GSDMD-Mediated Cardiomyocyte Pyroptosis Promotes Myocardial I/R Injury

Abstract

[Figure: see text].

Keywords: caspase; cell death; inflammation; necrosis; pyroptosis.

Figure 1.

Hypoxia/reoxygenation (H/R) induces cardiomyocyte pyroptosis through GSDMD (gasdermin D) activation. Adult cardiomyocytes from the ventricles of Myh6-Cre GSDMD +/+ mice (controls) or GSDMD-CKO (cardiomyocyte-specific GSDMD-deficient) male mice were isolated, and (A) H/R for 0.5/24 h induced the pyroptotic morphological manifestations of prominent bubble-like formations, as well as propidium iodide (PI)-positive staining of adult cardiomyocytes (black indicates necrotic cardiomyocytes and prominent bubble-like formations). B and C, GSDMD (n=5) and GSDME (gasdermin E) protein levels (n=5) and cleavage were assessed in cell lysates and supernatants from adult Myh6-Cre mouse cardiomyocytes that underwent H/R for different times. The target band was first normalized with β-actin and then calculated as fold changes vs H/R for 0 h (control groups). D, Representative GSDMD expression was detected in adult cardiomyocytes (subjected to H/R 0.5/24 h) by confocal laser scanning immunofluorescence. E, GSDMD-CKO adult mouse cardiomyocytes were isolated, subjected to the same H/R (0.5/24 h) treatment, and were photographed at the same time points as cardiac myocytes. F–I, PI-positive cardiomyocyte proportions (n=5), LDH (lactate dehydrogenase) release (n=5), cellular ATP levels (n=5), and IL (interleukin)-18 (n=5) as release measured by ELISA in adult mouse cardiac myocytes that underwent H/R 0.5/24 h, LDH and ATP levels were calculated as fold changes vs control group. Data are mean±SEM. P values were determined by using 1-way ANOVA with Bonferroni multiple comparisons test (B and C; P values adjusted for 5 comparisons), 2-way ANOVA with Bonferroni multiple comparisons test (F–I). cTnT indicates cardiac troponin T; NS, not significant; and WT, wild-type.

Figure 2.

GSDMD (gasdermin D) triggers cardiomyocyte pyroptosis due to the intrinsic pore-forming activity of its N-terminal domain. A–C, The full-length GSDMD (OV-GSDMD-FL, n=5), the GSDMD-N (OV-GSDMD-N, n=5) and GSDMD-C (OV-GSDMD-C, n=5) fragments, was transiently expressed in adult cardiomyocytes via plasmid transfection. The target band was first normalized with β-actin and then calculated as fold changes vs control groups. D, Hoechst 33342 (blue) and PI were added to detect the loss of plasma membrane integrity, and fluorescent images were obtained by confocal microscopy. Black arrows indicate pyroptosis in cardiomyocytes and prominent bubble-like formations. E (n=5), Propidium iodide (PI)-positive and total (Hoechst-positive) cells were counted in 5 randomly selected fluorescence microscopic visual fields. F and G, Cellular ATP levels (n=5) and LDH (lactate dehydrogenase) release (n=5) in the supernatants of adult mouse cardiac myocytes (subjected to hypoxia/reoxygenation [H/R] 0.5/24 h, LDH and ATP levels) were calculated as fold changes vs control group. Vector group (control plasmid), OV-GSDMD-FL (overexpressing plasmid pcDNA3.1-GSDMD), OV-GSDMD-N (overexpressing plasmid pcDNA3.1-GSDMD-N), OV-GSDMD-C (overexpressing plasmid pcDNA3.1-GSDMD-C). The data are expressed as the means±SEMs. P values were determined by using 1-way ANOVA with Bonferroni multiple comparisons test (A, B, and C, P values adjusted for 5 comparisons; E, F, and G, P values adjusted for 6 comparisons). NS indicates not significant.

Figure 3.

CASP11 (caspase-11) cleaves GSDMD (gasdermin D) in cardiomyocytes undergoing hypoxia/reoxygenation [H/R]-induced pyroptosis. Cardiomyocytes were stimulated with H/R for the indicated times. A (n=5), Western blot (WB) analysis was performed to measure caspase-11. Quantitative analysis of the WB results of CASP-11. B, Representative WB analysis of GSDMD and GSDMD-N in cardiomyocytes infected with Ad-β-gal (used as the control, n=5), Ad-CASP11 or Ad-CASP1 for 48 h and then treated with H/R for 0.5/24 h. C, Propidium iodide (PI) was added to detect the loss of plasma membrane integrity, and fluorescent images were obtained by confocal microscopy. D, n=5; PI-positive and total (Hoechst-positive) cells were counted in 5 randomly selected fluorescence microscopic visual fields. E–G (n=5), LDH (lactate dehydrogenase) release, cellular ATP levels, and IL (interleukin)-18 levels in supernatants as measured by ELISA (LDH and ATP levels were calculated as fold changes vs control group). The data are expressed as the means±SEMs. Fold changes in protein levels in the treatment groups compared with the control groups were determined (the target band was first normalized with β-actin and then calculated as fold changes vs control groups). P values were determined by using 1-way ANOVA with Bonferroni multiple comparisons test (A and B, P values adjusted for 4 comparisons), 2-way ANOVA with Bonferroni multiple comparisons test (D–G, P values adjusted for 4 comparisons). NS indicates not significant; and WT, wild-type.

Figure 4.

