EZH2-induced histone methylation in the Nrf2 promoter region mediates pyroptosis in inflammatory cardiomyocyte injury

Myocardial dysfunction is one of the most severe sepsis syndromes. EZH2 participates in regulating the inflammatory response in tissues; however, its role in septic myocarditis remains unclear. In this study, various concentrations of lipopolysaccharide (LPS) were used to treat H9C2 cells in order to mimic sepsis. Cell pyroptosis was detected by flow cytometry, and further confirmed by the expression of biomarkers and levels of cytokines. Caspase-1 activity was evaluated by flow cytometry and immunofluorescence assays. Gene expression was detected by reverse transcription-PCR (RT-PCR) and western blotting. Chromatin Immunoprecipitation – Quantitative PCR was used to detect the levels of histone methylation in the Nrf2 promoter region. Our results showed that LPS activated cell pyroptosis, promoted EZH2 expression, and inhibited Nrf2 expression in H9C2 cells. Overexpression of EZH2 enhanced LPS-induced cell pyroptosis, as shown by increased Caspase-1 activity, increased expression of N-GSDMD and NLRP3 proteins, and higher levels of IL-1β, IL-18, and LDH. Moreover, overexpression of EZH2 inhibited Nrf2 transcription. In contrast, knockdown of EZH2 suppressed pyroptosis and promoted Nrf2 expression in LPS-treated H9C2 cells. Results of chromatin immunoprecipitation – quantitative PCR verified that EZH2 regulated Nrf2 transcription via H3K27me3 modification. Furthermore, overexpression of Nrf2 inhibited cell pyroptosis and knockdown of Nrf2 promoted cell pyroptosis. Knockdown of Nrf2 reversed the cardioprotective effect of EZH2 knockdown. Collectively, our results suggest that EZH2 promotes cell pyroptosis by enhancing H3K27me3 expression and inhibiting Nrf2 transcription in cardiomyocytes under inflammatory conditions.