In vivo mitochondria-targeted protection against uterine artery vascular dysfunction and remodelling in rodent hypoxic pregnancy.

Gestational hypoxia adversely affects uterine artery function, increasing complications. However, an effective therapy remains unidentified. Here, we show in rodent uterine arteries that hypoxic pregnancy promotes hypertrophic remodelling, increases constrictor reactivity via protein kinase C signalling, and triggers compensatory dilatation via nitric oxide-dependent mechanisms and stimulation of large conductance Ca2+ -activated K+ -channels. Maternal in vivo oral treatment with the mitochondria-targeted antioxidant MitoQ in hypoxic pregnancy normalises uterine artery reactivity and prevents vascular remodelling. From days 6-20 of gestation (term ∼22 days), female Wistar rats were randomly assigned to normoxic or hypoxic (13-14% O2 ) pregnancy ± daily maternal MitoQ treatment (500 µm in drinking water). At 20 days of gestation, maternal, placental and fetal tissue was frozen to determine MitoQ uptake. The uterine arteries were harvested and, in one segment, constrictor and dilator reactivity was determined by wire myography. Another segment was fixed for unbiased stereological analysis of vessel morphology. Maternal administration of MitoQ in both normoxic and hypoxic pregnancy crossed the placenta and was present in all tissues analysed. Hypoxia increased uterine artery constrictor responses to norepinephrine, angiotensin II and the protein kinase C activator, phorbol 12,13-dibutyrate. Hypoxia enhanced dilator reactivity to sodium nitroprusside, the large conductance Ca2+ -activated K+ -channel activator NS1619 and ACh via increased nitric oxide-dependent mechanisms. Uterine arteries from hypoxic pregnancy showed increased wall thickness and MitoQ treatment in hypoxic pregnancy prevented all effects on uterine artery reactivity and remodelling. The data support mitochondria-targeted therapy against adverse changes in uterine artery structure and function in high-risk pregnancy. KEY POINTS: Dysfunction and remodelling of the uterine artery are strongly implicated in many pregnancy complications, including advanced maternal age, maternal hypertension of pregnancy, maternal obesity, gestational diabetes and pregnancy at high altitude. Such complications not only have immediate adverse effects on the growth of the fetus, but also they can also increase the risk of cardiovascular disease in the mother and offspring. Despite this, there is a significant unmet clinical need for therapeutics that treat uterine artery vascular dysfunction in adverse pregnancy. Here, we show in a rodent model of gestational hypoxia that in vivo oral treatment of the mitochondria-targeted antioxidant MitoQ protects against uterine artery vascular dysfunction and remodelling, supporting the use of mitochondria-targeted therapy against adverse changes in uterine artery structure and function in high-risk pregnancy.

Molecular mechanisms underlying adverse effects of dexamethasone and betamethasone in the developing cardiovascular system.

Antenatal glucocorticoids accelerate fetal lung maturation and reduce mortality in preterm babies but can trigger adverse effects on the cardiovascular system. The mechanisms underlying off-target effects of the synthetic glucocorticoids mostly used, Dexamethasone (Dex) and Betamethasone (Beta), are unknown. We investigated effects of Dex and Beta on cardiovascular structure and function, and underlying molecular mechanism using the chicken embryo, an established model system to isolate effects of therapy on the developing heart and vasculature, independent of effects on the mother or placenta. Fertilized eggs were treated with Dex (0.1 mg kg-1 ), Beta (0.1 mg kg-1 ), or water vehicle (Control) on embryonic day 14 (E14, term = 21 days). At E19, biometry, cardiovascular function, stereological, and molecular analyses were determined. Both glucocorticoids promoted growth restriction, with Beta being more severe. Beta compared with Dex induced greater cardiac diastolic dysfunction and also impaired systolic function. While Dex triggered cardiomyocyte hypertrophy, Beta promoted a decrease in cardiomyocyte number. Molecular changes of Dex on the developing heart included oxidative stress, activation of p38, and cleaved caspase 3. In contrast, impaired GR downregulation, activation of p53, p16, and MKK3 coupled with CDK2 transcriptional repression linked the effects of Beta on cardiomyocyte senescence. Beta but not Dex impaired NO-dependent relaxation of peripheral resistance arteries. Beta diminished contractile responses to potassium and phenylephrine, but Dex enhanced peripheral constrictor reactivity to endothelin-1. We conclude that Dex and Beta have direct differential detrimental effects on the developing cardiovascular system.

