Pivotal function of Nlrp3 inflammasome in myocardial IRI. (A-D) Kinetic analyses of infarct size and markers of inflammasome and apoptosis activation in hearts isolated at various time points after IRI (1-24 hours) compared with sham-operated mice. Representative images of triphenyl tetrazolium chloride (TTC) staining (infarct area encircled by dashed line; size bar, 20 µm) (A), dot plot summarizing data of infarct size (individual data points and mean ± SEM) (B), and representative immunoblots of inflammasome (cl-Casp1, cl–IL-1β) or apoptosis activation (cl-Casp3, cl-Casp7, BAX expression) markers, with glyceraldehyde-3-phosphate dehydrogenase [GAPDH] as loading control (C). (D) Line graph summarizing immunoblot data (mean ± SEM). (E) Experimental design. (F-L) Constitutively active Nlrp3^A350V abolishes the protective effect of aPC in myocardial IRI. (F) Induction of Nlrp3^A350V expression after tamoxifen injection into Nlrp3^V-ER mice compared with PBS-treated mice; representative immunoblots, with GAPDH as loading control. (G-H) aPC treatment fails to protect against myocardial IRI in Nlrp3^V-ER mice. Representative heart sections showing infarcted area detected by TTC staining (area encircled by dashed line; size bar, 20 µm) (G) and dot plot summarizing data (H). (I-L) aPC fails to reduce Nlrp3 expression, cl-Casp1 or cl–IL-1β, or plasma IL-1β and IL-18 levels in Nlrp3^V-ER mice after myocardial IRI. Representative immunoblots (I) and bar graphs (J) summarizing results, with GAPDH as loading control; arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β (I). (J) The active form was quantified. (K-L) Dot plots of plasma IL-1β and IL-18 levels. Sham-operated mice (sham) or mice with myocardial IRI without (cont; PBS) or with aPC pretreatment (aPC). Data shown represent mean ± SEM of at least 6 mice per group. *P < .05, **P < .01; Student t test (B,D) or analysis of variance (H,J-L).