LDHA exacerbates myocardial ischemia-reperfusion injury through inducing NLRP3 lactylation

Figures

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  Fig. 1. H/R treatment contributed to the pyroptosis and lactylation of H9c2 cells. (A) The LDH activity of two groups. (B-C) ELISA detect results of IL-1β and IL-18 expression in H9c2 cells treated or untreated with H/R. (D) Flow cytometry was carried out for estimation of cell pyroptosis rate. (E) Western blot analysis of the expression levels of pyroptosis biomarkers cleaved caspase-1 and GSDMD-N. (F) The role of H/R in lactylation was also determined by western blot. vs. the control group, ***P < 0.001, N = 3

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  Fig. 2. Promoting lactylation facilitated cell pyroptosis in H/R damage. (A-C) The concentrations of LDH, IL-1β and IL-18 were measured with matched kits. (D) The proportion of pyroptotic cardiomyocytes examined by flow cytometry. (E) Western blot detection of cleaved caspase-1 and GSDMD-N expression in different groups. vs. the control group, **P < 0.01, ***P < 0.001. vs. the H/R group, #P < 0.05, ##P < 0.01, ###P < 0.001, N = 3

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  Fig. 3. Knockdown of LDHA reversed the H/R-induced pyroptosis in H9c2 cells. (A) The qPCR analysis of LDHA expression in different groups after transfection. vs. the siNC group. (B) The LDH colorimetric assay kit was applied to test LDH activity of cardiomyocytes subjected to different treatments. (C-D) ELISA assay was conducted to assess the release of IL-1β and IL-18. (E) Flow cytometry analysis of cell pyroptosis rate in different groups. (F) Cleaved caspase-1 and GSDMD-N levels measured by Western bot. vs. the siNC group, ***P < 0.001. vs. the siLDHA#1 group, ##P < 0.01, ###P < 0.001, N = 3

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  Fig. 4. LDHA stabilized NLRP3 expression through lactylation. (A-B) The function of LDHA in NLRP3 lactylation was evaluated with western blot assay. (C) Potential lactation sites of NLRP3 predicted by bioinformation website (http://lin-group.cn/server/DeepKla/Serve.html). (D) Western blot was employed to identify the NLRP3 lactation site. (E) The impacts of LDHA on NLRP3 stability was also analyzed by western blot. vs. the control or siNC group, **P < 0.01, ***P < 0.001. vs. the H/R group, ###P < 0.001, N = 3

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  Fig. 5. Upregulation of NLRP3 eliminated the effects of LDHA knockdown on H/R damage. (A-C) The detection results of LDH activity and the expression of IL-1β and IL-18. (D) Flow cytometry assay was performed to verify the role of LDHA/NLRP3 axis in cell pyroptosis. (E) Western blot analysis of cleaved caspase-1 and GSDMD-N in H/R-induced cardiomyocytes following different treatment. vs. the siNC group, **P < 0.01, ***P < 0.001. vs. the siLDHA group, #P < 0.05, ##P < 0.01, ###P < 0.001, N = 3

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  Fig. 6. NLRP3 mediated the promoting function of LDHA in myocardial injury in vivo. (A) The TTC staining images of heart tissue in each group. (B) The quantification of cardiac infarct area. (C-E) The expression of myocardial enzymes LDH, CK-MB and cTnl in mice receiving different interventions. vs. the sham + KO-NC group. (F) The protein expression of LDHA in each group was analyzed by Western blot. vs. the sham + KO-NC group, ***P < 0.001. vs. the I/R + KO-NC group, ###P < 0.001. vs. the I/R + KO-LDHA group, &P < 0.05, &&&P < 0.01, N = 6