Embryonic Caffeine Exposure Acts via A1 Adenosine Receptors to Alter Adult Cardiac Function and DNA Methylation in Mice

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

Caffeine acts via A1 adenosine receptors to disrupt embryonic cardiac function.

Evidence suggests that adenosine acts via cardiac A1 adenosine receptors (A1ARs) to protect embryos against hypoxia. During embryogenesis, A1ARs are the dominant regulator of heart rate, and A1AR activation reduces heart rate. Adenosine action is inhibited by caffeine, which is widely consumed during pregnancy. In this study, we tested the hypothesis that caffeine influences developing embryos by altering cardiac function.Effects of caffeine and adenosine receptor-selective antagonists on heart rate were studied in vitro using whole murine embryos at E9.5 and isolated hearts at E12.5. Embryos were examined in room air (21% O(2)) or hypoxic (2% O(2)) conditions. Hypoxia decreased heart rates of E9.5 embryos by 15.8% and in E12.5 isolated hearts by 27.1%. In room air, caffeine (200 µM) had no effect on E9.5 heart rates; however, caffeine increased heart rates at E12.5 by 37.7%. Caffeine abolished hypoxia-mediated bradycardia at E9.5 and blunted hypoxia-mediated bradycardia at E12.5. Real-time PCR analysis of RNA from isolated E9.5 and E12.5 hearts showed that A1AR and A2aAR genes were expressed at both ages. Treatment with adenosine receptor-selective antagonists revealed that SCH-58261 (A2aAR-specific antagonist) had no affects on heart function, whereas DPCPX (A1AR-specific antagonist) had effects similar to caffeine treatment at E9.5 and E12.5. At E12.5, embryonic hearts lacking A1AR expression (A1AR-/-) had elevated heart rates compared to A1AR+/- littermates, A1AR-/- heart rates failed to decrease to levels comparable to those of controls. Caffeine did not significantly affect heart rates of A1AR-/- embryos.These data show that caffeine alters embryonic cardiac function and disrupts the normal cardiac response to hypoxia through blockade of A1AR action. Our results raise concern for caffeine exposure during embryogenesis, particularly in pregnancies with increased risk of embryonic hypoxia

Effects of A1AR expression on response to caffeine at E12.5.

<p>Isolated hearts lacking A1ARs (−/−) were treated with 200 µM caffeine (n = 16) or vehicle (n = 10). Hearts from A1AR +/− littermates were treated with 200 µM caffeine (n = 18) or vehicle (n = 20). Caffeine treatment elevated heart rates in cultured hearts expressing A1ARs (p = 0.03). In hearts lacking A1ARs, caffeine treatment had no significant effect on heart rate (p = 0.101). Heart rates were normalized to baseline. Mean ± SEM are shown. * p = 0.03 caffeine compared to vehicle, unpaired Student's t-test.</p

Effects of A1AR expression on response to hypoxia at E9.5 and E12.5.

<p>A: At E9.5, responses to hypoxia in A1AR −/− (n = 15) and A1AR +/− (n = 50) embryos were studied. In hypoxia, E9.5 A1AR +/− heart rates decreased then returned to baseline levels following recovery in room air (p<0.0001 and p<0.01, for t = 25 and t = 40 compared to t = 0, respectively). E9.5 A1AR −/− responses to hypoxia were impaired as compared to controls, where responses were characterized by initial heart rate reductions followed by paradoxical heart rate elevations above A1AR +/− levels. B: At E12.5, hypoxia decreased heart rates below baseline in both hearts lacking A1ARs (n = 31) and those of control A1AR +/− littermates (n = 45; p<0.0001). When heart rates were analyzed as a percent of baseline levels there was no statistically significant difference in overall response among the different genotypes (p = 0.612). Because lack of A1ARs resulted in increased baseline heart rates when compared to those of control (p<0.05), non-normalized was also analyzed (C). This observation revealed that although hypoxia caused proportional decreases in heart rate independent of A1ARs, without A1ARs heart rates remained elevated above controls (p = 0.004). Mean ± SEM are shown. * p<0.01, ** p<0.0001, A1AR−/− compared to A1AR+/− at each me point.</p

Exposure to 2% O₂ induces embryonic tissue hypoxia.

<p>E9.5 embryos (A and B) and E12.5 hearts (C and D) were incubated with Hypoxyprobe-1 for 1 h under hypoxic (2% O₂; B and D) or normoxic conditions (A and C). Immunohistochemistry against Hypoxyprobe-1 conjugates (green), which form at <10 mm Hg, demonstrated an increase in tissue hypoxia when embryonic tissues were exposed to 2% O₂ for 1 h. V, ventricle. Scale bar: 200 µM (A and B), 100 µM (C and D).</p

Expression of adenosine receptor subtypes in E9.5 and E12.5 hearts.

<p>Real-time PCR analysis was performed on RNA samples extracted from isolated hearts at (A) E9.5 and (B) E12.5. Adenosine receptor gene expression was compared to Rp113a expression to determine the mean normalized expression. Each analysis was performed in triplicate.</p

Effects of adenosine receptor-specific antagonists on responses to hypoxia at E9.5 and E12.5.

<p>A and B: Specimens were treated with DPCPX, an A1AR-specific antagonist at 10 nM (A: E9.5, n = 15; B: E12.5, n = 23), SCH-58261, an A2aAR-specific antagonist at 100 nM (A: E9.5, n = 15; B: E12.5, n = 19), or vehicle (A: E9.5, n = 27; B: E12.5, n = 42) at 15 min (arrows), and exposed to hypoxia (gray boxes). At both ages, heart rates were not affected by SCH-58261 treatment (A and B). In contrast, at both ages DPCPX elevated heart rates above vehicle levels in normoxic conditions and inhibited hypoxia-mediated heart rate decrease below baseline (A and B). Although E12.5 heart rates did not fall below baseline in hypoxia, heart rates decreased compared to levels following DPCPX addition in room air (p<0.01 and p<0.0001, for t = 50 min and t = 65 min compared to t = 20 min, respectively, B). C: Because heart rates in E12.5 DPCPX-treated hearts significantly decreased in hypoxia, responses to DPCPX were examined over time in normoxia. E12.5 hearts were treated with 10 nM DPCPX (n = 19) or vehicle (n = 19). DPCPX- mediated heart rate elevation did not significantly diminish over time compared to levels at t = 20 min. Heart rates were normalized to baseline. Mean ± SEM are shown. * p<0.05, ** p<0.001, DPCPX compared to vehicle at each time point; † p<0.05 compared to DPCPX at t = 20 min.</p

Caffeine concentration-response curves at E9.5 and E12.5.

<p>A and B: Specimens were treated with increasing concentrations of caffeine (E9.5, n = 14; E12.5, n = 20) or vehicle (E9.5, n = 13; E12.5, n = 17). In room air, caffeine had no effect at E9.5 (A) but caused a concentration-dependent increase in heart rates at E12.5 (B). C: A concentration-response curve at E12.5 was produced by treating individual hearts with one concentration of caffeine or vehicle (n = 9–10 per concentration). At E12.5, the E_max was at 40 µM where caffeine increased heart rates to 137.7% of baseline and the EC₅₀ was 118.4 µM. Heart rates were normalized to baseline. Mean ± SEM are shown. X-axes are logarithmically scaled. * p<0.05, ** p<0.01, *** p<0.001 caffeine compared to vehicle.</p