Hypoxic pregnancy promotes fetal growth restriction (FGR) and preterm birth, for which antenatal corticosteroids (ACS) are recommended to prevent respiratory distress. Adults born FGR or preterm are at greater risk of health conditions requiring medication(s), which are metabolised by hepatic cytochrome P450 (CYP) enzymes. We determined if ACS and/or hypoxic pregnancy alters fetal and adult offspring hepatic CYP activity. Ewes carrying singletons were randomly allocated to normoxic (Nx) or hypoxic (Hx; 11% O2) pregnancy from 105 to 138 dGA (term = 147 dGA). Dexamethasone (Dex, 12 mg IM) or vehicle (saline IV) was administered at 115 and 116 dGA. Ewes carrying male fetuses were humanely killed at 138 ± 2 dGA, while female fetuses lambed spontaneously and were humanely killed at 9 months (9 mo). Hepatic CYP activity was quantified using functional assays, and expression of glucocorticoid signalling and CYP regulating proteins was determined via Western blot. Hx increased fetal hepatic CYP2B6 and CYP2D6 activity, expression of 11β-HSD1&2 and reduced GRα-A nuclear expression, and cytosolic GRβ:α-A ratio. Dex reduced fetal CYP3A metabolism of testosterone to metabolites 6β-OHT and 2α-OHT, and GRα-A and GRβ cytosolic expression. Fetal CYP2B6 activity positively correlated with CAR in Nx, but not Hx. In 9 mo lambs, Hx reduced CYP2C19 activity and PPARα and GRβ cytosolic expression. Dex decreased CYP1A2, CYP2B6, CYP2E1 activity, and CYP3A testosterone metabolism to 6β-OHT. HxDex increased cytosolic and nuclear GRβ:α-A. Hx and Dex differentially effect hepatic CYP activity in offspring, and changes to CYP activity may be due to a loss of regulatory control.

Obesity during pregnancy is at pandemic proportions and predisposes women to pre- and postnatal cardiovascular dysfunction. The mechanisms underlying this maternal cardiovascular vulnerability remain unclear, partly due to a lack of translatable models capable of longitudinal in vivo cardiovascular monitoring. Here, we characterize a novel ovine model of maternal diet-induced obesity during pregnancy. Ewes were fed a control (CON) or obesogenic (OB; ad libitum concentrates) diet for 60 days pre-pregnancy and throughout gestation. Pregnant ewes were surgically instrumented with vascular catheters and Transonic flow probes using the wireless CamDAS system, which measured maternal cardiovascular function near term in free-moving ewes. Uterine artery vasoreactivity was assessed ex vivo by in vitro wire myography. OB ewes entered pregnancy 30% heavier than controls (P < 0.003) and were hyperglycaemic, hyperinsulinaemic and hyperlipidaemic during pregnancy, relative to CON ewes (all P < 0.05). OB ewes had elevated haematocrit and haemoglobin across pregnancy, and were hypertensive near term, with an increase in basal femoral artery blood flow, and elevated peripheral oxygen and glucose delivery (all P < 0.05). OB mothers carrying a female fetus showed increased uterine artery vascular resistance in vivo (P < 0.005) and reduced smooth muscle-dependent vasorelaxation ex vivo (P < 0.05) relative to CON. Conversely, OB mothers carrying a male fetus showed greater NO-independent mechanisms mediating the uterine vasodilator response to methacholine ex vivo (P < 0.001). Collectively, this study characterizes a robust model of maternal obesity during pregnancy that offers clinical translational potential and highlights fetal sex-specific changes to uterine artery function. KEY POINTS: Obesity during pregnancy is increasingly common and predisposes women to cardiovascular dysfunction during pregnancy and long after birth, but the specific mechanisms underlying this remain unclear. We developed a novel ovine model of diet-induced obesity during pregnancy that displays maternal hypertension, elevated haemoglobin, metabolic dysfunction, and alterations in uterine and peripheral blood flow and nutrient delivery near term. Mothers with obesity carrying a female fetus had elevated uterine vascular resistance in vivo and reduced uterine artery smooth muscle-dependent vasodilator reactivity ex vivo. Mothers with obesity carrying a male fetus showed no effect on uterine vascular resistance in vivo, but greater NO-independent mechanisms mediating the uterine vasodilator response to methacholine ex vivo. These findings highlight that fetal sex may influence maternal cardiovascular function during obese pregnancy.

