Fig. 1

Multiple AP drugs activated the NLRP3 inflammasome and myocyte pyroptotic death. a Schematic illustration of the study design. b Electrocardiography (ECG) monitoring of mice electrical activity in 0, 7, 14, and 21 days (n = 6 mice/group). c Gross images of vehicle (Veh, PBS) and Olz (5 mg/kg)-treated (21 days) mouse hearts. Black arrow indicated exudative patches over epicardium. d–f RNA sequencing of total RNAs from Veh (PBS) and Olz-treated mouse hearts (21 days). g qRT-PCR validation of gene expression including Nlrp3, Casp1, Asc, and Gsdmd in mouse hearts (n = 5 mice/group). h Western blot analysis of the NLRP3 inflammasome proteins in mouse HL-1 cells under Veh (PBS), low dose (LD, 1 μM), medium dose (MD, 4 μM), and high dose (HD, 16 μM) of Olz treatments for 24 h, or under constant Olz treatments (4 μM) for different hours. i Subcellular components were isolated from mouse HL-1 cells at indicated treatments for 24 h, and western blot analysis of GSDMD was performed. j Immunofluorescence assay analysis of GSDMD (green signal) localization. H-Ras (red signal) is a membrane marker, and cell membrane integrity was monitored by propidium iodide (PI, 3 μM) uptake. Scale bar = 100 μm. k, l Enzyme-linked immunosorbent assay (ELISA) analysis of mature IL-1β and IL-18 contents in supernatants of mouse HL-1 cells under Veh (PBS), quetiapine (Que, 4 μM), Olz (4 μM), and clozapine (Clz, 20 μM) treatments for 24 h. m Western blot analysis of the NLRP3 inflammasome and pyroptosis in primary mouse cardiomyocytes, mouse HL-1 cell line, and human AC-16 cells under Veh (PBS), Clz (20 μM), Olz (4 μM), and Que (4 μM) treatments for 24 h. n Immunofluorescence analysis of NLRP3 (red) and Casp1 (green) in mouse hearts receiving indicated treatments. Arrowheads indicated merged NLRP3 and Casp1 signals (yellow). Scale bar = 20 μm. o Time-lapse microscopy of Olz (4 μM) and Clz (20 μM)-treated rat H9c2 myocytes. Cell morphology was visualized by wide-field light microscopy (upper panel) and cell membrane integrity was monitored by PI uptake (lower panel). Arrowheads indicated pyroptotic cells with protrusions. Scale bar = 50 μm. Time duration = h: min: s. ms. p Scanning electron microscopy of Veh (PBS), Olz (4 μM), Clz (20 μM), and Que (4 μM)-treated rat H9c2 myocytes. Arrowheads indicated bubbling of pyroptotic cells. All quantification represents the mean and SEM of independent experiments. Two-way ANOVA was used for analysis in (b). The Student’s t-test was used for analysis in (g, k, l). ns, no significance. *p < 0.05; **p < 0.01; ***p < 0.001 as indicated