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Fetal growth restriction (FGR) is a common outcome in human suboptimal gestation and is related to prenatal origins of cardiovascular dysfunction in offspring. Despite this, therapy of human translational potential has not been identified. Using human umbilical and placental vessels and the chicken embryo model, we combined cellular, molecular, and functional studies to determine whether N-acetylcysteine (NAC) and hydrogen sulphide (H 2 S) protect cardiovascular function in growth-restricted unborn offspring. In human umbilical and placental arteries from control or FGR pregnancy and in vessels from near-term chicken embryos incubated under normoxic or hypoxic conditions, we determined the expression of the H 2 S gene CTH (i.e. cystathionine γ-lyase) (via quantitative PCR), the production of H 2 S (enzymatic activity), the DNA methylation profile (pyrosequencing) and vasodilator reactivity (wire myography) in the presence and absence of NAC treatment. The data show that FGR and hypoxia increased CTH expression in the embryonic/fetal vasculature in both species. NAC treatment increased aortic CTH expression and H 2 S production and enhanced third-order femoral artery dilator responses to the H 2 S donor sodium hydrosulphide in chicken embryos. NAC treatment also restored impaired endothelial relaxation in human third-to-fourth order chorionic arteries from FGR pregnancies and in third-order femoral arteries from hypoxic chicken embryos. This NAC-induced protection against endothelial dysfunction in hypoxic chicken embryos was mediated via nitric oxide independent mechanisms. Both developmental hypoxia and NAC promoted vascular changes in CTH DNA and NOS3 methylation patterns in chicken embryos. Combined, therefore, the data support that the effects of NAC and H 2 S offer a powerful mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy. KEY POINTS: Gestation complicated by chronic fetal hypoxia and fetal growth restriction (FGR) increases a prenatal origin of cardiovascular disease in offspring, increasing interest in antenatal therapy to prevent against a fetal origin of cardiovascular dysfunction. We investigated the effects between N-acetylcysteine (NAC) and hydrogen sulphide (H 2 S) in the vasculature in FGR human pregnancy and in chronically hypoxic chicken embryos. Combining cellular, molecular, epigenetic and functional studies, we show that the vascular expression and synthesis of H 2 S is enhanced in hypoxic and FGR unborn offspring in both species and this acts to protect their vasculature. Therefore, the NAC/H 2 S pathway offers a powerful therapeutic mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Chronic fetal hypoxaemia is a common pregnancy complication that increases the risk of infants experiencing respiratory complications at birth. In turn, chronic fetal hypoxaemia promotes oxidative stress, and maternal antioxidant therapy in animal models of hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development. However, whether antenatal antioxidant therapy confers any benefit on lung development in complicated pregnancies has not yet been investigated. Here, we tested the hypothesis that maternal antenatal treatment with MitoQ will protect the developing lung in hypoxic pregnancy in sheep, a species with similar fetal lung developmental milestones as humans. Maternal treatment with MitoQ during late gestation promoted fetal pulmonary surfactant maturation and an increase in the expression of lung mitochondrial complexes III and V independent of oxygenation. Maternal treatment with MitoQ in hypoxic pregnancy also increased the expression of genes regulating liquid reabsorption in the fetal lung. These data support the hypothesis tested and suggest that MitoQ as an antenatal targeted antioxidant treatment may improve lung maturation in the late gestation fetus. KEY POINTS: Chronic fetal hypoxaemia promotes oxidative stress, and maternal antioxidant therapy in hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development. MitoQ is a targeted antioxidant that uses the cell and the mitochondrial membrane potential to accumulate within the mitochondria. Treatment of healthy or hypoxic pregnancy with MitoQ, increases the expression of key molecules involved in surfactant maturation, lung liquid reabsorption and in mitochondrial proteins driving ATP synthesis in the fetal sheep lung. There were no detrimental effects of MitoQ treatment alone on the molecular components measured in the present study, suggesting that maternal antioxidant treatment has no effect on other components of normal maturation of the surfactant system., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The use of statins in complicated pregnancy is being considered, as they protect endothelial function in the mother and placenta. However, whether statins affect cardiovascular function in the fetus is completely unknown. Here, we have determined the effects of pravastatin