Hypermethylation of Hif3a and Ifltd1 is associated with atrial remodeling in pressure-overload murine model

Figures

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  Fig. 1. Pressure overload promotes atrial weight and fibrosis. (a) Operation protocol for animal experiments. The aortic constriction was released by reoperation 3 days or 7 days after the initial TAC operation (R3 or R7 groups). (b) The atrium (left and right)/body weight ratio and ventricle/body weight were compared among the 4 groups (Sham, R3, R7, and TAC). The atrium/body weight ratio was significantly increased at R7 and TAC, and a difference was present between R3 and R7. The ventricle/body weight ratio showed a significant increase only in TAC. (c) The right atrium/body weight ratio and left atrium/body weight were compared among the four groups (Sham, R3, R7, and TAC). (d) Representative image of Masson’s trichrome staining of left atrial tissue (left panel) and the summarized data of % fibrotic area in the left atrium (right panel). Similar to the change in the atrium/body weight ratio, % fibrosis showed a significant increase in R7 and TAC groups compared to sham. (e) The expression of Col1a2, Col3a1, and Mmp2 in the left atrium. The TAC group showed significantly high expression of Col1a2, Col3a1, and Mmp2. Similar to the change in % fibrosis, the expression of Col1a2 and…

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  Fig. 2. Comprehensive analysis of RNA expression and DNA methylation. (a) The scatter plot shows log2 fold changes (FC) in mRNA expression (y-axis) against log2 fold changes in MBD enrichment (x-axis). The dotted lines indicate a cutoff with log2FC (MBD-seq) > 1 or < -1, and log2FC (RNA-seq) > 0.75 or < − 0.75. (b) Heatmap of the results of MBD-seq (left panel) and RNA-seq (right panel) of candidate genes. The number of samples is two for TAC and the sham group. The color scale is shown on the right side of the heatmap.

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  Fig. 3. Candidate genes with DNA methylation at promoter region. (a) DNA methylation site by MBD-seq in Hif3a and Ifltd1. The position of the differentially methylated region is indicated with a red box, which shows significantly increased DNA methylation in the promoter region. (b) The expression of Hif3a and Ifltd1 left atrium is reduced in the TAC model and transient pressure overload. A significant reduction of Hif3a expression is achieved in the R3 group, and the expression of Hif3a is not different among the R3, R7, and TAC groups. The expression of Ifltd1 shows a gradual reduction in a time-dependent manner of pressure overload. Data are presented as mean ± SD. n = Sham; 6, R3; 7, R7; 7, TAC; 7. One-way analysis of variance followed by Bonferroni’s correction was performed. * p < 0.05.

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  Fig. 4. Bisulfite sequence of the promoter region of Hif3a and Ifltd1. Detailed DNA methylation in the promoter region (left panel) and summarized DNA methylation ratio of CpGs (right panel), in Hif3a (a) and Ifltd1 (b). Bisulfite sequencing identified an increase in DNA methylation of both genes in a time-dependent manner under pressure overload. Hif3a shows a significant increase in %methylation at R3, R7, and TAC groups. However, Ifltd1 exhibited a significant increase in methylation after R7 group. Data are presented as mean ± SE. n = 3 per group; * p < 0.05. A Chi-square test followed by Bonferroni’s correction was performed.

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  Fig. 5. Pressure overload model using Dnmt1mutmice. (a) The mRNA expression of Hif3a (left panel) and Ifltd1 (right panel) in the left atrium was compared between sham and TAC groups, using WT or Dnmt1mut mice. Dnmt1mut cancelled the reduced expression of both genes by TAC. (b) The atrium/body weight ratio (left panel) and ventricle/body weight ratio (right panel) were also compared between WT and Dnmt1mut mice. The increase in the atrium/body weight ratio was suppressed in the Dnmt1mut mouse. (c) The expression of Col1a2, Col3a1, and Mmp2 in the left atrium. The increase in the expression of Col1a2 and Col3a1 was suppressed in Dnmt1mut mice. Data are presented as mean ± SD. (a) n = WT-Sham; 5, WT-TAC; 5, Dnmt1mut-Sham; 5, Dnmt1mut-TAC; 6, (b) n = WT-Sham; 6, WT-TAC; 9, Dnmt1mut-Sham; 6, Dnmt1mut-TAC; 9, (c) n = 5 each; * p < 0.05. Unpaired t-tests were performed.