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

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, ß-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.

Intervention against hypertension in the next generation programmed by developmental hypoxia.

Evidence derived from human clinical studies and experimental animal models shows a causal relationship between adverse pregnancy and increased cardiovascular disease in the adult offspring. However, translational studies isolating mechanisms to design intervention are lacking. Sheep and humans share similar precocial developmental milestones in cardiovascular anatomy and physiology. We tested the hypothesis in sheep that maternal treatment with antioxidants protects against fetal growth restriction and programmed hypertension in adulthood in gestation complicated by chronic fetal hypoxia, the most common adverse consequence in human pregnancy. Using bespoke isobaric chambers, chronically catheterized sheep carrying singletons underwent normoxia or hypoxia (10% oxygen [O2]) ± vitamin C treatment (maternal 200 mg.kg-1 IV daily) for the last third of gestation. In one cohort, the maternal arterial blood gas status, the value at which 50% of the maternal hemoglobin is saturated with oxygen (P50), nitric oxide (NO) bioavailability, oxidative stress, and antioxidant capacity were determined. In another, naturally delivered offspring were raised under normoxia until early adulthood (9 months). Lambs were chronically instrumented and cardiovascular function tested in vivo. Following euthanasia, femoral arterial segments were isolated and endothelial function determined by wire myography. Hypoxic pregnancy induced fetal growth restriction and fetal oxidative stress. At adulthood, it programmed hypertension by enhancing vasoconstrictor reactivity and impairing NO-independent endothelial function. Maternal vitamin C in hypoxic pregnancy improved transplacental oxygenation and enhanced fetal antioxidant capacity while increasing NO bioavailability, offsetting constrictor hyper-reactivity and replenishing endothelial function in the adult offspring. These discoveries provide novel insight into mechanisms and interventions against fetal growth restriction and adult-onset programmed hypertension in an animal model of complicated pregnancy in a species of similar temporal developmental milestones to humans.

Molecular regulation of lung maturation in near-term fetal sheep by maternal daily vitamin C treatment in late gestation.

BACKGROUND: In the fetus, the appropriate balance of prooxidants and antioxidants is essential to negate the detrimental effects of oxidative stress on lung maturation. Antioxidants improve respiratory function in postnatal life and adulthood. However, the outcomes and biological mechanisms of antioxidant action in the fetal lung are unknown. METHODS: We investigated the effect of maternal daily vitamin C treatment (200 mg/kg, intravenously) for a month in late gestation (105-138 days gestation, term ~145 days) on molecular regulation of fetal lung maturation in sheep. Expression of genes and proteins regulating lung development was quantified in fetal lung tissue. The number of surfactant-producing cells was determined by immunohistochemistry. RESULTS: Maternal vitamin C treatment increased fetal lung gene expression of the antioxidant enzyme SOD-1, hypoxia signaling genes (HIF-2α, HIF-3α, ADM, and EGLN-3), genes regulating sodium movement (SCNN1-A, SCNN1-B, ATP1-A1, and ATP1-B1), surfactant maturation (SFTP-B and ABCA3), and airway remodeling (ELN). There was no effect of maternal vitamin C treatment on the expression of protein markers evaluated or on the number of surfactant protein-producing cells in fetal lung tissue. CONCLUSIONS: Maternal vitamin C treatment in the last third of pregnancy in sheep acts at the molecular level to increase the expression of genes that are important for fetal lung maturation in a healthy pregnancy. IMPACT: Maternal daily vitamin C treatment for a month in late gestation in sheep increases the expression of gene-regulating pathways that are essential for normal fetal lung development. Following late gestation vitamin C exposure in a healthy pregnancy, an increase in lung gene but not protein expression may act as a mechanism to aid in the preparation for exposure to the air-breathing environment after birth. In the future, the availability/development of compounds with greater antioxidant properties than vitamin C or more specific targets at the site of oxidative stress in vivo may translate clinically to improve respiratory outcomes in complicated pregnancies at birth.

Protective effects of pravastatin on the embryonic cardiovascular system during hypoxic development.

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% O2 ) 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.

Isolating adverse effects of glucocorticoids on the embryonic cardiovascular system.