and underlying mechanisms on the cardiovascular system of the hypoxic chicken embryo, a model system that permits the direct effects of pravastatin on the developing offspring to be isolated independently of additional effects on the mother and/or placenta. Chicken embryos were incubated under normoxia or hypoxia (14% O 2 ) from day 1 ± pravastatin (1 mg/kg/d) from day 13 of incubation (term is 21 days). On day 19 of incubation, hearts and vessels were isolated to determine changes in the cardiovascular structure and function. The data show that pravastatin protected the hypoxic chicken embryo against impaired cardiovascular dysfunction. Mechanisms involved in this protection included reduced oxidative stress, enhanced NO bioavailability, restored antioxidant defenses and normalized protein expression of RhoA in the embryonic heart, and improved NO-dependent vasodilator mechanisms in the peripheral circulation. Therefore, we show that the treatment of the chronically hypoxic chicken embryo with pravastatin from day 13 of incubation, equivalent to ca. 25 weeks of gestation in human pregnancy, has direct beneficial effects on the embryonic cardiovascular system. Therefore, pravastatin may be a candidate for human clinical translation to rescue fetal cardiovascular dysfunction in risky pregnancy., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

In mammals, pregnancy complicated by chronic hypoxia can program hypertension in the adult offspring. However, mechanisms remain uncertain because the partial contributions of the challenge on the placenta, mother, and fetus are difficult to disentangle. Here, we used chronic hypoxia in the chicken embryo-an established model system that permits isolation of the direct effects of developmental hypoxia on the cardiovascular system of the offspring, independent of additional effects on the mother or the placenta. Fertilized chicken eggs were exposed to normoxia (N; 21% O 2 ) or hypoxia (H; 13.5%-14% O 2 ) from the start of incubation (day 0) until day 19 (hatching, ≈day 21). Following hatching, all birds were maintained under normoxic conditions until ≈6 months of adulthood. Hypoxic incubation increased hematocrit (+27%) in the chicken embryo and induced asymmetrical growth restriction (body weight, -8.6%; biparietal diameter/body weight ratio, +7.5%) in the hatchlings (all P <0.05). At adulthood (181±4 days), chickens from hypoxic incubations remained smaller (body weight, -7.5%) and showed reduced basal and stimulated in vivo NO bioavailability (pressor response to NG-nitro-L-arginine methyl ester, -43%; phenylephrine pressor response during NO blockade, -61%) with significant hypertension (mean arterial blood pressure, +18%), increased cardiac work (ejection fraction, +12%; fractional shortening, +25%; enhanced baroreflex gain, +456%), and left ventricular wall thickening (left ventricular wall volume, +36%; all P <0.05). Therefore, we show that chronic hypoxia can act directly on a developing embryo to program hypertension, cardiovascular dysfunction, and cardiac wall remodeling in adulthood in the absence of any maternal or placental effects.

Integrating functional and molecular levels, we investigated the effects of maternal treatment with a xanthine oxidase inhibitor on the programming of cardiac dysfunction in adult offspring using an established rat model of hypoxic pregnancy. Female Wistar rats were divided into normoxic or hypoxic (13% O 2 ) pregnancy±maternal allopurinol treatment (30 mg kg -1 d -1 ). At 4 months, hearts were isolated from 1 male per litter per outcome variable to determine cardiac function and responses to ischemia-reperfusion in a Langendorff preparation. Sympathetic dominance, perfusate CK (creatine kinase) and LDH (lactate dehydrogenase) and the cardiac protein expression of the β 1 -adrenergic receptor, the M 2 Ach receptor (muscarinic type-2 acetylcholine receptor), and the SERCA2a (sarcoplasmic reticulum Ca 2+ ATPase 2a) were determined. Relative to controls, offspring from hypoxic pregnancy showed elevated left ventricular end diastolic pressure (+34.7%), enhanced contractility (dP/dt max , +41.6%), reduced coronary flow rate (-21%) and an impaired recovery to ischemia-reperfusion (left ventricular diastolic pressure, area under the curve recovery -19.1%; all P<0.05). Increased sympathetic reactivity (heart rate, +755.5%; left ventricular diastolic pressure, +418.9%) contributed to the enhanced myocardial contractility ( P<0.05). Perfusate CK (+431%) and LDH (+251.3%) and the cardiac expression of SERCA2a (+71.4%) were also elevated ( P<0.05), further linking molecular markers of cardiac stress and injury to dysfunction. Maternal allopurinol restored all functional and molecular indices of cardiac pathology. The data support a link between xanthine oxidase-derived oxidative stress in hypoxic pregnancy and cardiac dysfunction in the adult offspring, providing a target for early intervention in the developmental programming of heart disease.