The levels of proteins associated with pyroptosis in myocardial ischemia/reperfusion (I/R) injury in vivo. A–C, Myh6-Cre GSDMD+/+ mice (n=5 mice per group) were subjected to 30 min of ischemia followed by reperfusion for different times, and Western blot analysis was used to measure the levels of GSDMD (gasdermin D), GSDME (gasdermin E), and CASP11 (caspase-11). The target band was first normalized with β-actin and then calculated as fold changes vs sham groups. D, Representative GSDMD and caspase-11 expression and colocalization in cardiomyocytes as shown by immunofluorescence analysis in murine hearts after 30 min of ischemia followed by reperfusion for 24 h. The data are expressed as the means±SEMs, P values were determined by Kruskal-Wallis test (A–C, P values adjusted for 4 comparisons). cTnT indicates cardiac troponin T; and NS, not significant.

Figure 5.

GSDMD (gasdermin D) deficiency in cardiomyocytes blocks myocardial ischemia/reperfusion (I/R) injury. Wild-type (WT; n=10 per group), Myh6-Cre GSDMD +/+ (n=10 per group), GSDMDF/F (n=10 per group), and GSDMD-CKO (cardiomyocyte-specific GSDMD-deficient; n=10 mice per group) mice were subjected to I/R injury (30 min of ischemia/24 h of reperfusion). A, Representative photographs of cross-sections stained with triphenyl tetrazolium chloride (TTC) and Evans blue (EB) to determine the extent of I/R injury. B and C, The ratios (right) of infarct size (IF, n=10) to area at risk (AAR, n=10) in the hearts. D, Serum LDH (lactate dehydrogenase) levels (n=10) of mice with I/R injury (30 min of ischemia/24 h of reperfusion). E, Representative photographs and quantitative analysis of myocardial Evans blue dye (EBD, n=10) uptake, which shows infarcted cardiomyocytes; viable cardiomyocytes were labeled by cTnT antibodies in WT, Myh6-Cre, GSDMDF/F, and GSDMD-CKO (cardiomyocyte-specific GSDMD-deficient) mice (n=10 mice per group) subjected to I/R injury. The data are expressed as the means±SEM, the normal distribution was checked by the Shapiro-Wilk test, all P values were >0.05 indicated that the data were approximately normally distributed for each group. P values were determined by using 1-way ANOVA with Bonferroni multiple comparisons test (B, C, and F, P values adjusted for 3 comparisons), 2-way ANOVA with Bonferroni multiple comparisons test (D). cTnT indicates cardiac troponin T; and NS, not significant.

Figure 6.

Oxidative stress-induced pyroptosis in cardiomyocytes is mediated by GSDMD (gasdermin D). Adult cardiomyocytes from the ventricles of Myh6-Cre GSDMD^+/+ mice (controls) or GSDMD-CKO (cardiomyocyte-specific GSDMD-deficient) mice were isolated. A, Representative adult cardiomyocytes treated with PBS or H₂O₂ (final concentration 200 μmol/L) for 2 h exhibited the pyroptotic morphological manifestations of prominent bubble-like formations, as well as propidium iodide (PI)-positive staining (black arrows indicate necrotic cardiomyocytes and prominent bubble-like formations). PI (red) was added to detect the loss of plasma membrane integrity, and fluorescent images were obtained by confocal microscopy. B (n=5), PI-positive and total (Hoechst-positive) cells were counted in 5 randomly selected fluorescence microscopic visual fields. C (n=5) and D (n=5), Cellular ATP levels and LDH (lactate dehydrogenase) (n=5) release in cardiomyocytes with the same treatment (LDH and ATP levels were calculated as fold changes vs control group). E (n=5), Adult cardiomyocytes were treated with vehicle or H₂O₂ (200 μmol/L) as indicated, and Western blot analysis was performed to measure GSDMD. The data are expressed as the means±SEMs, fold changes in protein levels in the treatment groups compared with the control groups were determined (the target band was first normalized with β-actin and then calculated as fold changes vs control groups). P values were determined by using 2-way ANOVA with Bonferroni multiple comparisons test (B, C, and D); 1-way ANOVA with Bonferroni multiple comparisons test (E); (E, P values adjusted for 4 comparisons). NS indicates not significant.

Figure 7.

GSDMD (gasdermin D) levels in the culture supernatants of cardiomyocytes and serum samples of patients after percutaneous coronary intervention (PCI). Hypoxia/reoxygenation (H/R)-induced GSDMD release in adult cardiomyocytes, and cardiomyocytes were stimulated with H/R for 0.5/24 h. A, Culture supernatants were concentrated and analyzed by Western blotting. B (n=5), GSDMD levels in the culture supernatants at different reoxygenation times were measured by ELISA. C, GSDMD levels in the serum of patients with ST-segment–elevation myocardial infarction at different timepoints after PCI or in stable patients with coronary artery disease (CAD) by ELISA. The data are expressed as the means±SEMs. Data were analyzed using 1-way ANOVA with Bonferroni multiple comparisons test (B), repeated measures 2-way ANOVA with Bonferroni multiple comparisons test statistics obtained by ANOVA (C). MIRI indicates myocardial ischemia reperfusion injury.

Figure 8.

Schematic illustrating the mechanism of cardiomyocyte pyroptosis and related signaling during myocardial ischemia/reperfusion (I/R) injury. We propose that oxidative stress triggers GSDMD (gasdermin D) cleavage via CASP-11 (caspase-11). GSDMD-N oligomerizes and perforates the plasma membrane to mediate pyroptotic cell death. GSDMD, GSDMD-N, GSDMD-C, and IL (interleukin)-18 are released into the supernatant or peripheral blood. RIP3 indicates receptor-interacting protein 3.