Embryonic caffeine exposure acts via A1 adenosine receptors to alter adult cardiac function and DNA methylation in mice

Abstract

Evidence indicates that disruption of normal prenatal development influences an individual's risk of developing obesity and cardiovascular disease as an adult. Thus, understanding how in utero exposure to chemical agents leads to increased susceptibility to adult diseases is a critical health related issue. Our aim was to determine whether adenosine A1 receptors (A1ARs) mediate the long-term effects of in utero caffeine exposure on cardiac function and whether these long-term effects are the result of changes in DNA methylation patterns in adult hearts. Pregnant A1AR knockout mice were treated with caffeine (20 mg/kg) or vehicle (0.09% NaCl) i.p. at embryonic day 8.5. This caffeine treatment results in serum levels equivalent to the consumption of 2-4 cups of coffee in humans. After dams gave birth, offspring were examined at 8-10 weeks of age. A1AR+/+ offspring treated in utero with caffeine were 10% heavier than vehicle controls. Using echocardiography, we observed altered cardiac function and morphology in adult mice exposed to caffeine in utero. Caffeine treatment decreased cardiac output by 11% and increased left ventricular wall thickness by 29% during diastole. Using DNA methylation arrays, we identified altered DNA methylation patterns in A1AR+/+ caffeine treated hearts, including 7719 differentially methylated regions (DMRs) within the genome and an overall decrease in DNA methylation of 26%. Analysis of genes associated with DMRs revealed that many are associated with cardiac hypertrophy. These data demonstrate that A1ARs mediate in utero caffeine effects on cardiac function and growth and that caffeine exposure leads to changes in DNA methylation.

Figure 1. Embryonic caffeine exposure leads to increased weight in adulthood.

Male mice treated in utero with caffeine were weighed every week starting 2 weeks after birth. Between 3–8 weeks of age, caffeine/A1AR+/+ mice were significantly heavier than vehicle/A1AR+/+ mice. Two-way ANOVA with Bonferroni post-test comparison was performed. *P≤0.05, **P≤0.01, ***P≤0.001. N = 8.

Figure 2. Embryonic caffeine exposure leads to thickening of left ventricular walls.

Cardiac morphology was analyzed in adult mice by echocardiography at 8–10 weeks of age. (A) Caffeine-treated A1AR+/+ left ventricles were heavier than vehicle treated controls. Caffeine treatment of A1AR+/+ mice also caused increased thickness of the left ventricular posterior wall (LVPW) in both diastole (B) and systole (C), and increased thickness of the interventricular septum (IVS) in both diastole (D) and systole (E) when compared to vehicle controls. Vehicle/A1AR+/+, N = 6; Vehicle/A1AR +/−, N = 23; Vehicle/A1AR −/−, N = 8; caffeine/A1AR+/+, N = 6, caffeine/A1AR+/−, N = 15; caffeine/A1AR−/−, N = 10. Two-way ANOVA with Bonferroni post-test comparison was performed. *P≤0.05.

Figure 3. Embryonic caffeine exposure leads to altered cardiac function.

Cardiac function of mice exposed to in utero caffeine was analyzed by echocardiography between 8–10 weeks of age. Analysis revealed that A1AR+/+ caffeine-treated mice had reduced stroke volume (A), increased % fractional shortening (B), reduced left ventricular internal diameter (LVID) in both diastole (C) and systole (D), and reduced left ventricle volume at both diastole (E) and systole (F). Vehicle/A1AR+/+, N = 6; Vehicle/A1AR+/−, N = 23; Vehicle/A1AR−/−, N = 8; caffeine/A1AR+/+, N = 6, caffeine/A1AR+/−, N = 15; caffeine/A1AR−/−, N = 10. Two-way ANOVA with Bonferroni post-test comparison was performed. *P≤0.05.

Figure 4. No effect on connective tissue deposition in adult hearts was observed with caffeine treatment.

