Mapping DNA methylation to cardiac pathologies induced by beta-adrenergic stimulation in a large panel of mice

Figures

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  Figure 1.. DNA methylation changes from ISO in the HMDP. a) total heart Weight as a percentage of day 0 body weight across 90 strains of the HMDP. b) Volcano plot showing differential methylation of CpGs with and without ISO. Green points are CpGs whose methylation shifts by at least 3% between conditions, while blue are CpGs that pass a 1% FDR threshold and red points are the 397 CpGs that meet both criteria. c) Gene set enrichment of genes proximal to significantly differentially methylated CpGs, with blue representing sites that are hypermethylated while red reflects sites that are hypomethylated.

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  Figure 2.. Heatmap of the top 1% of differentially methylated loci across the HMDP cohort. X axis is organized by hierarchical clustering and shows the separation of the panel into distinct cohorts based on their sub-panel origin, with the BXD, AXB/BXA and BxC RI panels clustering together (see bottom edge) while ISO treatment was not a major driver of clustering (top edge). CpGs were clustered along the Y axis based on similarity and genes proximal to these CpGs were analyzed for GO enrichments, seen along the right edge of the heatmap and annotated in the legend. Larger version is available as Supplemental Figure 2.

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  Figure 3.. Representative Manhattan plots from the EWAS study. in each case, the X axis represents the position of a CpG across the genome and the Y axis is the negative log10 of the association p-value as determined by the MACAU algorithm. The red line indicates our calculated genome-wide significance threshold of p = 4.17 × 10−7, while the blue line denotes our suggestive threshold of p = 4.17 × 10−6. Genes of interest are highlighted and detailed further in Table 1. a) Treated CpGs affecting isoproterenol cardiac fibrosis b) Treated CpGs affecting isoproterenol posterior wall thickness. c) Untreated CpGs affecting control adrenal gland Weight d) Treated CpGs affecting treated adrenal gland Weight.

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  Figure 4.. In vitro NRVM studies of candidate gene knockdown. a) Representative image of 20x resolution NRVMs b) percentage of siRNA-targeted gene expression compared to scramble controls. N = 9, representing 3 independent trials with 3 technical replicates each c-e) NRVM cross-sectional areas for scramble and siRNA-treated cells in both control and ISO-treated (60 uM) conditions. p values are indicated, with greyed-out p values deemed not significant. N for Anks1aand Mospd3 studies was 60 cells per siRna/condition combination. N for Tsc2 was 200 for scramble control and scramble ISO, 175 for Tsc2 control and Tsc2 ISO.

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  Figure 5.. Concomitant DNMT inhibitor (RG108) treatment in ISO-treated mice de-methylates hypermethylated genes in severe-responder mouse strain BTBRT and is associated with an improvement in phenotype response. a) H&E cross-sections of hearts from ‘mild-responder’ C57BL/6 and ‘severe-responder’ BTBRT mice treated with SAL, ISO, and ISO+RG108 for 3 weeks. b) Boxplot on C57BL/6 and BTBRT mice cardiac phenotype measurements LVIDd and %ejection fraction after 3 weeks of RG108 treatment. c) Most significantly differentially expressed genes in BTRBT strain after RG108 treatment. Downregulated genes in ISO (blue) were upregulated in saline and ISO+RG108 (yellow). Similarly, the upregulated genes in ISO showed the opposite with downregulation in saline and ISO+RG108. Colors are row-normalized to enhance contrast D) global DNA methylation distribution at the promoter, gene body and intergenic regions of the differentially expressed genes detected in Figure 3c. Corresponding figure for differential methylation and gene expression in B6 can be found in Supplemental Figure 4. N = 12 per group.