Ischemic Preconditioning Confers Epigenetic Repression of Mtor and Induction of Autophagy Through G9a‐Dependent H3K9 Dimethylation

Figures

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  Figure 1. Cardiomyocyte chromatin is enriched for H3K9me2 after ischemic preconditioning. Western blot analysis of histone modifications in animals subjected to ischemic preconditioning (IPC) or sham controls reveals a significant increase in H3K9me2 in myocardial area at risk (AAR) compared to remote myocardium (RM) (*P<0.05 using 2‐way ANOVA with Bonferroni post hoc analysis of AAR vs RM, n=6).

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  Figure 2. The Mtor gene is subject to H3K9me2‐mediated epigenetic regulation in ischemic preconditioning. A, Overview of experimental and bioinformatic procedures. B, Upper: UCSC genome browser view of the mouse Mtor gene. Regions of differential H3K9me2‐enrichment in the AAR called by SICER (red) and CisGenome (orange) are indicated. Overlapping regions are indicated by green bars. Lower: Integrative Genomics Viewer (IGV) visualizations of ChIP‐Seq reads in regions 1 to 4 of the AAR and RM in representative IPC mice. C, ChIP‐qPCR validation confirms that H3K9me2 is more highly associated with the Mtor gene in ischemic preconditioned AAR vs RM. Left panel shows individual regions (*P<0.05 using 2‐way ANOVA with Bonferroni post hoc analysis, n=6, Region 3 did not amplify) and right panel shows the cumulative H3K9me2‐levels in Regions 1, 2, and 4. D, qPCR analysis of Mtor expression in the AAR and RM of IPC mice (n=6, *P<0.05 using Student t test). Mtor expression was normalized to Gapdh and expressed relative to the mean of the RM samples. I/R, ischemia/reperfusion; IPC, ischemic preconditioning; qPCR, quantitative polymerase chain reaction.

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  Figure 3. G9a/Ehmt2 regulates Mtor H3K9me2‐levels and gene expression. A, Analysis of Ehmt2 expression in HL‐1 cells transfected with scrambled negative control siRNA (Scr siRNA) or Ehmt2 siRNA with qPCR. Ehmt2 expression was normalized to Gapdh and expressed relative to the mean of the Scr siRNA samples (**P<0.01 using Student t test). Analysis of (B) G9a/Ehmt2 and (C) H3K9me2 levels in HL‐1 cells transfected with Scr or Ehmt2 siRNA using Western blot (**P<0.01, ***P<0.001 using Student t test). Beta Tubulin was used as loading control. Results are derived from 3 separate experiments with 3 biological replicates in each experiment. A representative blot from 1 experiment is shown. The long and short isoforms of G9a are both visible on the blot. The combined density of both bands was used for quantification. D, H3K9me2 ChIP‐qPCR of HL‐1 cells transfected with Scr or Ehmt2 siRNA. Primers corresponding to regions of differential H3K9 dimethylation in the Mtor gene of IPC mice were used. Primers for the Myod promoter were used as a positive control (*P<0.05 using Student t test). Results are derived from 3 separate experiments. E, Analysis of Mtor gene expression in HL‐1 cells transfected…

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  Figure 4. G9a/Ehmt2 regulates cardiomyocyte autophagy and sensitivity to hypoxia. HL‐1 cells transfected with scrambled negative control siRNA (Scr siRNA) or Ehmt2 siRNA were treated with 5 μmol/L rapamycin for 16 hours or subjected to serum starvation (SS) in the presence of 10 μmol/L chloroquine for 4 hours. Autophagic activity was measured using a fluorescent lysosomal probe and the mean fluorescence intensity (MFI) was analyzed with flow cytometry. A, Representative overlay histograms of HL‐1 cells transfected with Scr siRNA (blue) or Ehmt2 siRNA (red) and subjected to SS. B, There was a significant increase in autophagic activity in control cells after treatment with either 5 μmol/L rapamycin (### P<0.001) or (SS) (## P<0.01). Transfection with Ehmt2 siRNA significantly reduced the levels of autophagy in the baseline (*P<0.05) as well as when treated with 5 μmol/L rapamycin (**P<0.001) or SS/10 μmol/L chloroquine (**P<0.01). Two‐way ANOVA with Bonferroni correction for multiple comparisons was used to calculate statistical significance. Results are derived from 3 separate experiments with 3 biological replicates in each experiment. C and D, Representative confocal images of HL‐1 cells…

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  Figure 5. Inhibtion of Mtor transcription is sufficient for induction of autophagy in cardiomyocytes. A, Western blot analysis of Mtor protein in animals subjected to IPC, comparing the area at risk (AAR) to remote myocardium (RM), n=6. B, siRNA‐mediated knock down of Mtor in HL‐1 cells and its effect on autophagy. Upper, relative expression of Mtor in HL‐1 cells at different time points after transfection with Mtor siRNA or negative control siRNA measured with qPCR. Data are normalized to Gapdh and expressed relative to the mean of Scr siRNA‐treated cells at each time point. A 2‐way ANOVA with Bonferroni correction was used to calculate statistical significance between control cells and cells transfected with Mtor siRNA at each time point (***P<0.001, **P<0.01). Lower, autophagic activity in HL‐1 cells at different time points after transfection with Mtor siRNA or negative control siRNA measured with a fluorescent lysosomal probe. A 2‐way ANOVA with Bonferroni correction was used to calculate statistical significance between control cells and cells transfected with Mtor siRNA at each time point (*P<0.05, ***P<0.001). Results are derived from 2 separate experiments with 6 biological…

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  Figure 6. Working model. The Mtor gene is actively transcribed in cardiomyocytes, which provides constitutive inhibition of autophagy. Upon IPC‐treatment, G9a is activated and provides dimethylation of H3K9 throughout the Mtor gene, conferring transcriptional repression and initiation of autophagic activity. IPC, ischemic preconditioning.