Embryonic caffeine exposure induces adverse effects in adulthood

Abstract

The purpose of this study was to determine both the short-term effects on cardiac development and embryo growth and the long-term effects on cardiac function and body composition of in utero caffeine exposure. Pregnant mice (C57BL/6) were exposed to hypoxia (10% O(2)) or room air from embryonic days (E) 8.5-10.5, and treated with caffeine (20 mg/kg, i.p.) or vehicle (normal saline, 0.9% NaCl). This caffeine dose results in a circulating level that is equivalent to 2 cups of coffee in humans. Hypoxic exposure acutely reduced embryonic growth by 30%. Exposure to a single dose of caffeine inhibited cardiac ventricular development by 53% in hypoxia and 37% in room air. Caffeine exposure resulted in inhibition of hypoxia-induced HIF1alpha protein expression in embryos by 40%. When offspring from dams treated with a single dose of caffeine were studied in adulthood, we observed that caffeine treatment alone resulted in a decrease in cardiac function of 38%, as assessed by echocardiography. We also observed a 20% increase in body fat with male mice exposed to caffeine. Caffeine was dissolved in normal saline, so it was used as a control. Room air controls were used to compare to the hypoxic mice. Exposure to a single dose of caffeine during embryogenesis results in both short-term effects on cardiac development and long-term effects on cardiac function.

Figure 1.

Hypoxia is primarily responsible for growth retardation in embryos. Pregnant dams were exposed to either room air (A, B) or 10% oxygen (C, D) from E8.5–10.5. Dams were injected once at E8.5 with normal saline (A, C) or 20 mg/kg caffeine. The crown-rump (C-R) length was measured at the end of experiment at E10.5. NorNS (A) embryos were indistinguishable from NorCf (B) embryos in crown-rump length (E). Both HyNS (C) and HyCf (D) were significantly smaller than their room air controls (E). HyCf embryos were significantly slightly smaller then HyNS embryos (E). No gross structural anomalies were observed, *P ≤ 0.001; one-way ANOVA with Bonferroni posttest. **P ≤ 0.05; Student’s t test. Scale bars = 1 mm. n = number of embryos measured.

Figure 2.

Hypoxia and caffeine treatment lead to reduced ventricular myocardial tissue. Embryos were exposed to hypoxia and caffeine from E8.5-E10.5. Embryos were collected at E10.5 and processed for histological analysis. Hematoxylin and eosin (H&E)-stained saggital sections were used to measure the cross-sectional area of the ventricular myocardium and AV canal cushions at the level of the cardiac cushions. Compared to NorNS (A, n=3), ventricular myocardial area was decreased by 37.3% in the NorCf (B, E; n=3) group. Exposure to hypoxia caused a more substantial decrease in myocardial area, including 55.7% in the HyNS group (C, E; n=5) and 53.3% in the HyCf group (D, E; n=5). Hypoxia caused a decrease in AV canal area under both hypoxia conditions, but not caffeine alone (E). V, ventricle; ★, AVC cushions (A–D). *P ≤ 0.001, **P ≤ 0.01; one-way ANOVA with Bonferroni posttest. Scale bars = 100 μm.

Figure 3.

Caffeine treatment inhibited HIF1α protein accumulation in hypoxic embryos. Dams were injected with normal saline or 20 mg/kg caffeine and then placed immediately in hypoxia (10% O₂) or left in room air (21% O₂). Embryos were collected after 6 h, and whole embryo protein was isolated. Western blot analysis of HIF1α protein expression demonstrated a 40% reduction in stabilized HIF1α protein in caffeine-treated embryos exposed to hypoxia. β-Actin protein was examined on the same Western blots as a loading control.

Figure 4.

Caffeine treatment leads to increased body fat in adult male mice. Dams were treated with a single dose of normal saline or 20 mg/kg caffeine at E8.5 and then placed in hypoxia (10% O₂) or left in room air until E10.5. Dams and pups were then reared in room air, and body fat analysis was performed at 8 wk of age. Adult NorCf males are significantly larger than the NorNS males. *P ≤ 0.02; Spearman test.

Figure 5.

Adult cardiac function is reduced by in utero exposure to both hypoxia and caffeine. Dams were treated with normal saline or caffeine and exposed to hypoxia or room air, as described in Materials and Methods. Following in utero treatment, pups were reared in room air, and echocardiography was performed on 8-to 10-wk-old male and female mice. No difference was observed between sexes, so data were combined. Percentage fractional shortening, a measure of cardiac function, was reduced in the HyNS group compared to NorNS (A). Both the NorCf and HyCf groups had significantly reduced fractional shortening compared to their normal saline controls (A). Along with reduced function, both caffeine-treated groups had increased LVIDs during diastole compared to their normal saline controls; this effect was not seen with hypoxia treatment alone (B). *P ≤ 1 × 10⁻¹⁴; one-way ANOVA with Bonferroni posttest. **P ≤ 0.003; Student’s t test. NorNS, n = 21; NorCf, n = 22; HyNS, n = 19; HyCf, n = 15.

Figure 6.

No effects on muscle structure or connective tissue deposition were observed in adult hearts. Adult hearts from mice exposed to hypoxia and caffeine in utero were collected and examined with histological stains. No differences were observed in the myocardial structure with H&E staining (A–D). In addition, heart sections were stained with trichrome, and no differences in connective tissue accumulation were observed (E–H). NorNS (A, E), NorCf (B, F), HyNS (C, G), and HyCf (D, H). Scale bars = 100 μM.