Ventricular arrhythmias are the leading cause of sudden cardiac death. It is well-established that environmental factors contribute to the origin and penetrance of ventricular arrhythmic disorders. However, to our knowledge, no studies have considered the role of the intrauterine environment. In this study, we investigated the long-term effects of fetal hypoxia on ventricular arrhythmia susceptibility. Pregnant Wistar rats were assigned to normoxia (21% O2) or hypoxia (13% O2 between gestational days 6-20), and offspring were raised to 6 months. Hearts were isolated and loaded with the fluorescent calcium- and voltage-sensitive indicators, Rhod-2 and RH237, respectively. Optical mapping was performed while the left ventricle was burst paced (10-20hz) to induce arrhythmias. Hearts isolated from adult offspring exposed to fetal hypoxia were more susceptible to arrythmia during burst pacing, compared to controls. This phenotype was associated with prolonged Ca2+ transients and action potentials, an increased frequency of Ca2+ waves and delayed after depolarisations, as well as lower gene and protein expression of the sarcoplasmic reticulum Ca2+ ATPase. Collectively, our data shows that fetal hypoxia can programme ventricular arrhythmia sensitivity in adulthood, driven by abnormalities in excitation-contraction coupling. This is the first evidence that some ventricular arrhythmias may have a developmental origin, highlighting pregnancy as a potential window for early preventive intervention. ### Competing Interest Statement The authors have declared no competing interest. British Heart Foundation, https://ror.org/02wdwnk04, PG/18/5/33527, PG/23/11296

Abstract Metabolic dysfunction-associated steatotic liver disease (MASLD) afflicts more than one-third of adults globally, contributing significantly to an increased cardiovascular disease risk. Further, patients with severe liver disease experience muscle weakness (sarcopenic obesity) and fatigue. Hypoxia-inducible factor 2α (HIF2α) accumulates in the livers of MASLD patients and has been implicated in disease progression. Here we sought to understand the role of hepatic HIF2α in mediating hepatic and extra-hepatic features of MASLD. Using a well-validated obese mouse model of MASLD, we investigated the impact of hepatocyte-specific HIF2α deletion (hHIF2α -/- ) on hepatic, cardiac and skeletal muscle metabolism, and cardiac function. Over 28 weeks, mice were exposed to a high-fat, high-fructose, high-cholesterol (GAN) diet, which induced obesity alongside hepatic steatosis, fibrosis and inflammation. In contrast to observations in lean mouse models of liver disease, hHIF2α -/- did not protect against MASLD, despite greater hepatic NADH-supported mitochondrial respiration and higher intracellular sphingomyelin levels. Instead, in the hearts of GAN-fed mice, hHIF2α -/- caused diacylglycerol accumulation independent of diet, accumulation of long-chain acyl-carnitines and exacerbation of ceramide accumulation. Langendorff-perfused hearts from hHIF2α -/- mice showed systolic and diastolic dysfunction, including 24% lower left ventricular developed pressure and 34% lower maximal rate of relaxation (dP/dt min ). However, isolated hearts from hHIF2α -/- mice were protected against MASLD-associated sympathetic dominance, determined using autonomic receptor agonist stimulation. Both GAN-feeding and hHIF2α -/- were associated with lower lean mass (14% and 5.4% lower than respective controls), whilst hHIF2α -/- enhanced OXPHOS-associated protein levels in gastrocnemius muscle. Overall, hHIF2α -/- resulted in detrimental extra-hepatic effects, including myocardial lipid accumulation, impaired cardiac function, and loss of whole-body lean mass, with no apparent protection against MASLD disease progression.

