NLRP3 Activation and Calcium-Dependent Contractile Function in Rat Right Ventricle Cardiomyocytes (rvcms) Are Sexually Dimorphic and Controlled by 17Β-Estradiol-via Estrogen Receptor-α

Introduction: NLRP3 inflammasome activation promotes right ventricle (RV) contractile dysfunction in pulmonary hypertension (PH). However, the role of NLRP3 signaling in RVCMs has not been studied. 17β-estradiol (E2) improves RVCM function in PH. Hypothesis: E2, via estrogen receptor-α (ERα), prevents RVCM NLRP3 inflammasome activation and contractile dysfunction in experimental PH. Methods: RV failure in male and female Sprague Dawley rats were induced with monocrotaline (MCT). Co-localization of NLRP3 and its partner ASC was assessed. RVCMs isolated from healthy male or female wildtype (WT) or ERα loss of function mutant (ERα mut ) rats were treated with NLRP3 activators lipopolysaccharide (LPS 1 μg/mL, 4 h) and ATP (2 mM, 10 min) ± E2 (1 nM, 24 h) or NLRP3 inhibitor MCC950 (1 μM, 30 min). RVCM contractility and Ca 2+ levels were evaluated via IONOPTIX system. Protein levels of NLRP3 and its targets were assessed. ERα binding to the NLRP3 promoter was assessed by immunoprecipitation. RNA-seq was performed in RV tissues from male and intact or ovariectomized female rats with RV failure. Results: RNA-seq demonstrated that transcripts involved in NLRP3 activation are E2-regulated. RVCMs from male, but not female, MCT rats showed increased NLRP3 and ASC co-localization/activation (p<0.05). In addition, male MCT rats RVs exhibited increased activation of NLRP3 downstream mediators caspase-1 and IL-1β (p<0.05). Cultured male RVCMs treated with LPS+ATP demonstrated more pronounced NLRP3 activation and decreased contractility and Ca 2+ levels than female RVCMs (p<0.05). NLRP3 activation in RVCMs from male rats resulted in reduced contractility; this was abolished with NLRP3 inhibition (p<0.05). E2 treatment in male RVCMs reduced NLRP3 and ASC interaction and increased contractility Ca 2+ levels in male WT but not in ERα mut RVCMs (p<0.05). IP demonstrated ERα binding to the NLRP3 promoter. Conclusion: NLRP3 signaling in RVCMs from MCT-PH rats is sexually dimorphic, with more pronounced activation and contractile dysfunction in males. E2 prevents RVCM NLRP3 activation and contractile dysfunction in an ERα-dependent manner, likely mediated by ERα binding to the NLRP3 promoter. NLRP3 activation may be therapeutically targetable in PH patients of either sex.