Cardiomyocyte GSDME Drives Doxorubicin-Induced Cardiotoxicity by Targeting the CCL2-CCR2 Axis

Figures

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  Figure 1. Global Gsdme knockout prevents DIC. A, Diagram of the experimental design for the establishment of a chronic doxorubicin-induced cardiotoxicity (DIC) mouse model. B, Western blot and quantitative analysis of full-length GSDME (GSDME-F) and cleaved N-terminal GSDME (GSDME-N) in the hearts of DOX-treated WT mice at the indicated times (days 0, 3, 10, 14, 28, 42, and 49); n = 6 per group. C, heart weight (HW) normalized to tibia length (TL); n = 6 per group. D, Representative hematoxylin and eosin (H&E) staining of WT or Gsdme-/- murine hearts on day 49 after DOX treatment. Scale bar = 1000 μm. E, Representative wheat germ agglutinin (WGA) staining and quantification of the cardiomyocyte cross-sectional area of WT or Gsdme-/- murine hearts on day 49 after DOX treatment. Scale bar = 20 μm; n = 6 per group. F, Representative left ventricular M-mode images and echocardiographic measurements of the ejection fraction (EF) and fractional shortening (FS) of WT or Gsdme-/- mice on day 49 after DOX treatment; n = 6 per group. G, Representative Masson's trichrome staining and relative quantification of the myocardial fibrosis area in WT or Gsdme-/- murine hearts on day 49 after DOX treatment.…

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  Figure 2. Gsdme knockout in cardiomyocytes, but not in myeloid cells, prevents DIC. A and B, Western blot and relative quantification of full-length GSDME and cleaved N-terminal GSDME in isolated adult mouse cardiomyocyte (AMCM) fraction A, and in cardiac noncardiomyocyte fraction B, from DOX-treated WT mice; n = 6 per group. C, Representative fluorescence staining of GSDME (red), cTnT (green), and F4/80 (green) in mouse hearts on day 14 after DOX treatment. Insets indicate an enlarged view. Scale bar = 50 μm. D, Ratio of HW to TL; n = 6 per group. E, Representative H&E staining of hearts from GsdmeCon, GsdmeCKO, and GsdmeMKO mice after DOX administration. Scale bar = 1000 μm. F, WGA staining and measurements of cardiomyocyte cross-sectional area in heart sections from DOX-treated GsdmeCon, GsdmeCKO, and GsdmeMKO mice. Scale bar = 20 μm; n = 6 per group. G, Representative echocardiographic images and quantitative analysis of cardiac contractile function, including left ventricular EF and FS, in GsdmeCon, GsdmeCKO, and GsdmeMKO mice receiving DOX therapy; n = 6 per group. H, Masson's trichrome staining and quantification of cardiac fibrosis after DOX treatment on GsdmeCon, GsdmeCKO, and…

