KDM3A Attenuates Myocardial Ischemic and Reperfusion Injury by Ameliorating Cardiac Microvascular Endothelial Cell Pyroptosis

Figures

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  Figure 1. H/R treatment resulted in CMEC injury and dysfunction. (a) The proliferative capacity of CMECs was assessed by the CCK-8 assay, and the OD value was the quantitative indicator (n = 6, ∗p < 0.05, relative to the Con group). The migration capacity of CMECs was measured by the transwell chamber assay (b) and wound healing assay (c), respectively. The number of cells on the lower membrane surface (scale bar = 50 μm) and wound closure rate of each group were calculated (n = 6, ∗p < 0.05, relative to the Con group). The neovascularization capacity of CMECs was measured by tube formation (d) and sprouting assays (e). Total tube length and total loops (scale bar = 50 μm), as well as cumulative sprouts and sprout counts for each group, were evaluated (n = 6, ∗p < 0.05, relative to the Con group). (f) To detect the synthesis and secretion capacities of CMECs, eNOS and NO levels in supernatants of every group were determined (n = 6, ∗p < 0.05, relative to the Con group).

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  Figure 2. H/R treatment induced CMEC pyroptosis accompanied by downregulation of KDM3A and p-Akt. (a) The Western blot assay was conducted to evaluate the levels of pyroptosis-related proteins, including NLRP3, GSDMD-N, cleaved-caspase-1, ASC, IL-1β, and IL-18 (n = 3, ∗p < 0.05, relative to the Con group). (b) The expressions of KDM3A and p-Akt before and after H/R treatments were also detected by Western blot assay (n = 3, ∗p < 0.05, relative to the Con group). (c) Hoechst 33342/PI double fluorescent staining was performed to detect cell death in vitro, and the percentage of PI-positive cells was calculated in each group (n = 6, ∗p < 0.05, relative to the Con group).

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  Figure 3. KDM3A knockout exaggerated H/R-induced CMEC injury and dysfunction. (a) The CCK-8 assay was used to estimate the proliferative abilities of WT CMECs and KDM3A-KO CMECs during H/R (n = 6, ∗p < 0.05, compared to the H/R+WT group). (b, c) Transwell chamber and wound healing assays were conducted to assess the migration capacities of CMECs before and after KDM3A knockout during H/R (n = 6, ∗p < 0.05 relative to the H/R+WT group). (d, e) Tube formation and sprouting assays were performed to investigate the neovascularization capacities of CMECs in the H/R+WT and H/R+KO groups (n = 6, ∗p < 0.05, relative to the H/R+WT group). (f) Levels of eNOS and NO in supernatants were measured to assess the synthesis and secretion capacities of CMECs in every group after H/R treatment (n = 6, ∗p < 0.05, relative to the H/R+WT group).

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  Figure 4. KDM3A knockout promoted H/R-induced CMEC pyroptosis and suppressed p-Akt levels. (A) Western blotting was performed to determine the levels of pyroptosis-related proteins, such as NLRP3, GSDMD-N, cleaved-caspase-1, ASC, IL-1β, and IL-18 (n = 3, ∗p < 0.05 relative to the H/R+WT group). (b) Western blotting was also performed to verify the effects of KDM3A knockout and to detect p-Akt levels after KDM3A knockout during H/R stimulation (n = 3, ∗p < 0.05, relative to H/R+WT group). (c) CMECs that had lost their vitality in the H/R+WT and H/R+KO groups were analyzed by Hoechst 33342/PI double fluorescent staining (n = 6, ∗p < 0.05, compared to the H/R+WT group).

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  Figure 5. KDM3A overexpression ameliorated H/R-induced CMEC injury and dysfunctions, while suppression of the PI3K/Akt signaling pathway reversed these beneficial effects. (a) The CCK-8 assay was conducted to measure the proliferative abilities of CMECs in each group (n = 6, ∗p < 0.05, relative to the H/R+Ad-GFP group, #p < 0.05, compared to the H/R+Ad-KDM3A group). (b, c) Migration abilities of CMECs after KDM3A overexpression or/and PI3K/Akt inhibition were detected by transwell chamber and wound healing assays (n = 6, ∗p < 0.05, compared to the H/R+Ad-GFP group, #p < 0.05, compared to the H/R+Ad-KDM3A group). (d, e) Images of tube formation and sprouting assay for each group. Neovascularization capacities of CMECs after KDM3A overexpression or/and PI3K/Akt signaling pathway inhibition were evaluated. (f) eNOS and NO levels in supernatants of each group were measured to determine the synthesis and secretion capacities of CMECs (n = 6, ∗p < 0.05, compared to the H/R+Ad-GFP group; #p < 0.05, compared to the H/R+Ad-KDM3A group).

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  Figure 6. KDM3A overexpression mitigated H/R-induced CMEC pyroptosis through activating PI3K/Akt signaling pathway. (a) Protein levels of NLRP3, ASC, GSDMD-N, cleaved-caspase-1, IL-1β, and IL-18 were analyzed by Western blot assay. KDM3A overexpression significantly downregulated the expressions of pyroptosis-associated proteins while LY294002 treatment ameliorated these beneficial effects in the setting of H/R (n = 3, ∗p < 0.05, compared to the H/R+Ad-GFP group, #p < 0.05, compared to the H/R+Ad-KDM3A group). (b) Western blot assay was also performed to verify the transfection effects of Ad-KDM3A and to detect the expressions of p-Akt after KDM3A overexpression or/and PI3K/Akt inhibition under the condition of H/R stimulation. (c) Hoechst 33342/PI double fluorescent staining was conducted to assess cell death in each group (n = 6, ∗p < 0.05, compared to the H/R+Ad-GFP group, #p < 0.05, compared to the H/R+Ad-KDM3A group).

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  Figure 7. KDM3A knockout further exacerbates the deteriorated cardiac function and myocardial injury induced by I/R injury as well as reduces the perfusion of coronary microcirculation. (a) The changes in cardiac function, including LVEF and LVFS, were evaluated by echocardiography in rats of different groups (n = 6, ∗p < 0.05, as compared to the SO group; #p < 0.05, as compared to the H/R+WT group). (b) Representative images of the myocardial infarction area of each group, and the ratio of the infarction area to the total left ventricular area was calculated (n = 4, ∗p < 0.05, as compared to the SO group; #p < 0.05, as compared to the H/R+WT group). (c) Representative images of thioflavin-S and Evan's blue dual-staining were performed to detect the no-reflow area after I/R injury of each group (n = 4, ∗p < 0.05, as compared to the SO group; #p < 0.05, as compared to the H/R+WT group). (d) Representative images of CD31 fluorescence staining were conducted to estimate the capillary density. Scale bar = 20 μm. (e) Representative images and averaged data of the apoptotic index in rat heart tissue of each group. TUNEL staining (green) indicates apoptotic nuclei; DAPI counterstaining (blue)…