DNA Methylation Analysis Identifies Novel Epigenetic Loci in Dilated Murine Heart upon Exposure to Volume Overload

Figures

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  Figure 1. Long-term VO (16 weeks post-shunt) resulted in pathological cardiac remodeling and HF progression. (A) Workflow for DNA methylation analysis in VO mice. (B–D) Echocardiographic parameters: HR, heart rate (B); LVEDD, LV end-diastolic diameter (C); EF, ejection fraction (D). (E) Kaplan–Meier survival analysis, log-rank test, n = 9 sham and 19 shunt mice. (F,G) Morphometric parameters: LVW/TL, LV weight-to-tibia length (F); LungW/TL, lung weight-to-tibia length (G). (H) Representative images of WGA- (upper panels) and picrosirius red- (lower panels) stained myocardial cross-sections. (I) Quantitative real time PCR analysis for the expression of fetal cardiac stress genes. Values are mean ± SEM, n = 3–6 hearts/group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham, two-tailed unpaired Student’s t-test.

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  Figure 2. Differential promoter DNA methylation identified by RRBS. Heatmap showing the differential DNA methylation level for the candidate genes from 3 sham and 6 shunt mouse hearts. Log2FC shows the log2 value of fold change between the mean value of the sham and the mean value of the shunt. Padj shows the adjusted p-value between the sham and shunt hearts.

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  Figure 3. Gene-specific promoter DNA methylation assessment with bisulfite sequencing and MeDIP-qPCR. (A) Histogram showing the CpGs content at the gene promoter region. The locations for bisulfite sequencing and MeDIP-qPCR primers are indicated on top of the histogram. (B) Bisulfite sequencing analysis showing the methylation status of CpGs at the selected candidate genes. Every panel shows the sequencing results derived from four different animals. Closed circles indicate methylated, open circles indicate unmethylated, and gray circles indicate undetermined CpGs. (C) MeDIP-qPCR results showing the promoter methylation level for the four selected genes. Values are mean ± SEM, n = 4–7 hearts/group. * p < 0.05, *** p < 0.001 vs. sham, two-tailed unpaired Student’s t-test.

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  Figure 4. Promoter DNA methylation in the blood of shunt mice. MeDIP-qPCR results showing the promoter methylation level for the four selected genes from the DNA isolated from 16-week-shunt mice blood. Values are mean ± SEM, n = 4–5 mice/group. * p < 0.05, ** p < 0.01 vs. sham, two-tailed unpaired Student’s t-test.

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  Figure 5. qPCR analysis showing the mRNA expression level of the four selected candidate genes in 16-week-shunt mouse hearts. Values are mean ± SEM, n = 3–4 hearts/group. * p < 0.05, ** p < 0.01 vs. sham, two-tailed unpaired Student’s t-test. n.s., not significant.

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  Figure 6. Promoter DNA methylation assessment in the hearts and blood isolated from short-term VO-exposed mice (1 week post-shunt). (A–D) Echocardiographic and morphometric parameters: LVEDD, LV end-diastolic diameter (A); EF, ejection fraction (B); LVW/TL, LV weight-to-tibia length (C); LungW/TL, lung weight-to-tibia length (D). (E,F) MeDIP-qPCR to assess DNA methylation in the hearts (E) and blood (F). Values are mean ± SEM, n = 3–7 mice/group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham, two-tailed unpaired Student’s t-test.

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  Figure 7. mRNA expression of the selected genes at 1 week after shunt. (A) qPCR analysis showing the mRNA expression level of the four selected candidate genes. (B) Pie chart summarizing the percentage of upregulated (5.84%), downregulated (8.25%), and unchanged (85.9%) genes in 1-week-shunt vs. sham mouse hearts. (C) Volcano plot shows downregulation of Jph2, Vapb and Ipmk in 1-week-shunt vs. sham mouse hearts. The selected candidate genes are shown with blue dots. Filter criteria: fold Change: >1 or <−1 and p-value < 0.05. (D) Barplot showing the Affymetrix signal intensity for the Ipmk, Vapb, and Jph2 genes compared between 1-week-sham and shunt mouse hearts. Values are mean ± SEM, n = 4–5 hearts/group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham, two-tailed unpaired Student’s t-test.

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  Figure 8. Quantitative real-time PCR of Dnmts and Tets enzymes. Values are mean ± SEM, n = 3–5 hearts/group. * p < 0.05 vs. sham, two-tailed unpaired Student’s t-test. ns, not significant.