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  Figure 3. Gsdme knockout reduces CCL2-CCR2 signaling and cardiac inflammation. A, Cytokine array evaluation of the levels of inflammatory factors in the hearts of GsdmeCon mice treated with DOX; n = 4 per group. B, Levels of five inflammatory factors in the hearts of GsdmeCKO mice compared with those in the hearts of GsdmeCon mice after DOX treatment; n = 4 per group. C, Representative immunoblotting and relative quantitative analysis of CCL2 expression in mouse hearts; n = 6 per group. D and E, RT‒qPCR analysis of the transcription levels of Ccl2 D), Tnf-α, Il-6, and Il-1β E) in mouse hearts; n = 6 per group. F, Representative flow cytometry images and quantitative analysis of mouse heart CD45+CD11b+F4/80+CCR2+ macrophages; n = 3 per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 4. The CCL2 inhibitor bindarit prevents DIC. A, Bindarit intervention strategy. B, Representative immunoblotting and relative quantitative analysis of mouse heart CCL2 expression; n = 6 per group. C, RT‒qPCR analysis of Ccl2 transcription levels in mouse hearts; n = 6 per group. D, Representative flow cytometry images and quantitative analysis of CD45+CD11b+F4/80+CCR2+ macrophages in the heart; n = 3 per group. E, RT‒qPCR analysis of the transcription levels of Tnf-α, Il-6, and Il-1β in mouse hearts; n = 6 per group. F, Ratio of HW to TL; n = 6 per group. G, Representative H&E staining of mouse hearts. Scale bar = 1000 μm. H, Representative WGA staining of mouse hearts and quantitative analysis of the cross-sectional area of cardiomyocytes. Scale bar = 20 μm; n = 6 per group. I, Representative echocardiography of mouse hearts and measurements of EF and FS; n = 6 per group. J, Representative Masson's trichrome staining and quantitative results of mouse hearts. Scale bar = 50 μm; n = 6 per group. K, Representative immunoblotting and relative quantitative analysis of collagen I, collagen III, α-SMA, TGF-β, p-SMAD2, SMAD2, p-SMAD3 and SMAD3 expression in mouse hearts; n = 6 per group.…

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  Figure 5. CCR2+ macrophage depletion prevents DIC. A, Schematic illustration of CCR2+ macrophage depletion by DT injection every two days for 4 consecutive weeks in a DIC model. B, RT‒qPCR analysis of the mRNA levels of Tnf-α, Il-6, and Il-1β in mouse hearts; n = 6 per group. C, HW/TL; n =6 per group. D, Representative H&E staining of hearts from WT and Ccr2-DTR mice upon DOX treatment. Scale bar = 1000 μm. E, Representative images of WGA staining and quantification of cardiomyocyte cross-sectional area in heart sections from WT and Ccr2-DTR mice upon DOX administration. Scale bar = 20 μm; n = 6 per group. F, Representative M-mode echocardiography of mouse heart and measurements of left ventricular EF and FS in WT and Ccr2-DTR mice upon DOX treatment; n = 6 per group. G, Representative images of Masson's trichrome staining and quantification of cardiac fibrosis in heart sections from WT and Ccr2-DTR mice receiving DOX. Scale bar = 50 μm; n = 6 per group. H, Representative immunoblot and summary data showing the expression levels of collagen I, collagen III, α-SMA, TGF-β, phospho-SMAD2, total-SMAD2, phospho-SMAD3, and total-SMAD3 in the hearts of WT and Ccr2-DTR mice upon DOX…

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  Figure 6. Gsdme knockout protects against DOX-induced mitochondrial damage and mtDNA release. A, Representative immunofluorescence staining of GSDME and TOM20 in WT AMCMs 24 hours after DOX stimulation. Scale bar = 20 μm. B, Representative immunoblots and quantification of the intensity of cleaved N-terminal GSDME and Cyt c bands from cytosolic and mitochondrial fractions from WT and Gsdme-KO AMCMs treated with DOX for 24 hours; n = 6 per group. C, Representative fluorescence images of TMRE staining in WT and Gsdme-KO AMCMs 24 hours after DOX treatment. Scale bar = 20 μm; n = 6 per group. D, Representative fluorescence images of MitoSOX Red-stained WT and Gsdme-KO AMCMs 24 hours after DOX treatment. Scale bar = 20 μm; n = 6 per group. E, Representative images of extra-mitochondrial (PK Mito Red) mtDNA (SYBRTM Gold) in WT and Gsdme-KO AMCMs stimulated with DOX for 24 hours. Scale bar = 10 μm. The white arrowheads represent mtDNA inside the mitochondria, whereas the blue arrowheads indicate mtDNA outside the mitochondria; n = 6 per group. F and G, Cytosolic mtDNA levels in DOX-treated WT and Gsdme-KO AMCMs detected by primers for D-loop or Nd1 using PCR F) and by a primer for Cox1 using qPCR…

