Long-term exposure to high altitude hypoxia during pregnancy increases fetal heart susceptibility to ischemia/reperfusion injury and cardiac dysfunction

Abstract

Background: High altitude hypoxia (HAH) exposure affects fetal development. However, the fetal cardiovascular responses to the HAH are not well understood. We have tested the hypothesis that long-term HAH exposure alters the hypoxia/ischemia-sensitive gene expressions, leading to an increase in fetal heart susceptibility to ischemia/reperfusion (I/R) injury and cardiac dysfunction.

Methods: Time-dated pregnant sheep were exposed to high-altitude (3820 m) or were maintained at sea level (~300 m) for 110 days. Fetal hearts were isolated from the near-term ewes and subjected to I/R in a Langendorff preparation.

Results: HAH decreased the fetal body and heart weights in the female but not male fetuses. HAH had no effect on the left ventricle (LV) function at baseline, but increased the LV infarct size and attenuated the post-ischemic recovery of LV function in both male and female fetuses, as compared with the normoxic groups. HAH increased the protein levels of hypoxia-inducible factor (HIF)-1α and DNA methyltransferases type 3b (DNMT3b), but attenuated protein kinase C epsilon (PKCε) levels in the fetal hearts. AHA induced a 4.3 fold increase of miR-210 in the males and a 2.9 fold increase in female hearts. In addition, HAH had no effect on mTOR protein and phosphorylation levels but increased the autophagy biomarker, LC3B-II protein levels and LC3B-II/LC3B-I ratio in the fetal hearts.

Conclusion: The results suggest that gestational HAH exposure induces in utero programming of the hypoxia/ischemia-sensitive gene expression pattern in the developing heart and increases cardiac susceptibility to I/R injury.

Keywords: Fetal heart; High altitude hypoxia; Ischemia/reperfusion injury.

Figure 1.. Effect of HAH on post ischemic recovery of LV function in both male and female fetuses.

Hearts were isolated from the male and female fetuses. The hearts were subjected to 20 min of ischemia and 60 min of reperfusion in a Langendorff preparation. Post-ischemic recoveries of the left ventricular diastolic pressures (LVDP) in male (A) and female (E). dP/dpmax in male (B) and female (F). dP/dpmin in male (C) and female (G). Heart rate in male and female (H). Data are means ± SEM of animals from each group. Data were analyzed by 2-way repeated measures ANOVA (*P < 0.05 vs. control group). Then, compare the two group at every time point using multiple t-test comparison (# P < 0.05 vs. control at each time point).

Figure 2.. Effect of HAH on I/R-induced coronary flow rate (CF), LVEDP and myocardial infarction in both male and female fetuses.

Hearts were isolated from the male and female fetuses. The hearts were subjected to 20 min of ischemia and 60 min of reperfusion in a Langendorff preparation. During ischemia/reperfusion (I/R), the pulmonary artery effluent was collected from both male (A) and female (D) as an index of coronary flow (milliliters per minute per gram of heart wet weigh). Post-ischemic recovery of the left ventricular end-diastolic pressures (LVEDP) was determined during the course of reperfusion in both male (B) and female (E) fetuses. The left ventricular tissue were collected from both male (C) and female fetuses at the end of reperfusion, and the myocardial infarct size was determined with 1% triphenyltrazolium chloride (TTC) staining and expressed as a percentage of the total ventricular weight. Data are means ± SEM of animals from each group. Data for CF and LEVDP were analyzed with 2-way repeated measures ANOVA (*P < 0.05 vs. control group). Then, compare the two group at every time point using multiple t-test comparison (# P < 0.05 vs. control at each time point). Data for infarct size were analyzed by student t-test. *P < 0.05 vs control (normoxia).

Figure 3.. HAH-mediated changes of hypoxic biomarkers and protein expressions.

Heart were isolated from fetuses from near-term pregnant sheep maintained at sea level (control) or exposed to high altitude hypoxia (HAH). Protein abundances in the left ventricle (LV) tissues were determined by Western blot analyses. The protein levels of HIF-1α in male (A) and female (E) LV tissues. The protein levels of DNMT3b in male (C) and female (G) LV tissues. The protein levels of PKCε in male (D) and female (H) LV tissues. The protein levels are expressed as fold of GAPDH (loading control). MiRNA-210 levels in the LV tissues isolated from male and female (F) fetuses were measured by qRT-PCR analysis, as described under Material and Methods. The expression of miR-210 are expressed as percentage of SNORD61 (internal control). Data are means ± SEM of animals from each group and were analyzed by student t-test. *P < 0.05 vs control (normoxia).

Figure 4.. Effect of HAH on autophagy-related protein expressions.

Heart were isolated from fetuses from near-term pregnant sheep maintained at sea level (control) or exposed to high altitude hypoxia (HAH). Protein abundances in the left ventricle (LV) tissues were determined by Western blot analyses. The protein levels of phosphor-mTOR in male (A) and female (D) LV tissues. The total protein levels of mTOR in male (B) and female (E) LV tissues. The ratio of LC3B-II to LC3B-I protein levels in male (C) and female (F) LV tissues. Data are means ± SEM of animals from each group and were analyzed by student t-test. *P < 0.05 vs control (normoxia).