Fetal chronic hypoxia is a common pregnancy complication associated with fetal growth restriction. Growth-restricted offspring have a higher risk for liver metabolic disease. Our objective was to better understand how chronic hypoxia impacts the developing fetal liver. We hypothesized that hypoxia promotes hepatocellular injury, shifts nutrient metabolism, and activates energetic and oxidative stress in the fetal liver. We used an ovine model of chronic hypoxia where pregnant ewes were housed under normoxic (CON) or hypoxic (HOX) conditions for 30 days in late gestation. Fetal liver was obtained, histologically analysed and profiled using bulk-RNA sequencing and metabolomics. Nutrient and oxidative stress signalling pathways were also measured. HOX fetuses had greater hepatic periportal collagen deposition. Metabolomics and transcriptomics predicted disruptions in central carbon metabolism, mitochondrial dysfunction and decreased oxidative phosphorylation. In support, we found potentiation of the gluconeogenic pathway and increased lactate production, pyruvate oxidation and AMPK activation. By contrast to the predicted effects, hypoxic livers maintained mitochondrial oxidation and antioxidant capacity. Interestingly, acylcarnitines were increased, yet hepatic triglyceride content was similar. Although there was little activation of oxidative stress markers, such as lipid peroxidation or oxidized glutathione, we uncovered a unique profile of liver stress-related metabolites in association with periportal collagen. Thus, hypoxic pregnancy increased fetal hepatic collagen deposition, indicating liver injury, in association with a unique profile of liver stress metabolites and adaptations in central carbon metabolism. These results provide new insight into how chronic fetal hypoxia may initiate fibrotic and metabolic liver disease risk in offspring of adverse pregnancy. KEY POINTS: Chronic exposure to hypoxic pregnancy increased fetal hepatic collagen deposition, indicating hepatocellular injury. Hypoxic fetal livers had a unique profile of stress metabolites and adaptations in central carbon metabolism. This provides new insight into how hypoxia, a common pregnancy complication associated with fetal growth restriction, may initiate fibrotic and metabolic liver disease risk.

BACKGROUND:Chronic hypoxemia is a common cause of fetal growth restriction and can have significant effects on the developing fetal lung. Maternal antioxidant treatment in hypoxic pregnancy protects against offspring cardiovascular dysfunction. The effects of antenatal antioxidants on lung development in the chronically hypoxic growth restricted fetus is unknown. METHODS:We investigated the effect of maternal daily Vitamin C (200 mg/kg i.v. vs. Saline) for a month in late gestation on molecular markers regulating lung maturation between normoxic normally grown and hypoxic growth-restricted fetal sheep. Chronic fetal hypoxia and fetal growth restriction were induced by exposure to maternal chronic hypoxia (10% O2 vs. Normoxia=21% O2) from 105-138 d gestation (term=145 d). RESULTS:The data show a differential effect of antenatal Vitamin C treatment on regulation of genes involved in surfactant maturation, sodium movement and hypoxia signaling. Limited responsiveness to antenatal Vitamin C exposure in the lung of the hypoxic fetus, compared to responsiveness to antenatal Vitamin C in the normoxic fetus, suggests a maximal upregulation of the molecular signaling pathways in response to the chronic hypoxic insult alone. CONCLUSION:We provide molecular insight into the heterogeneity of antenatal Vitamin C treatment on development of the normoxic and growth restricted hypoxic fetal lung. IMPACT:The effect of maternal Vitamin C on molecular markers of lung maturation between normoxic normally grown and hypoxic growth restricted fetal sheep was unknown. We show a differential effect of Vitamin C with a greater increase in molecular markers of lung maturation in normoxic compared with hypoxic fetuses. Limited responsiveness in the hypoxic fetal lung is likely due to maximal upregulation by the hypoxic insult alone, thus added exposure to Vitamin C is unable to upregulate the system further. The work highlights the need to understand differential effects of antenatal interventions in healthy and complicated pregnancy, prior to clinical translation.

