Overexpression of GSDMD-N blunts cardioprotective and anti-inflammatory effect of SerpinB1. (A) Schematic of the study. R26-LSL-SerpinB1/α-MHC^MerCreMer double transgenics (Ctg-SerpinB1) mice were treated with AAV9-cTnT-GSDMD-N via intravenous tail injection for 3 weeks, subjected to intraperitoneal tamoxifen injections of every other day thrice. Subsequently, the mice underwent either sham or TAC surgery, followed by echocardiographic analyses at 4 weeks after surgery and histological analyses at 6 weeks after surgery. (B–G) Echocardiographic analyses of left ventricular structure and function. (B) Representative M-mode echocardiographic images. Scale bars = 2 mm (vertical) and 0.1 s (horizontal). Quantification of end-diastolic diameter (LVEDd, C), end-systolic diameter (LVESd, D), end-diastolic posterior wall thicknesses (LVPWd, E), ejection fraction (LVEF, F), and fractional shortening (LVFS, G) (n = 6 mice per group). Representative WGA staining images (H) and quantification of cardiomyocyte cross-sectional area (I) (scale bars = 20 μm; n = 6 mice per group). Masson trichrome staining of heart sections (J) and quantification of interstitial fibrotic area (K) (scale bars = 100 μm; n = 6 mice per group). Representative western blots (L) and quantitative results of cleavage status of interleukin-1β (IL-1β, M) and IL-18 (N) (n = 5 mice per group). (O–P) Representative immunofluorescence images (O) of CD45 staining in heart sections. The nuclei were counterstained with DAPI (scale bars = 50 μm). White arrows indicate CD45-positive cells. Quantification of CD45-positive cells per HPF at ×600 magnification (P) is shown (n = 5 mice per group). Data are represented as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison test.