Antenatal glucocorticoid therapy reduces mortality in the preterm infant, but evidence suggests off-target adverse effects on the developing cardiovascular system. Whether deleterious effects are direct on the offspring or secondary to alterations in uteroplacental physiology is unclear. Here, we isolated direct effects of glucocorticoids using the chicken embryo, a model system in which the effects on the developing heart and circulation of therapy can be investigated, independent of effects on the mother and/or the placenta. Fertilized chicken eggs were incubated and divided randomly into control (C) or dexamethasone (Dex) treatment at day 14 out of the 21-day incubation period. Combining functional experiments at the isolated organ, cellular and molecular levels, embryos were then studied close to term. Chicken embryos exposed to dexamethasone were growth restricted and showed systolic and diastolic dysfunction, with an increase in cardiomyocyte volume but decreased cardiomyocyte nuclear density in the left ventricle. Underlying mechanisms included a premature switch from tissue accretion to differentiation, increased oxidative stress, and activated signaling of cellular senescence. These findings, therefore, demonstrate that dexamethasone treatment can have direct detrimental off-target effects on the cardiovascular system in the developing embryo, which are independent of effects on the mother and/or placenta.

Sildenafil therapy for fetal cardiovascular dysfunction during hypoxic development: studies in the chick embryo.

KEY POINTS: Common complications of pregnancy, such as chronic fetal hypoxia, trigger a fetal origin of cardiovascular dysfunction and programme cardiovascular disease in later life. Sildenafil treatment protects placental perfusion and fetal growth, but whether the effects of sildenafil transcend the placenta to affect the fetus is unknown. Using the chick embryo model, here we show that sildenafil treatment directly protects the fetal cardiovascular system in hypoxic development, and that the mechanisms of sildenafil protection include reduced oxidative stress and increased nitric oxide bioavailability; Sildenafil does not protect against fetal growth restriction in the chick embryo, supporting the idea that the protective effect of sildenafil on fetal growth reported in mammalian studies, including humans, is secondary to improved placental perfusion. Therefore, sildenafil may be a good candidate for human translational antioxidant therapy to protect the chronically hypoxic fetus in adverse pregnancy. ABSTRACT: There is a need for developing clinically translatable therapy for preventing fetal origins of cardiovascular disease in pregnancy complicated by chronic fetal hypoxia. Evidence shows that sildenafil protects placental perfusion and fetal growth. However, whether beneficial effects of sildenafil transcend onto the fetal heart and circulation in complicated development is unknown. We isolated the direct effects of sildenafil on the fetus using the chick embryo and hypothesised that sildenafil also protects fetal cardiovascular function in hypoxic development. Chick embryos (n = 11 per group) were incubated in normoxia or hypoxia (14% O2 ) from day 1 and treated with sildenafil (4 mg kg-1  day-1 ) from day 13 of the 21-day incubation. Hypoxic incubation increased oxidative stress (4-hydroxynonenal, 141.1 ± 17.6% of normoxic control), reduced superoxide dismutase (60.7 ± 6.3%), increased phosphodiesterase type 5 expression (167 ± 13.7%) and decreased nitric oxide bioavailability (54.7 ± 6.1%) in the fetal heart, and promoted peripheral endothelial dysfunction (70.9 ± 5.6% AUC of normoxic control; all P < 0.05). Sildenafil treatment after onset of chronic hypoxia prevented the increase in phosphodiesterase expression (72.5 ± 22.4%), protected against oxidative stress (94.7 ± 6.2%) and normalised nitric oxide bioavailability (115.6 ± 22.3%) in the fetal heart, and restored endothelial function in the peripheral circulation (89.8 ± 2.9%). Sildenafil protects the fetal heart and circulation directly in hypoxic development via mechanisms including decreased oxidative stress and enhanced nitric oxide bioavailability. Sildenafil may be a good translational candidate for human antioxidant therapy to prevent fetal origins of cardiovascular dysfunction in adverse pregnancy.

Maternal chronic hypoxia increases expression of genes regulating lung liquid movement and surfactant maturation in male fetuses in late gestation.

