Mitochondria antioxidant protection against cardiovascular dysfunction programmed by early-onset gestational hypoxia

Abstract

Mitochondria-derived oxidative stress during fetal development increases cardiovascular risk in adult offspring of pregnancies complicated by chronic fetal hypoxia. We investigated the efficacy of the mitochondria-targeted antioxidant MitoQ in preventing cardiovascular dysfunction in adult rat offspring exposed to gestational hypoxia, integrating functional experiments in vivo, with those at the isolated organ and molecular levels. Rats were randomized to normoxic or hypoxic (13%-14% O₂ ) pregnancy ± MitoQ (500 μM day^-1 ) in the maternal drinking water. At 4 months of age, one cohort of male offspring was chronically instrumented with vascular catheters and flow probes to test in vivo cardiovascular function. In a second cohort, the heart was isolated and mounted onto a Langendorff preparation. To establish mechanisms linking gestational hypoxia with cardiovascular dysfunction and protection by MitoQ, we quantified the expression of antioxidant system, β-adrenergic signaling, and calcium handling genes in the fetus and adult, in frozen tissues from a third cohort. Maternal MitoQ in hypoxic pregnancy protected offspring against increased α₁ -adrenergic reactivity of the cardiovascular system, enhanced reactive hyperemia in peripheral vascular beds, and sympathetic dominance, hypercontractility and diastolic dysfunction in the heart. Inhibition of Nfe2l2-mediated oxidative stress in the fetal heart and preservation of calcium regulatory responses in the hearts of fetal and adult offspring link molecular mechanisms to the protective actions of MitoQ treatment of hypoxic pregnancy. Therefore, these data show the efficacy of MitoQ in buffering mitochondrial stress through NADPH-induced oxidative damage and the prevention of programmed cardiovascular disease in adult offspring of hypoxic pregnancy.

Keywords: IUGR; cardiovascular; fetus; hypoxia; mitochondria; oxidative stress; programming.

Figure 1

Postnatal experimental design. Pregnant Wistar dams were exposed to normoxia (N; 21% O₂) or (hypoxia (H; 13%-14% O₂) from 6 to 20 days gestation (dGA) ± MitoQ (NM and HM, respectively) provided at 500 μM day⁻¹ in the maternal drinking water. Offspring were born and maintained in normoxia until 4 months of age. One male per litter was randomly selected for either conscious in vivo cardiovascular testing in chronically instrumented preparations or ex vivo analysis of cardiac function using an isolated heart Langendorff preparation. For in vivo testing, offspring were instrumented with femoral vascular catheters and a femoral transonic flow probe placed in the contralateral leg. Basal and stimulated in vivo cardiovascular function was determined following 4-5 days of postsurgical recovery

Figure 2

Effect of hypoxic pregnancy and MitoQ on basal cardiovascular function in the adult offspring. Values are means ± SEM for heart rate (HR; A), mean arterial pressure (MAP; B), systolic blood pressure (SBP; C), diastolic blood pressure (DBP; D), the rate-pressure product (RPP; E), and femoral blood flow amplitude (FBF Amp; F) in adult offspring of normoxic (N, n = 6-10), hypoxic (H, n = 5-8), hypoxic treated with MitoQ (HM, n = 6-8), or normoxic treated with MitoQ (NM, n = 6-9) pregnancies. Statistical differences are (P < .05): *main effect of hypoxia; hypoxia x MitoQ interaction indicated by brackets (Two-Way ANOVA with Tukey’s post hoc comparison)

Figure 3

Effect of hypoxic pregnancy and MitoQ on stimulated cardiovascular function in the adult offspring. Values are means ± SEM. for the percent change from baseline in mean arterial pressure (MAP; A), systolic blood pressure (SBP; B), diastolic blood pressure (DBP; C), and the fall in the amplitude of femoral blood flow (FBF Amp; D) in response to in vivo treatment with an intravenous dose of 80 μg kg^-1 phenylephrine (PE) in conscious adult offspring of normoxic (N, n = 6-8), hypoxic (H, n = 4-8), hypoxic treated with MitoQ (HM, n = 6-7), or normoxic treated with MitoQ (NM, n = 7) pregnancies. Panels E and F show the reactive hyperemic response in femoral blood flow post-PE infusion in terms of time to peak and the peak amplitude of the femoral dilator response, respectively. Statistical differences are (P < .05): * main effect of hypoxia; † main effect of MitoQ; hypoxia x MitoQ interaction indicated by brackets (Two-Way ANOVA with Tukey’s post hoc comparison)

Figure 4

Effect of hypoxic pregnancy and MitoQ on isolated cardiac function in the adult offspring. Values are means ± SEM. for the contractility index (A), the inotropic sympathetic dominance (B), left ventricular developed pressure (LVDP; C), the maximum first derivative of the left ventricular pressure (dP/dtmax; D), left ventricular end diastolic pressure (LVEDP; E), and the minimum first derivative of the left ventricular pressure (dP/dtmin; F) in adult offspring of normoxic (N, n = 5-6), hypoxic (H, n = 8), hypoxic treated with MitoQ (HM, n = 6), or normoxic treated with MitoQ (NM, n = 7-8) pregnancies. The inotropic sympathetic dominance was calculated as the ratio of the LVDP response to a maximal dose of isoprenaline relative to a maximal dose of carbachol. Statistical differences are (P < .05): * main effect of hypoxia; hypoxia x MitoQ interaction indicated by brackets (Two-Way ANOVA with Tukey’s post hoc comparison)

Figure 5

Effect of hypoxic pregnancy and MitoQ on mitochondrial oxidative stress and endogenous antioxidant mRNA expression in the heart of fetal and adult offspring. Values are for the mRNA expression in hearts of fetal (A-D) and adult (E-H) offspring of hypoxic (H; grey, n = 5-6), hypoxic MitoQ (HM; red, n = 6-7), and normoxic MitoQ (NM; blue, n = 5-9) relative to normoxic (N, n = 6-8) groups, and of HM relative to H (striped) groups. Data are normalized to the geometric mean of the two most stable reference genes and are reported as fold change (95% Confidence Interval, CI). Where CIs do not include 1.0, mRNA expression is statistically different from N (*: H, HM, and NM), or H (†: HM) at the 5% level

Figure 6

Effect of hypoxic pregnancy and MitoQ on calcium regulation and β-adrenergic signaling mRNA expression in the heart of fetal and adult offspring. Values are for the mRNA expression in hearts of fetal (A-D) and adult (E-H) offspring of hypoxic (H; grey, n = 5-6), hypoxic MitoQ (HM; red, n = 6-7), and normoxic MitoQ (NM; blue, n = 5-9) relative to normoxic (N, n = 6-8) groups, and of HM relative to H (striped) groups. Data are normalized to the geometric mean of the two most stable reference genes and are reported as fold change (95% Confidence Interval, CI). Where CIs do not include 1.0, mRNA expression is statistically different from N (*: H, HM, and NM) at the 5% level