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  Figure 7. STING/NFκB mediates regulation of CCL2 production by GSDME. A, Representative western blot and quantitative analysis of STING/NFκB-related proteins in DOX-treated WT and Gsdme-KO AMCMs; n = 6 per group. B, Representative immunoblot images and quantification of NFκB p65 in cytosolic and nuclear fractions from DOX-treated WT and Gsdme-KO AMCMs; n = 6 per group. C and D, RT‒qPCR C) and immunoblotting D) analysis of CCL2 in NFκB activator 1-treated AMCMs; n = 6 per group E, NFκB-binding sites on the promoter region of Ccl2 predicted using the JASPAR database and luciferase reporter plasmids with cloning of the WT or mutant Ccl2 promoter sequence. F, Luciferase activity after the cotransfection of NFκB overexpression plasmids and WT or mutant Ccl2 promoter luciferase reporter plasmids. Firefly luciferase activity was normalized to that of Renilla luciferase; n = 6 per group. G and H, Representative immunoblots and summary of STING/NFκB-related proteins and CCL2 in AMCMs treated with H-151 G) or SR-717 H); n = 6 per group. **P < 0.01, ***P < 0.001.

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  Figure 8. The STING inhibitor H-151 prevents DIC. A, Diagram of the H-151 intervention strategy. B, Representative western blots and statistical results of p-STING/STING, p-TBK1/TBK1, p-IRF3/IRF3, p-NFκB p65/NFκB p65 and CCL2 in the hearts of H-151-treated DIC mice; n = 6 per group. C, RT‒qPCR analysis of Ccl2 transcriptional levels in the hearts of H-151-treated DIC mice; n = 6 per group. D, Representative flow cytometry images and quantitative analysis of CD45+CD11b+F4/80+CCR2+ macrophages in the hearts of H-151-treated DIC mice; n = 3 per group. E, RT‒qPCR analysis of the transcriptional levels of Tnf-α, Il-6, and Il-1β in the hearts of H-151-treated DIC mice; n = 6 per group. F, Ratio of HW to TL; n = 6 per group. G, Representative H&E staining of the hearts of H-151-treated DIC mice. Scale bar = 1000 μm. H, Representative WGA staining of mouse hearts and statistical results of the cardiomyocyte cross-sectional area. Scale bar = 20 μm; n = 6 per group. I, Representative M-mode images and echocardiographic measurements of the left ventricular EF and FS in H-151-treated DIC mice; n = 6 per group. J, Representative Masson's trichrome staining and quantitative results of H-151-treated…

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  Figure 9. Pharmacological blockade of GSDME alleviates DIC. A, Diagram of the experimental design of DMF administration via oral gavage in a chronic DIC model. B, Representative western blots and statistical histograms of cleaved GSDME in the hearts of DMF-treated DIC mice; n = 6 per group. C, Representative transmission electron microscopy images of mouse cardiomyocytes. Scale bar = 2 μm. D, Representative western blots and gray-intensity measurements of p-STING/STING, p-TBK1/TBK1, p-IRF3/IRF3, p-NFκB p65/NFκB p65 and CCL2 in the hearts of DMF-treated DIC mice; n = 6 per group. E, RT‒qPCR detection of Ccl2 mRNA levels in the hearts of DMF-treated DIC mice; n = 6 per group. F, Representative flow cytometric images and statistical histograms of CD45+CD11b+F4/80+CCR2+ macrophages in the hearts of DMF-treated DIC mice; n = 3 per group. G, RT-qPCR detection of the mRNA levels of Tnf-α, Il-6, and Il-1β in the hearts of DMF-treated DIC mice; n = 6 per group. H, Ratio of HW to TL; n = 6 per group. I, Representative H&E staining of hearts from DMF-treated DIC mice. Scale bar = 1000 μm. J, Representative WGA staining and quantitative analysis of the cardiomyocyte cross-sectional area in the hearts…