This renewal proposal builds on the scientific premise that even though right ventricle (RV) function and female sex are major determinants of survival in pulmonary hypertension (PH), no RV-specific or sex steroid-directed therapies exist. While it is known that macrophage NLRP3 inflammasome activation leads to RV failure, we now demonstrate for the first time that the NLRP3 inflammasome is also active in RVCMs to promote contractile dysfunction. The goal of this proposal is to identify novel and therapeutically targetable mechanisms by which the female sex steroid 17b-estradiol (E2), via its receptor ERα, inhibits NLRP3 inflammasome activation in RVCMs to enhance contractile function and improve RV-pulmonary artery (PA) coupling. We put forward the novel hypothesis that RVCM NLRP3 inflammasome activation is sexually dimorphic, and that ERα improves RV- PA coupling in PH by inhibiting RVCM NLRP3 activation and preventing inflammasome-mediated RVCM contractile dysfunction. We propose the following specific aims: 1) To identify sexual dimorphisms in RVCM NLRP3 inflammasome activation and downstream signaling as mediators of sex differences in RV adaptation and RV-PA coupling; 2) To establish that E2 inhibits RVCM NLRP3 inflammasome activation via ERα; and 3) To establish NLRP3 inflammasome inhibition as a therapeutic strategy to improve RV-PA coupling in RV failure in low endogenous estrogen states. We will employ rat models for comprehensive in vivo and ex vivo studies, complement these studies with mechanistic experiments in isolated primary rats RVCMs as well as in human chambered muscle pumps generated with inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) exposed to pressure overload, and validate our findings in RV tissue from PH patients. Endpoints investigated will include RV function and structure, exercise capacity, NLRP3 signaling, contractile signaling, as well as ATAC-seq followed by transcription factor enrichment analysis (TFEA). Contractile function will be assessed by pressure volume loops and echocardiography, an isolated perfused heart model, and by measuring contractility in iPSC-CMs and RVCMs. The proposed studies are significant, because they will 1) identify NLRP3 signaling as a sexually dimorphic modulator of RV function that is mediating RV resilience in pre-menopausal females, 2) establish a novel and therapeutically targetable ERα-NLRP3-calcium signaling axis in the RV, and 3) establish optimal strategies and conditions to inhibit NLRP3 in the RV-PA unit. Conceptual innovation is provided by identification of RVCM NLRP3 inflammasome activation as a novel, sexually dimorphic, and ERα-regulated modifier of RV adaptation in PH. Technical innovation is provided by use of 1) human chambered muscle pumps generated with iPSC-CMs, 2) ATAC-seq with TFEA in RVCMs, 3) novel and highly selective NLRP3 inhibitors, and 4) a large animal model of RVF. Upon completion of the proposed studies, we will have established sexu...