Epigenetic modification mechanism of histone demethylase KDM1A in regulating cardiomyocyte apoptosis after myocardial ischemia-reperfusion injury

Abstract

Hypoxia and reoxygenation (H/R) play a prevalent role in heart-related diseases. Histone demethylases are involved in myocardial injury. In this study, the mechanism of the lysine-specific histone demethylase 1A (KDM1A/LSD1) on cardiomyocyte apoptosis after myocardial ischemia-reperfusion injury (MIRI) was investigated. Firstly, HL-1 cells were treated with H/R to establish the MIRI models. The expressions of KDM1A and Sex Determining Region Y-Box Transcription Factor 9 (SOX9) in H/R-treated HL-1 cells were examined. The cell viability, markers of myocardial injury (LDH, AST, and CK-MB) and apoptosis (Bax and Bcl-2), and Caspase-3 and Caspase-9 protein activities were detected, respectively. We found that H/R treatment promoted cardiomyocyte apoptosis and downregulated KDM1A, and overexpressing KDM1A reduced apoptosis in H/R-treated cardiomyocytes. Subsequently, tri-methylation of lysine 4 on histone H3 (H3K4me3) level on the SOX9 promoter region was detected. We found that KDM1A repressed SOX9 transcription by reducing H3K4me3. Then, HL-1 cells were treated with CPI-455 and plasmid pcDNA3.1-SOX9 and had joint experiments with pcDNA3.1-KDM1A. We disclosed that upregulating H3K4me3 or overexpressing SOX9 reversed the inhibitory effect of overexpressing KDM1A on apoptosis of H/R-treated cardiomyocytes. In conclusion, KDM1A inhibited SOX9 transcription by reducing the H3K4me3 on the SOX9 promoter region and thus inhibited H/R-induced apoptosis of cardiomyocytes.

Keywords: Bax; Bcl-2; Cardiomyocyte apoptosis; H3K4me3; Histone demethylation; Hypoxia-reoxygenation; KDM1A; SOX9.

Figure 1. H/R treatment promotes cardiomyocyte apoptosis and downregulates KDM1A.

HL-1 cells were subjected to hypoxia-reoxygenation treatment (the H/R group), with HL-1 cells untreated as the control (the Blank group). (A–B) qRT-PCR and Western blot were adopted to detect the levels of KDM1A in HL-1 cells; (C) CCK-8 was used to verify HL-1 cell viability; (D) ELISA was performed to detect the concentrations of myocardial injury markers in HL-1 cells; (E) qRT-PCR was used to detect the mRNA levels of apoptotic proteins in HL-1 cells; (F) Flow cytometry was conducted to detect the apoptosis rate of HL-1 cells; (G) Colorimetric method was used to examine the activities of Caspase-3 and Caspase-9. The cell experiment was repeated three times independently; data were expressed as mean ± standard deviation. Data in (A–B) and (D–G) were tested by independent t test; data in (C) were analyzed by two-way ANOVA, followed by Tukey’s post-hoc test. LDH, Lactate Dehydrogenase; AST, Aspartate Aminotransferase; CK-MB, Creatine Kinase-MB.

Figure 2. Overexpressing KDM1A reduces apoptosis of H/R-treated cardiomyocytes.

Plasmid pcDNA3.1-KDM1A (oe-KDM1A) was transfected into HL-1 cells, with pcDNA3.1-NC (oe-NC) as the control. (A) qRT-PCR was used to measure the transfection efficiency; (B) Western blot was performed to examine the protein level of KDM1A in HL-1 cells; (C) CCK-8 assay was adopt to detect cell viability; (D) ELISA was conducted to the concentrations of myocardial injury markers in HL-1 cells; (E) qRT-PCR was performed to verify the mRNA levels of apoptotic proteins in HL-1 cells; (F) Flow cytometry was used to detect the apoptosis rate; (G) Colorimetric method was conducted to measure the activities of Caspase-3 and Caspase-9. The cell experiment was repeated three times independently; data were expressed as mean ± standard deviation. Data in (C) were analyzed using two-way ANOVA and data in (A–B) and (D–G) were analyzed using one-way ANOVA, followed by Tukey’s post-hoc test. LDH, Lactate Dehydrogenase; AST, Aspartate Aminotransferase; CK-MB, Creatine Kinase-MB.

Figure 3. KDM1A inhibits the transcription of SOX9 by reducing H3K4me3.

(A) Western blot was used to detect the level of H3K4me3 in HL-1 cells. (B) ChIP analysis was used to detect the changes of H3K4me3 on the SOX9 promoter and exon; (C) qRT-PCR to detect the level of SOX9 mRNA in HL-1 cells; The cell experiment was repeated three times independently; data were expressed as mean ± standard deviation. Data in (B) were analyzed by two-way ANOVA and data in (A) and (C) were analyzed by one-way ANOVA, and followed by Tukey’s post-hoc test.

Figure 4. Upregulating H3K4me3 reverses the effect of overexpressing KDM1A on alleviating H/R-induced cardiomyocyte apoptosis.

H3K4me3 in H/R cells was promoted using CPI-455 (solvent control: DMSO) and then the cells were combined with oe-KDM1A. (A) Western blot was used to detect the levels of H3K4me3 in HL-1 cells; (B) qRT-PCR was performed to examine the mRNA expression of SOX9 in HL-1 cells; (C) CCK-8 was adopted to detect cell viability; (D) ELISA was performed to verify the concentrations of myocardial injury markers in HL-1 cells; (E) qRT-PCR was conducted to examine the mRNA levels of apoptotic proteins in HL-1 cells; (F) Flow cytometry was used to detect the apoptotic rate; (G) Colorimetric method was to conducted to test the activities of Caspase-3 and Caspase-9 in the cells. The cell experiment was repeated three times independently; data were expressed as mean ± standard deviation. Data in (C) were analyzed by two-way ANOVA and data in (A–B) and (D–G) were analyzed by one-way ANOVA, and followed by Tukey’s post-hoc test. LDH, Lactate Dehydrogenase; AST, Aspartate Aminotransferase; CK-MB, Creatine Kinase-MB.

Figure 5. Overexpressing SOX9 reverses the alleviative effect of overexpressing KDM1A on H/R-induced cardiomyocyte apoptosis.

pcDNA3.1-SOX9 (oe-SOX9) was transfected into HL-1 cells using oe-NC as the control. (A) qRT-PCR was adopted to detect the transfection efficiency. Then, the H/R-treated HL-1 cells overexpressing SOX9 were combined with oe-KDM1A. (B) CCK-8 was used to detect cell viability; (C) ELISA was used to detect the concentrations of myocardial injury markers in HL-1 cells; (D) qRT-PCR was conducted to verify the mRNA levels of apoptotic proteins in HL-1 cells; (E) Flow cytometry was used to examine the apoptotic rate; (F) Colorimetric method was adopted to test that activities of Caspase-3 and Caspase-9 proteins in HL-1 cell. The cell experiment was repeated 3 times independently; data were expressed as mean ± standard deviation. Data in (B) were analyzed by two-way ANOVA and data in (A) and (C–F) were analyzed by one-way ANOVA, followed by Tukey’s post-hoc test. LDH, Lactate Dehydrogenase; AST, Aspartate Aminotransferase; CK-MB, Creatine Kinase-MB.