Fig 2: MIAT knockdown significantly ameliorated cardiac pyroptosis in vivo(A) Representative immunohistochemistry heart images of CASP1 and IL-1ß in mice with indicated treatments.(B–E) The relative gene expression levels of MIAT (B), miR-214-3p (C), CASP1 (D), and IL-1ß (E) in control, DM, DM + LV-siMIAT, and DM + CASP1 I groups.(F) The relative protein expression levels of CASP1, IL-1ß, IL-18, and GSDMD in control, DM, DM + LV-siMIAT, and DM + CASP1 I groups were detected by western blotting. Densitometry analysis was performed and demonstrated as fold change, normalized to GAPDH.(G) is Densitometry analysis was performed and demonstrated as fold change, normalized to GAPDH.(H) ICa,L was detected by patch clamping The baseline and peak fluorescence intensity were demonstrated as line charts. Scale bar: 20 µm. n = 5 for each experiment. Treatment using verapamil (20umol/L) to block calcium channel was used as a positive control **p < 0.01, ***p < 0.001 versus the control group. #p < 0.05, ##p < 0.01 versus DM, ###p < 0.001 versus DM.

Fig 3: High glucose treatment causes inflammatory damage and calcium overload in cardiomyocytes(A) The primary cardiomyocyte was treated with either NG or HG for indicated hours. The relative protein expression levels of pro-IL-1ß, IL-1ß, pro-CASP1, CASP1, pro-IL-18, IL-18, and GSDMD in cardiomyocytes were detected by western blotting.(B) Densitometry analysis was performed and demonstrated as fold change.(C) Representative transmission electron microscopy images. Scale bar: 2 µm, three different sections were captured, and the area of each cell were calculated. ***p < 0.001.(D) The baseline and peak fluorescence in primary human cardiomyocytes with either NG or HG treatment were captured via confocal microscopy.(E) The baseline and peak fluorescence intensity were demonstrated as a line chart. Scale bar: 20 µm. n = 5 for each experiment. All experiments are performed at least 3 times.

Fig 4: MIAT regulated pyroptosis in cardiomyocytes via MIAT-miR-214-3p-CASP1 axis(A and B) Primary cardiomyocytes treated with si-NC + AMO-NC, HG + si-NC + AMO-NC, HG + si-MIAT + AMO-NC, HG + si-NC + AMO-miR-214-3p, respectively, were harvested for western blotting. The protein expression levels of CASP1, IL-1ß, IL-18, and GSDMD were detected by immunofluorescence. Densitometry analysis was performed and demonstrated as fold change, normalized to GAPDH.(C and D) Representative transmission electron microscopy images of primary cardiomyocytes with indicated treatments. Scale bar: 2 µm, three different sections were captured, and the area of each cell was calculated.(E) The images of fluorescence intensity of [Ca2+i in primary cardiomyocytes with each indicated treatment was captured by confocal microscopy, and [Ca2+i was labeled by arrows.(F) Fluorescence measurement of [Ca2+i in primary cardiomyocytes with indicated treatments. The baseline and peak fluorescence intensity were demonstrated as a line chart. Scale bar: 20 µm. All experiments are performed at least 3 times. **p < 0.01, ***p < 0.001 versus si-NC + AMO-NC; ##p < 0.01, ###p < 0.001 versus HG + si-NC + AMO-NC; $$p < 0.01, $$$p < 0.001 versus HG + si-MIAT + AMO-NC.

Fig 5: Inhibited cGAS-STING pathway effectively reduced cardiomyocyte pyroptosis and hypertrophy induced by PA treatment.a Western blot analysis of NLRP3 and GSDMD-N/GSDMD after treatment with PA with or without siRNA of cGAS or siRNA of STING in H9C2 cells normalized to ß-actin. n = 3 in each group. b Relative mRNA levels of NLRP3, TNF-a, IFN-ß, IL-1ß, and IL-18 in H9C2 cells. n = 3 in each group. c Representative flow cytometry image and the corresponding quantification showing the number of PI+ cells as the pyroptosis population. n = 4 in each group. d LDH release from each group of H9C2 cells. n = 8 in each group. e H9C2 cells were incubated with PA with or without siRNA of cGAS or siRNA of STING for 24 h and then evaluated by cell counting kit-8. n = 8 in each group. f, g Representative images and quantitative analysis of caspase-1 (red) and TUNEL (green) double-positive cells in PA-induced NMCMs. n = 5 in each group. h, i Quantitative analysis of the myocyte cross-sectional area by a-actinin staining in NMCMs. n = 6 in each group. j mRNA levels of ANP, BNP, and ß-MHC in H9C2 cells. n = 3 in each group. Values are the mean ± SEM. *P < 0.05 vs. NC, #P < 0.05 vs. PA-treated cells. NC negative control, PA palmitic acid, si-cGAS small interfering RNA of cGAS, si-STING small interfering RNA of STING.