Figure 1.
Caffeine concentration-response curves at E9.5 and E12.5.
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 Emax 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.
Figure 2.
Exposure to 2% O2 induces embryonic tissue hypoxia.
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).
Figure 3.
Caffeine effects on embryonic heart function at E9.5 and E12.5 under normoxic and hypoxic (2% O2) conditions.
In room air, E9.5 embryos (A) and E12.5 hearts (B) were treated with 200 µM caffeine (E9.5, n = 20; E12.5, n = 17) or vehicle (E9.5, n = 24; E12.5, n = 17) at 15 min, as indicated by arrows. At both ages, heart rates were stable for the duration of the experiment, as demonstrated by lack of heart rate variability in vehicle-treated specimens (E9.5, p = 0.394; E12.5, p = 0.263). At E9.5 (A), there were no differences in heart rates between caffeine and vehicle-treated groups. At E12.5 (B), caffeine elevated heart rates above vehicle levels. Following addition of caffeine, heart rates in E12.5 specimens did not vary over time, suggesting the caffeine effects did not significantly diminish in the time required for experiments (p = 0.093). C and D: The effects of hypoxia were determined at both ages in the presence of 200 µM caffeine (E9.5, n = 20; E12.5, n = 22) or vehicle (E9.5, n = 19; E12.5, n = 22). At both ages, heart rates decreased in hypoxia (gray boxes) and increased following recovery in room air (C and D, vehicle, p<0.0001). At E9.5 (C), caffeine treatment completely inhibited hypoxia-mediated decrease in heart rates (p = 0.038). At E12.5 (D), caffeine treatment elevated heart rates and blunted hypoxia-mediated decrease in heart rate (p = 0.002). Heart rates were normalized to baseline. Mean ± SEM are shown. * p<0.05, ** p<0.001, caffeine compared to vehicle at each time point.
Figure 4.
Expression of adenosine receptor subtypes in E9.5 and E12.5 hearts.
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.
Figure 5.
Effects of adenosine receptor-specific antagonists on responses to hypoxia at E9.5 and E12.5.
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.
Table 1.
Mean baseline heart rates in A1AR transgenic embryos.
Figure 6.
Effects of A1AR expression on response to caffeine at E12.5.
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.
Figure 7.
Effects of A1AR expression on response to hypoxia at E9.5 and E12.5.
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.
Figure 8.
Schematic of influences of adenosine and hypoxia on embryonic heart rate regulation.
Hypoxia induces increased levels of adenosine, which in turn activate A1ARs to lower heart rate. Reduction in heart rate will affect embryonic tissue perfusion, which in turn will influence embryonic development. At E9.5, effects of hypoxia on heart rate appear to be exclusively mediated via A1ARs. At E12.5, hypoxia also can influence heart rate independent of A1ARs. Caffeine, an adenosine antagonist, will perturb this physiological cascade.