The carotid body (CB) chemoreceptors mediate rapid cardiorespiratory reflexes to hypoxia, which mature peri-natally and are vital for fetal hypoxia tolerance and post-natal ventilatory control. This maturation is associated with an increase in the sensitivity of the CB electrophysiological response to hypoxia (chemosensitivity): a process that is incompletely understood but critical to systemic oxygen homeostasis. Hypothesizing that perinatal CB gene expression changes would reveal candidate mechanisms for oxygen chemosensitivity, we studied the CB transcriptome in sheep, where peri-natal CB physiology is well-characterised. CB-mediated cardiovascular reflexes are detectable at fetal day 120, and robust by term (day 145), while hypoxic ventilatory responses are established by post-natal day 15. We performed RNA sequencing on sheep CBs at each of these stages, and adults, along with the superior cervical ganglion (SCG) as an oxygen-insensitive control. This allowed us to define tissue-specific changes in the CB transcriptome correlating with chemosensitivity maturation. Striking, progressive CB enrichment is observed in genes implicated in murine CB chemosensitivity, including potassium channels ( KCNK9 ), mitochondrial complex IV regulators ( NDUFA4L2, HIGD1C ), and HIF-2α ( EPAS1 ). Genes with this expression pattern are also enriched for regulators of diacylglycerol (DAG), particularly the DAG kinase DGKH: one of the most abundant CB transcripts increasing in parallel with chemosensitivity. Across developmental stages, the CB also exhibits marked down-regulation of metabolic pathways and ATP/GTP consuming processes, potentially providing a state permissive to metabolic signal detection. Together, this builds a detailed picture of the CB transcriptional signature, with core features established in fetal life and conserved across species. Key points ### Competing Interest Statement P. J. R. is a non-executive director of Immunocore Holdings PLC. Wellcome Trust, https://ror.org/029chgv08, 106241/Z/14/Z, 226354/Z/22/Z MRC Centre, MR/V03362X/1 BHF, RG/17/8/32924, FS/PhD/25/29633 Ludwig Cancer Research The Francis Crick Institute, https://ror.org/04tnbqb63, FC001501

IntroductionChronic fetal hypoxia is commonly associated with fetal growth restriction and can predispose to respiratory disease at birth and in later life. Antenatal antioxidant treatment has been investigated to overcome the effects of oxidative stress in utero to improve respiratory outcomes. We aimed to determine if the effects of chronic fetal hypoxia and antenatal antioxidant administration persist in the lung in early adulthood.MethodsChronically catheterised pregnant sheep were exposed to normoxia (N; n = 20) or hypoxia (H; n = 18; 10% O2) ± maternal daily i. v. saline (N = 11; H = 8) or Vitamin C (VC; NVC = 9; HVC = 10) from 105 to 138 days (term, ∼145 days). Lungs were collected from female lambs 9 months after birth (early adulthood). Lung tissue expression of genes and proteins regulating oxidative stress, mitochondrial function, hypoxia signalling, glucocorticoid signalling, surfactant maturation, inflammation and airway remodelling were measured.ResultsChronic fetal hypoxia upregulated lung expression of markers of prooxidant, surfactant lipid transport and airway remodelling pathways in early adulthood. Antenatal Vitamin C normalized prooxidant and airway remodelling markers, increased endogenous antioxidant, vasodilator and inflammatory markers, and altered regulation of hypoxia signalling and glucocorticoid availability.ConclusionThere are differential effects of antenatal Vitamin C on molecular markers in the lungs of female lambs from normoxic and hypoxic pregnancy in early adulthood.

