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.