Experimental studies in animal models supporting protective effects on the fetus of melatonin in adverse pregnancy have prompted clinical trials in human pregnancy complicated by fetal growth restriction. However, the effects of melatonin on the fetal defense to acute hypoxia, such as that which may occur during labor, remain unknown. This translational study tested the hypothesis, in vivo, that melatonin modulates the fetal cardiometabolic defense responses to acute hypoxia in chronically instrumented late gestation fetal sheep via alterations in fetal nitric oxide (NO) bioavailability. Under anesthesia, 6 fetal sheep at 0.85 gestation were instrumented with vascular catheters and a Transonic flow probe around a femoral artery. Five days later, fetuses were exposed to acute hypoxia with or without melatonin treatment. Fetal blood was taken to determine blood gas and metabolic status and plasma catecholamine concentrations. Hypoxia during melatonin treatment was repeated during in vivo NO blockade with the NO clamp. This technique permits blockade of de novo synthesis of NO while compensating for the tonic production of the gas, thereby maintaining basal cardiovascular function. Melatonin suppressed the redistribution of blood flow away from peripheral circulations and the glycemic and plasma catecholamine responses to acute hypoxia. These are important components of the fetal brain sparing response to acute hypoxia. The effects of melatonin involved NO-dependent mechanisms as the responses were reverted by fetal treatment with the NO clamp. Melatonin modulates the in vivo fetal cardiometabolic responses to acute hypoxia by increasing NO bioavailability., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Fetal hypoxia is a common complication of pregnancy. It has been shown to programme cardiac and endothelial dysfunction in the offspring in adult life. However, the mechanisms via which this occurs remain elusive, precluding the identification of potential therapy. Using an integrative approach at the isolated organ, cellular and molecular levels, we tested the hypothesis that oxidative stress in the fetal heart and vasculature underlies the molecular basis via which prenatal hypoxia programmes cardiovascular dysfunction in later life. In a longitudinal study, the effects of maternal treatment of hypoxic (13% O(2)) pregnancy with an antioxidant on the cardiovascular system of the offspring at the end of gestation and at adulthood were studied. On day 6 of pregnancy, rats (n = 20 per group) were exposed to normoxia or hypoxia ± vitamin C. At gestational day 20, tissues were collected from 1 male fetus per litter per group (n = 10). The remaining 10 litters per group were allowed to deliver. At 4 months, tissues from 1 male adult offspring per litter per group were either perfusion fixed, frozen, or dissected for isolated organ preparations. In the fetus, hypoxic pregnancy promoted aortic thickening with enhanced nitrotyrosine staining and an increase in cardiac HSP70 expression. By adulthood, offspring of hypoxic pregnancy had markedly impaired NO-dependent relaxation in femoral resistance arteries, and increased myocardial contractility with sympathetic dominance. Maternal vitamin C prevented these effects in fetal and adult offspring of hypoxic pregnancy. The data offer insight to mechanism and thereby possible targets for intervention against developmental origins of cardiac and peripheral vascular dysfunction in offspring of risky pregnancy.

Key points: In mammals, pregnancy complications can trigger an embryonic or fetal origin of cardiac dysfunction. However, underlying mechanisms remain uncertain because the partial contributions of the challenge on the mother, placenta or offspring are difficult to disentangle.The avian embryo permits isolation of the direct effects of suboptimal conditions during development on the cardiac function of the offspring, independent of additional effects on the mother and/or the placenta.Therefore, the objectives of this work were to adapt the isolated Langendorff technique using the chicken embryo to study the physiology of the developing heart.Here, we introduce a novel technique and show the utility of the technique for exploring cardioprotective roles of H2S in the chicken embryo heart. This work lays the foundation for studying the direct effects of H2S therapy on the embryonic heart independent of effects on the mother and the placenta in adverse development. This study adapted the isolated Langendorff preparation to study the chicken embryo heart in response to ischaemia‐reperfusion (IR) injury. The utility of the technique was tested by investigating cardioprotective effects of hydrogen sulphide (H2S) and underlying mechanisms. Embryonic hearts (19 out of 21 days of incubation) mounted on a Langendorff preparation were exposed to IR (30 min ischaemia) after 4 treatments administered randomly, all as a 1 mm bolus, into the perfusate: saline vehicle (control); sodium hydrogen sulphide (NaHS); NaHS plus glibenclamide, an antagonist of KATP opening (NaHS Glib), and Glib alone (Glib). Relative to controls, NaHS treatment improved cardiac function