Fetal hypoxaemia promotes oxidative stress and increases risk of chronic diseases in later life. The medications for managing chronic diseases are often metabolised by hepatic cytochrome P450 (CYP) enzymes; however, it is unknown if oxidative stress in utero programs altered CYP activity. Maternal treatment with the antioxidant MitoQ protects against hypertension in adult offspring, programmed by fetal hypoxaemia. We hypothesised that fetal hypoxaemia alters offspring CYP activity and MitoQ would rescue this. From 105-138 days gestational age (dGA), ewes carrying singletons were randomly allocated to normoxic (n = 34, 21 % O2) or hypoxic (n = 36, 11 % O2) pregnancy with maternal vehicle (n = 34) or MitoQ (n = 36, 6 mg kg-1 MS010 IV) daily bolus. Male fetuses were humanely killed at 137 ± 2 dGA while female fetuses lambed spontaneously and were humanely killed at 9 months of age (9mo). Hepatic CYP activity and protein abundance were quantified using functional assays and Western blot. Hypoxic pregnancy increased fetal ATF-6 expression, decreased 9mo CYP2B6 and CYP2E1 activity, and decreased mitochondrial abundance at 9mo. MitoQ decreased fetal CYP3A activity, while at 9mo CYP3A activity increased and CYP1A2 activity decreased. MitoQ decreased fetal mitochondrial abundance and expression of mitochondrial complexes I and IV, but at 9mo complex II and IV expression increased. MitoQ also increased fetal and 9mo ATF-6 expression and 9mo HNF-4ɑ and PPARɑ expression. Fetal hypoxaemia and maternal MitoQ treatment independently programmed hepatic CYP activity in fetal sheep and 9mo lambs. These effects may impact therapeutic efficacy and safety for treatment of chronic diseases in offspring from complicated pregnancies.

Background/Objectives: In pregnancy threatened by preterm birth, antenatal corticosteroids (ACS) are administered to accelerate fetal lung maturation. However, they have side effects, including the production of reactive oxygen species that can impact cytochrome P450 (CYP) activity. We hypothesised that antioxidants could protect a fetus treated with ACS during gestation and prevent the programming of altered hepatic CYP activity in the offspring. The primary outcome of our study was the impact of different maternal treatments on the activity of hepatic drug-metabolising enzymes in offspring. Methods: At 100 ± 1 days gestational age (dGA, term = 147 dGA), 73 ewes were randomly allocated to the following: saline (5 mL IV daily 105–137 ± 2 dGA, n = 17), ACS (Dexamethasone (Dex); 12 mg IM at 115 and 116 dGA; n = 25), MitoQ (6 mg/kg MS010 IV, daily bolus 105–137 ± 2 dGA; n = 17) or Dex and MitoQ (Dex+MitoQ; n = 14). CYP activity and protein abundance were assessed using functional assays and Western blot. Results: Dex decreased the hepatic activity of fetal CYP3A (−56%, PDex = 0.0322), and 9 mo lamb CYP1A2 (−22%, PDex = 0.0003), CYP2B6 (−36%, PDex = 0.0234), CYP2C8 (−34%, PDex = 0.0493) and CYP2E1 (−57%, PDex = 0.0009). For all, except CYP1A2, activity returned to control levels with Dex+MitoQ in 9 mo lambs. In 9 mo lambs, MitoQ alone increased activity of CYP2B6 (+16%, PMitoQ = 0.0011) and CYP3A (midazolam, +25%, PMitoQ = 0.0162) and increased CAT expression (PMitoQ = 0.0171). Dex+MitoQ increased CYP3A4/5 activity (testosterone, +65%, PIntx < 0.0003), decreased CYP1A2 activity (−14%, PIntx = 0.0036) and decreased mitochondrial abundance (PIntx = 0.0051). All treatments decreased fetal hepatic DRP1, a regulator of mitochondrial fission (PDex = 0.0055, PMitoQ = 0.0006 and PIntx = 0.0034). Conclusions: Antenatal Dex reduced activity of only one CYP in the fetus but programmed the reduced activity of several hepatic CYPs in young adult offspring, and this effect was ameliorated by combination with MitoQ.

