Long-term epigenetic and metabolomic changes in the mouse ventricular myocardium after exertional heat stroke

Abstract

Evidence from human epidemiological studies suggests that exertional heat stroke (EHS) results in an elevated risk of long-term cardiovascular and systemic disease. Previous results using a preclinical mouse model of EHS demonstrated severe metabolic imbalances in ventricular myocardium developing at 9-14 days of recovery. Whether this resolves over time is unknown. We hypothesized that the long-term effects of EHS on the heart reflect retained maladaptive epigenetic responses. In this study, we evaluated genome-wide DNA methylation, RNA-Seq, and metabolomic profiles of the left ventricular myocardium in female C57BL/6 mice, 30 days after EHS (exercise in 37.5°C; n = 7-8), compared with exercise controls. EHS mice ran to loss of consciousness, reaching core temperatures of 42.4 ± 0.2°C. All mice recovered quickly. After 30 days, the left ventricles were rapidly frozen for DNA methyl sequencing, RNA-Seq, and untargeted metabolomics. Ventricular DNA from EHS mice revealed >13,000 differentially methylated cytosines (DMCs) and >900 differentially methylated regions (DMRs; ≥5 DMCs with ≤300 bp between each CpG). Pathway analysis using DMRs revealed alterations in genes regulating basic cell functions, DNA binding, transcription, and metabolism. Metabolomics and mRNA expression revealed modest changes that are consistent with a return to homeostasis. Methylation status did not predict RNA expression or metabolic state at 30 days. We conclude that EHS induces a sustained DNA methylation memory lasting over 30 days of recovery, but ventricular gene expression and metabolism return to a relative homeostasis at rest. Such long-lasting alterations to the DNA methylation landscape could alter responsiveness to environmental or clinical challenges later in life.

Keywords: DMRs; DNA methylation; environmental epigenetics.

Graphical abstract

Figure 1.

Genome-wide DNA methylation analyses of individual differentially methylated cytosines (DMCs). A: number of hypomethylated (blue) vs. hypermethylated (red) DMCs. Threshold inclusion was > 10% change in methylation and Q < 0.05. B: fraction of DMCs within each genomic region.

Figure 2.

Gene location of individual differentially methylated cytosines (DMCs) and regions (DMRs). A: scatterplot of individual DMCs, hypermethylation (red) or hypomethylation (black), and DMRs, hypermethylation (green) or hypomethylation (blue) as a function of distance from the transcription start site (TSS). B: histogram of the frequency of DMCs and DMRs as a function of distance from the TSS. Note this is an expansion of the axis from “A” as indicated by the black dotted arrows.

Figure 3.

Genome-wide DNA methylation analyses of differentially methylated regions (DMRs). A: frequency of hypomethylation (blue) vs. hypermethylation (red). B: genomic location of a given DMR. Threshold inclusion was > ± 10% average change in methylation and Q < 0.05. C: box plot of actual fractional methylation levels for each group of samples across genomic features affected by DMRs. White dots represent mean; the black horizontal lines represent medians; the boxes are interquartile ranges. **P ≤ 0.005 by Mann–Whitney U test. DMC, differentially methylated cytosine; EXC, exercise control; EHS, exertional heat stroke.

Figure 4.

Multivariate analysis of differentially methylated regions (DMRs). A: heat map/hierarchical clustering of all significant DMRs identified in the left ventricle. All DMRs in each row represent those with Q < 0.05 and ± 10% change in methylation. The change in methylation is scaled from −2 to +2-fold across all samples (columns, 4/group) with the mean level depicted in black, hypermethylated DMRs (403) in red, and hypomethylated DMRs (544) in cyan. B: unbiased PCA plot of individual samples demonstrating clustering of exercise control (EXC) and exertional heat stroke (EHS) treatments. PCA, principal component analysis.

Figure 5.

Gene Ontology (GO) enrichment analysis. Significantly enriched GO terms from DMR results using ShinyGO (y-axis) are plotted with the fold enrichment along the x-axis. The color of the dots represents the −log10(Q value) and the size of the dots are based on the number of genes with DMRs assigned to the enriched GO term on the left. DMR, differentially methylated region; FDR, false discovery rate.

Figure 6.

Integrative Genomics Viewer (IGV) tracks of Ndufa8 (A) and Uggt2 (B) after 30 days of recovery from EHS or EXC. The sequence coordinates of the promoters of Ndufa8 (chromosome 2; divergently transcribed relative to Mom5) and Uggt2 (chromosome 14) are indicated at the top of each panel. The first eight tracks indicate the sequencing coverage, i.e., the number of times that each CpG was sequenced (combined top and bottom strands) in each sample (EXC, blue bars; EHS, red bars). Only CpGs sequenced ≥10× are shown, with all coverage scales set from 0 to 60 reads. The % methylation tracks are all scaled from 0 to 100%. EXC, exercise control; EHS, exertional heat stroke.

Figure 7.

RNA-Seq results of EHS vs. EXC (n = 4/group). A: volcano plot of the log of fold changes in expression to all 14,696 RNA detected. Red outlined points and gene names are the six mRNAs that reached an FDR Q < 0.05. All were significantly downregulated. The post hoc power analysis revealed levels of β > 0.98 for all six identified mRNAs. B: heat map of the hierarchical expression of mRNA of all genes that exhibited ±1.5-fold differences between EHS and EXC. The color code is normalized to bright red (+2) representing the highest fold changes and cyan representing the lowest fold changes (−2). C: unbiased PCA plot of mRNA results using the genes that exhibited the highest overall variance compared to the mean (coefficient of determination) of all 8 samples (EXC + EHS) for a given gene. EXC, exercise control; EHS, exertional heat stroke; PCA, principal component analysis.

Figure 8.

Significantly different left ventricular metabolites. All data are expressed as a fraction of the mean exercise control value for that metabolite. Red bars: exertional heat stroke (EHS) vs. white bars: matched sham exercise controls (EXC); n = 7/group, means ± SD. Only 16-hydroxypalmitate was within a Benjamini–Hochberg FDR threshold of Q < 0.2. Uncorrected P values ***P < 0.001, **< 0.01, *< 0.05. The average post hoc power calculations for these 11 significant comparisons was 0.84, ranging from 0.57 to 0.99. FDR, false discovery rate.