after ischaemia (mean ± SD for area under the curve, AUC, for left ventricular developed pressure, LVDP: 1767.3 ± 929.5 vs. 492.7 ± 308.1; myocardial contractility, dP/dtmax: 2748.9 ± 1514.9 vs. 763.7 ± 433.1) and decreased infarct size (22.7 ± 8.0 vs. 43.9 ± 4.2%) and cardiac damage (% change in creatinine kinase, 49.3 ± 41.3 vs. 214.6 ± 155.1; all P < 0.05). Beneficial effects of NaHS were blocked by Glib. Glib alone had no effects. NaHS increased coronary flow rate (CFR) during baseline (mean ± SD for AUC: 134.3 ± 91.6 vs. 92.2 ± 35.8) and post IR (1467 ± 529.5 vs. 748.0 ± 222.1; both P < 0.05). However, this effect was not prevented by Glib. Therefore, the chicken embryo heart is amenable for study via the Langendorff preparation under basal conditions and during IR. The data show that H2S confers embryonic cardiac protection via opening of myocardial KATP channels and not via increasing CFR. H2S may prove a useful therapeutic agent to protect the human fetal heart against IR injury, as may occur in complicated labour. Key points: In mammals, pregnancy complications can trigger an embryonic or fetal origin of cardiac dysfunction. However, underlying mechanisms remain uncertain because the partial contributions of the challenge on the mother, placenta or offspring are difficult to disentangle.The avian embryo permits isolation of the direct effects of suboptimal conditions during development on the cardiac function of the offspring, independent of additional effects on the mother and/or the placenta.Therefore, the objectives of this work were to adapt the isolated Langendorff technique using the chicken embryo to study the physiology of the developing heart.Here, we introduce a novel technique and show the utility of the technique for exploring cardioprotective roles of H2S in the chicken embryo heart. This work lays the foundation for studying the direct effects of H2S therapy on the embryonic heart independent of effects on the mother and the placenta in adverse development. [ABSTRACT FROM AUTHOR]

Key points: We introduce a technique to test whether intrinsic fetal heart rate variability (iFHRV) exists and we show the utility of the technique by testing the hypothesis that iFHRV is affected by chronic fetal hypoxia, one of the most common adverse outcomes of human pregnancy complicated by fetal growth restriction.Using an established late gestation ovine model of fetal development under chronic hypoxic conditions, we identify iFHRV in isolated fetal hearts and show that it is markedly affected by hypoxic pregnancy.Therefore, the isolated fetal heart has intrinsic variability and carries a memory of adverse intrauterine conditions experienced during the last third of pregnancy. Fetal heart rate variability (FHRV) emerges from influences of the autonomic nervous system, fetal body and breathing movements, and from baroreflex and circadian processes. We tested whether intrinsic heart rate variability (iHRV), devoid of any external influences, exists in the fetal period and whether it is affected by chronic fetal hypoxia. Chronically catheterized ewes carrying male singleton fetuses were exposed to normoxia (n = 6) or hypoxia (10% inspired O2, n = 9) for the last third of gestation (105–138 days of gestation (dG); term ∼145 dG) in isobaric chambers. At 138 dG, isolated hearts were studied using a Langendorff preparation. We calculated basal intrinsic FHRV (iFHRV) indices reflecting iFHRV's variability, predictability, temporal symmetry, fractality and chaotic behaviour, from the systolic peaks within 15 min segments in each heart. Significance was assumed at P < 0.05. Hearts of fetuses isolated from hypoxic pregnancy showed approximately 4‐fold increases in the Grid transformation as well as the AND similarity index (sgridAND) and a 4‐fold reduction in the scale‐dependent Lyapunov exponent slope. We also detected a 2‐fold reduction in the Recurrence quantification analysis, percentage of laminarity (pL) and recurrences, maximum and average diagonal line (dlmax, dlmean) and the Multiscale time irreversibility asymmetry index. The iHRV measures dlmax, dlmean, pL and sgridAND correlated with left ventricular end‐diastolic pressure across both groups (average R2 = 0.38 ± 0.03). This is the first evidence that iHRV originates in fetal life and that chronic fetal hypoxia significantly alters it. Isolated fetal hearts from hypoxic pregnancy exhibit a time scale‐dependent higher complexity in iFHRV. Key points: We introduce a technique to test whether intrinsic fetal heart rate variability (iFHRV) exists and we show the utility of the technique by testing the hypothesis that iFHRV is affected by chronic fetal hypoxia, one of the most common adverse outcomes of human pregnancy complicated by fetal growth restriction.Using an established late gestation ovine model of fetal development under chronic hypoxic conditions, we identify iFHRV in isolated fetal hearts and show that it is markedly affected by hypoxic pregnancy.Therefore, the isolated fetal heart has intrinsic variability and carries a memory of adverse intrauterine conditions experienced during the last third of pregnancy. [ABSTRACT FROM AUTHOR]