Adult hearts from mice exposed to in utero caffeine examined with trichrome stain to reveal heart muscle structure and connective tissue deposition. No differences in the amount of connective tissue deposition were observed between (A) veh+/+ and (B) caff+/+ adult left ventricles. However, caff+/+ left ventricles were thicker than veh+/+. N = 3. Scale bars = 100 µM.

Figure 5. Embryonic caffeine exposure caused a change in DNA methylation patterns.

Vehicle and caffeine treated mice with the same genotype were compared to identify differentially methylated regions (DMRs) in the genome (A). The veh+/+ vs. caff+/+ comparison identified the most differentially methylated regions in the genome following in utero caffeine exposure. Both (B) hypermethylated and (C) hypomethylated DMRs were detected throughout the genome with each comparison. Most DMRs were detected within the gene or promoter regions of the genome. The number of DNA samples per treatment group analyzed was 2.

Figure 6. Distribution of the differentially methylated regions.

(A) A Venn diagram indicates the number of DMRs that are shared by different comparison groups. The majority of DMRs within a comparison group are unique to that group with few regions detected in multiple comparison groups, and only 69 DMRs present in all three comparisons. (B) This chart illustrates locations in the genome for the DMRs identified from the veh+/+ vs. caff+/+ comparison. Analysis demonstrates that promoter regions from −1 to −3000 and intron regions contain the greatest percentage of DMRs. (C) In this chart, pink bars represent the percent of the genome that each chromosome contains and the blue bars are the percent of the total number of DMRs that are located on each chromosome. This chart identifies chromosomes 2, 7, and 11 as having the highest percentage of DMRs. N = 2.

Figure 7. Significantly enriched cardiovascular related pathways.

Gene set enrichment analysis was done with the differentially methylated genes between A1AR+/+ mice treated with or without caffeine. The analysis was conducted with MetaCore Enrichment Analysis using the ontologies of Diseases (by Biomarkers), Map Folders, Pathway Maps, and Process Networks. Bars represent the percentage of altered methylation genes (in black) within a pathway. The numbers of altered genes and genes in a pathway are listed next to the bars, which represent the percentage of altered genes within a pathway. Dots indicate the negative log 10 of the P-values. Larger –log(P-value) means that the pathway is more significant. The threshold for significance is marked in the graph as a dotted-line at 1.3 (−log(0.05)). N = 2.

Figure 8. Significantly enriched body weight related pathways.

Gene set enrichment analysis was performed with the differentially methylated genes between A1AR+/+ mice treated with or without caffeine. The analysis was conducted with MetaCore Enrichment Analysis using the ontologies of Diseases (by Biomarkers), Map Folders, Pathway Maps, and Process Networks. Bars represent the percentage of altered methylation genes (in black) within a pathway. The numbers of altered genes and genes in a pathway are listed next to the bars. Dots indicate the negative log 10 of the P-values. Larger –log(P-value) means that the pathway is more significant. The threshold for significance is marked in the graph as a dotted-line at 1.3 (−log(0.05)). N = 2.

Figure 9. Relation between gene expression and DNA methylation.

Expression of critical genes in the cardiac hypertrophy signaling pathway was compared to their DNA methylation status measured by DNA methylation array or bisulfite sequencing. A1AR+/+ mice treated in utero with either caffeine or vehicle were compared. Expression or methylation differences are shown as fold-change of caffeine treatments divided by normal saline controls. Gene expression results represent data from three repeats of qPCR measurements (N = 3 per group, genomic DNA and mRNA were extracted from left ventricles of the same animals). Student's t-test used and error bars are SEM. * indicates P≤0.05.

Figure 10. Embryonic caffeine exposure leads to a decrease in global DNA methylation.

DNA was isolated from adult left ventricles of mice treated in utero with caffeine. (A) The percentage of 5-methylcytosine decreased in caff+/+ hearts compared to veh+/+ controls, but no change in global DNA methylation levels was observed in caff+/− hearts compared to veh+/− hearts. (B) But there was no change in the percentage of 5-hydroxymethylcytosine in the left ventricular DNA in either the caff+/+ or caff+/− hearts. N = 3 per group, each sample was measured 3–4 times, student t-test, ** P = 0.0053).