm-Šídák post-test; (C, F) *p<0.05 vs. male WT or ERαmut control, #p<0.05 for male MCT E2 or MCC950 vs. male WT MCT by Log-rank [Mantel-Cox] test. Each data point represents one rat (means ± SEM).

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  Fig 12:. E2 improves RV-pulmonary artery (PA) coupling and survival in female OVX MCT-PH rats. Intact or OVX female wild-type (WT) MCT-PH rats were treated with E2 as outlined in Fig. 2. End-systolic elastance (Ees) and arterial elastance (Ea) were measured using pressure-volume loops. Additional animals were utilized for survival studies. (A) Ees/Ea in all experimental groups. Individual values for Ees and Ea are listed in the table in (B). (C) Survival curves. Animal numbers at risk are shown below x-axis. Note that curve for E2 group overlaps with curve of control group due to lack of mortality in either group. (A) *p<0.05 by one-way ANOVA with Holm-Šídák post-test; (B) *p<0.05, ***p<0.005 vs. control, #p<0.05 vs. OVX MCT by one-way ANOVA with Holm-Šídák post-test; (C) *p<0.05 vs. control, #p<0.05 for MCT E2 vs. OVX MCT by Log-rank [Mantel-Cox] test. Each data point represents one rat (means ± SEM).