KEY POINTS: Chronic fetal hypoxaemia is a common pregnancy complication associated with intrauterine growth restriction that may influence respiratory outcome at birth. We investigated the effect of maternal chronic hypoxia for a month in late gestation on signalling pathways regulating fetal lung maturation and the transition to air-breathing at birth using isobaric hypoxic chambers without alterations to maternal food intake. Maternal chronic hypoxia in late gestation increases fetal lung expression of genes regulating hypoxia signalling, lung liquid reabsorption and surfactant maturation, which may be an adaptive response in preparation for the successful transition to air-breathing at birth. In contrast to other models of chronic fetal hypoxaemia, late gestation onset fetal hypoxaemia promotes molecular regulation of fetal lung maturation. This suggests a differential effect of timing and duration of fetal chronic hypoxaemia on fetal lung maturation, which supports the heterogeneity observed in respiratory outcomes in newborns following exposure to chronic hypoxaemia in utero. ABSTRACT: Chronic fetal hypoxaemia is a common pregnancy complication that may arise from maternal, placental and/or fetal factors. Respiratory outcome of the infant at birth likely depends on the duration, timing and severity of the hypoxaemic insult. We have isolated the effect of maternal chronic hypoxia (MCH) for a month in late gestation on fetal lung development. Pregnant ewes were exposed to normoxia (21% O2 ) or hypoxia (10% O2 ) from 105 to 138 days of gestation (term ∼145 days). At 138 days, gene expression in fetal lung tissue was determined by quantitative RT-PCR. Cortisol concentrations were determined in fetal plasma and lung tissue. Numerical density of surfactant protein positive cells was determined by immunohistochemistry. MCH reduced maternal PaO2 (106 ± 2.9 vs. 47 ± 2.8 mmHg) and fetal body weight (4.0 ± 0.4 vs. 3.2 ± 0.9 kg). MCH increased fetal lung expression of the anti-oxidant marker CAT and decreased expression of the pro-oxidant marker NOX-4. MCH increased expression of genes regulating hypoxia signalling and feedback (HIF-3α, KDM3A, SLC2A1, EGLN-3). There was no effect of MCH on fetal plasma/lung tissue cortisol concentrations, nor genes regulating glucocorticoid signalling (HSD11B-1, HSD11B-2, NR3C1, NR3C2). MCH increased expression of genes regulating sodium (SCNN1-B, ATP1-A1, ATP1-B1) and water (AQP-4) movement in the fetal lung. MCH promoted surfactant maturation (SFTP-B, SFTP-D, ABCA3) at the molecular level, but did not alter the numerical density of surfactant positive cells in lung tissue. MCH in late gestation promotes molecular maturation of the fetal lung, which may be an adaptive response in preparation for the successful transition to air-breathing at birth.

Melatonin rescues cardiovascular dysfunction during hypoxic development in the chick embryo.

There is a search for rescue therapy against fetal origins of cardiovascular disease in pregnancy complicated by chronic fetal hypoxia, particularly following clinical diagnosis of fetal growth restriction (FGR). Melatonin protects the placenta in adverse pregnancy; however, whether melatonin protects the fetal heart and vasculature in hypoxic pregnancy independent of effects on the placenta is unknown. Whether melatonin can rescue fetal cardiovascular dysfunction when treatment commences following FGR diagnosis is also unknown. We isolated the effects of melatonin on the developing cardiovascular system of the chick embryo during hypoxic incubation. We tested the hypothesis that melatonin directly protects the fetal cardiovascular system in adverse development and that it can rescue dysfunction following FGR diagnosis. Chick embryos were incubated under normoxia or hypoxia (14% O2) from day 1 ± melatonin treatment (1 mg/kg/day) from day 13 of incubation (term ~21 days). Melatonin in hypoxic chick embryos rescued cardiac systolic dysfunction, impaired cardiac contractility and relaxability, increased cardiac sympathetic dominance, and endothelial dysfunction in peripheral circulations. The mechanisms involved included reduced oxidative stress, enhanced antioxidant capacity and restored vascular endothelial growth factor expression, and NO bioavailability. Melatonin treatment of the chick embryo starting at day 13 of incubation, equivalent to ca. 25 wk of gestation in human pregnancy, rescues early origins of cardiovascular dysfunction during hypoxic development. Melatonin may be a suitable antioxidant candidate for translation to human therapy to protect the fetal cardiovascular system in adverse pregnancy.

Heart disease link to fetal hypoxia and oxidative stress.