The mechanisms that drive placental dysfunction in pregnancies complicated by hypoxia and fetal growth restriction remain poorly understood. Changes to mitochondrial respiration contribute to cellular dysfunction in conditions of hypoxia and have been implicated in the pathoaetiology of pregnancy complications, such as pre-eclampsia. We used bespoke isobaric hypoxic chambers and a combination of functional, molecular and imaging techniques to study cellular metabolism and mitochondrial dynamics in sheep undergoing hypoxic pregnancy. We show that hypoxic pregnancy in sheep triggers a shift in capacity away from beta-oxidation and complex I-mediated respiration, while maintaining total oxidative phosphorylation capacity. There are also complex-specific changes to electron transport chain composition and a switch in mitochondrial dynamics towards fission. Hypoxic placentas show increased activation of the non-canonical mitochondrial unfolded protein response pathway and enhanced insulin like growth factor 2 signalling. Combined, therefore, the data show that the hypoxic placenta undergoes significant metabolic and morphological adaptations to maintain cellular energy balance. Chronic hypoxia during pregnancy in sheep activated placental mitochondrial stress pathways, leading to alterations in mitochondrial respiration, mitochondrial energy metabolism and mitochondrial dynamics, as seen in the placenta of women with pre-eclampsia.

Chronic fetal hypoxia is one of the most common complications of pregnancy and can programme cardiac abnormalities in adult offspring including ventricular remodelling, diastolic dysfunction and sympathetic dominance. However, the underlying mechanisms at the level of the cardiomyocyte are unknown, preventing the identification of targets for therapeutic intervention. Therefore, we aimed to link echocardiographic data with cardiomyocyte function to reveal cellular mechanism for cardiac dysfunction in rat offspring from hypoxic pregnancy. Further, we investigated the potential of maternal treatment with melatonin as antenatal antioxidant therapy. Wistar rats were randomly allocated into normoxic (21% O2) or hypoxic (13% O2) pregnancy with or without melatonin treatment (5 µg/ml; normoxic melatonin in the maternal drinking water from gestational day 6 to 20 (term = 22 days). After delivery, male and female offspring were maintained to adulthood (16 weeks). Cardiomyocytes were isolated from the left and right ventricles, and calcium (Ca2+) handling was investigated in field-stimulated myocytes. Systolic and diastolic function was negatively impacted in male and female offspring of hypoxic pregnancy demonstrating biventricular systolic and diastolic dysfunction and compensatory increases in cardiac output. Ca2+ transients from isolated cardiomyocytes in offspring of both sexes in hypoxic pregnancy displayed diastolic dysfunction with a reduced rate of [Ca2+]i recovery. Cardiac and cardiomyocyte dysfunction in male and female adult offspring was ameliorated by maternal antenatal treatment with melatonin in hypoxic pregnancy. Therefore, cardiomyocyte Ca2+ mishandling provides a cellular mechanism explaining functional deficits in hearts of male and female offspring in pregnancies complicated by chronic fetal hypoxia. KEY POINTS: This study identified significant changes in Ca2+ handling within cardiomyocytes isolated from offspring of hypoxic pregnancy including reduced systolic Ca2+ transients, impaired diastolic recovery of [Ca2+]i and a greater increase in systolic [Ca2+]i amplitude to β-adrenergic stimulation. These changes in cardiomyocyte Ca2+ handling help to explain dysregulation of biventricular systolic and diastolic dysfunction determined by echocardiography. The data show protection against maladaptive cardiomyocyte calcium handling and thereby improvement in cardiac function in adult offspring of hypoxic pregnancy treated with melatonin with doses lower than those recommended for overcoming jet lag in humans. Melatonin treatment alone in healthy pregnancy did cause some alterations in cardiac structure. Therefore, maternal treatment with melatonin should only be given to pregnancies affected by chronic fetal hypoxia.

Timothy syndrome, an extremely rare disease, is closely associated with a mutation in CACNA1C gene, which encodes the cardiac L-type voltage-gated calcium channel (Cav1.2). In this study, we generated a human induced pluripotent stem cell (iPSC) line from a Timothy syndrome infant carrying heterozygous CACNA1C mutation (transcript variant NM_000719.7c.1216G>A: p.G406R). The generated iPSC line showed typical stem cell morphology, positively expressed pluripotency and proliferation markers, normal karyotype, and trilineage differentiation potential. Therefore, this patient-specific iPSC can be of great significance in investigating the mechanisms underlying Timothy syndrome, and hence establishing effective intervention strategies