The quality of the intrauterine environment interacts with our genetic makeup to shape the risk of developing disease in later life. Fetal chronic hypoxia is a common complication of pregnancy. This chapter reviews how fetal chronic hypoxia programmes cardiac and endothelial dysfunction in the offspring in adult life and discusses the mechanisms via which this may occur. Using an integrative approach in large and small animal models at the in vivo, isolated organ, cellular and molecular levels, our programmes of work have raised the hypothesis that oxidative stress in the fetal heart and vasculature underlies the mechanism via which prenatal hypoxia programmes cardiovascular dysfunction in later life. Developmental hypoxia independent of changes in maternal nutrition promotes fetal growth restriction and induces changes in the cardiovascular, metabolic and endocrine systems of the adult offspring, which are normally associated with disease states during ageing. Treatment with antioxidants of animal pregnancies complicated with reduced oxygen delivery to the fetus prevents the alterations in fetal growth, and the cardiovascular, metabolic and endocrine dysfunction in the fetal and adult offspring. The work reviewed offers both insight into mechanisms and possible therapeutic targets for clinical intervention against the early origin of cardiometabolic disease in pregnancy complicated by fetal chronic hypoxia.

Growth capacity of a Wharton's Jelly derived mesenchymal stromal cells tissue engineered vascular graft used for main pulmonary artery reconstruction in piglets.

Background: Surgical treatment of congenital heart defects affecting the right ventricular outflow tract (RVOT) often requires complex reconstruction and multiple reoperations due to structural degeneration and lack of growth of currently available materials. Hence, alternative approaches for RVOT reconstruction, which meet the requirements of biocompatibility and long-term durability of an ideal scaffold, are needed. Through this full scale pre-clinical study, we demonstrated the growth capacity of a Wharton's Jelly derived mesenchymal stromal cells (WJ-MSC) tissue engineered vascular graft used in reconstructing the main pulmonary artery in piglets, providing proof of biocompatibility and efficacy. Methods: Sixteen four-week-old Landrace pigs were randomized to undergo supravalvar Main Pulmonary Artery (MPA) replacement with either unseeded or WJ-MSCs-seeded Small Intestinal Submucosa-derived grafts. Animals were followed up for 6 months by clinical examinations and cardiac imaging. At termination, sections of MPAs were assessed by macroscopic inspection, histology and fluorescent immunohistochemistry. Results: Data collected at 6 months follow up showed no sign of graft thrombosis or calcification. The explanted main pulmonary arteries demonstrated a significantly higher degree of cellular organization and elastin content in the WJ-MSCs seeded grafts compared to the acellular counterparts. Transthoracic echocardiography and cardiovascular magnetic resonance confirmed the superior growth and remodelling of the WJ-MSCs seeded conduit compared to the unseeded. Conclusion: Our findings indicate that the addition of WJ-MSCs to the acellular scaffold can upgrade the material, converting it into a biologically active tissue, with the potential to grow, repair and remodel the RVOT.

The effect of cardioplegic supplementation with sildenafil on cardiac energetics in a piglet model of cardiopulmonary bypass and cardioplegic arrest with warm or cold cardioplegia.

Cardioplegic cardioprotection strategies used during paediatric open-heart surgery remain suboptimal. Sildenafil, a phosphodiesterase 5 (PDE-5) inhibitor, has been shown to be cardioprotective against ischemia/reperfusion injury in a variety of experimental models and this study therefore tested the efficacy of supplementation of cardioplegia with sildenafil in a piglet model of cardiopulmonary bypass and arrest, using both cold and warm cardioplegia protocols. Piglets were anaesthetized and placed on coronary pulmonary bypass (CPB), the aorta cross-clamped and the hearts arrested for 60 min with cardioplegia with or without sildenafil (10 nM). Twenty minutes after removal of cross clamp (reperfusion), attempts were made to wean the pigs from CPB. Termination was carried out after 60 min reperfusion. Throughout the protocol blood and left ventricular tissue samples were taken for analysis of selected metabolites (using HPLC) and troponin I. In both the cold and warm cardioplegia protocols there was evidence that sildenafil supplementation resulted in faster recovery of ATP levels, improved energy charge (a measure of metabolic flux) and altered release of hypoxanthine and inosine, two purine catabolites. There was no effect on troponin release within the studied short timeframe. In conclusion, sildenafil supplementation of cardioplegia resulted in improved cardiac energetics in a translational animal model of paediatric CPB surgery.

Outcome monitoring and risk stratification after cardiac procedure in neonates, infants, children and young adults born with congenital heart disease: protocol for a multicentre prospective cohort study (Children OMACp).

INTRODUCTION: Congenital heart disease (CHD) represents the most common birth defect, affecting from 0.4% to 1.2% of children born in developed countries. The survival of these patients has increased significantly, but CHD remains one of the major causes of neonatal and childhood death. The aetiology of CHD is complex, with some evidence of both genetic and environmental causes. However, there is still lack of knowledge regarding modifiable risk factors and molecular and genetic mechanisms underlying the development of CHD. This study aims to develop a prospective cohort of patients undergoing cardiac procedures that will bring together routinely collected clinical data and biological samples from patients and their biological mothers, in order to investigate risk factors and predictors of postoperative-outcomes, as well as better understanding the effect of the surgical intervention on the early and long-term outcomes. METHODS AND ANALYSIS: Children OMACp (OMACp, outcome monitoring after cardiac procedure in congenital heart disease) is a multicentre, prospective cohort study recruiting children with CHD undergoing a cardiac procedure. The study aims to recruit 3000 participants over 5 years (2019-2024) across multiple UK sites. Routine clinical data will be collected, as well as participant questionnaires collecting sociodemographic, NHS resource use and quality of life data. Biological samples (blood, urine and surgical waste tissue from patients, and blood and urine samples from biological mothers) will be collected where consent has been obtained. Follow-up outcome and questionnaire data will be collected for 5 years. ETHICS AND DISSEMINATION: The study was approved by the London-Brent Research Ethics Committee on 30 July 2019 (19/SW/0113). Participants (or their parent/guardian if under 16 years of age) must provide informed consent prior to being recruited into the study. Mothers who wish to take part must also provide informed consent prior to being recruited. The study is sponsored by University Hospitals Bristol and Weston Foundation Trust and is managed by the University of Bristol. Children OMACp is adopted onto the National Institute for Health Research Clinical Research Network portfolio. Findings will be disseminated through peer-reviewed publications, presentation at conference, meetings and through patient organisations and newsletters. TRIAL REGISTRATION NUMBER: ISRCTN17650644.

Determination of Agrin and Related Proteins Levels as a Function of Age in Human Hearts.

Background: Mature cardiomyocytes are unable to proliferate, preventing the injured adult heart from repairing itself. Studies in rodents have suggested that the extracellular matrix protein agrin promotes cardiomyocyte proliferation in the developing heart and that agrin expression is downregulated shortly after birth, resulting in the cessation of proliferation. Agrin based therapies have proven successful at inducing repair in animal models of cardiac injury, however whether similar pathways exist in the human heart is unknown. Methods: Right ventricular (RV) biopsies were collected from 40 patients undergoing surgery for congenital heart disease and the expression of agrin and associated proteins was investigated. Results: Agrin transcripts were found in all samples and their levels were significantly negatively correlated to age (p = 0.026), as were laminin transcripts (p = 0.023), whereas no such correlation was found for the other proteins analyzed. No significant correlations for any of the proteins were found when grouping patients by their gender or pathology. Immunohistochemistry and western blots to detect and localize agrin and the other proteins under analysis in RV tissue, confirmed their presence in patients of all ages. Conclusions: We show that agrin is progressively downregulated with age in human RV tissue but not as dramatically as has been demonstrated in mice; highlighting both similarities and differences to findings in rodents. Our results lay the groundwork for future studies exploring the potential of agrin-based therapies in the repair of damaged human hearts.

Effect of cardioplegic arrest and reperfusion on left and right ventricular proteome/phosphoproteome in patients undergoing surgery for coronary or aortic valve disease.

Our earlier work has shown inter­disease and intra­disease differences in the cardiac proteome between right (RV) and left (LV) ventricles of patients with aortic valve stenosis (AVS) or coronary artery disease (CAD). Whether disease remodeling also affects acute changes occuring in the proteome during surgical intervention is unknown. This study investigated the effects of cardioplegic arrest on cardiac proteins/phosphoproteins in LV and RV of CAD (n=6) and AVS (n=6) patients undergoing cardiac surgery. LV and RV biopsies were collected during surgery before ischemic cold blood cardioplegic arrest (pre) and 20 min after reperfusion (post). Tissues were snap frozen, proteins extracted, and the extracts were used for proteomic and phosphoproteomic analysis using Tandem Mass Tag (TMT) analysis. The results were analysed using QuickGO and Ingenuity Pathway Analysis softwares. For each comparision, our proteomic analysis identified more than 3,000 proteins which could be detected in both the pre and Post samples. Cardioplegic arrest and reperfusion were associated with significant differential expression of 24 (LV) and 120 (RV) proteins in the CAD patients, which were linked to mitochondrial function, inflammation and cardiac contraction. By contrast, AVS patients showed differential expression of only 3 LV proteins and 2 RV proteins, despite a significantly longer duration of ischaemic cardioplegic arrest. The relative expression of 41 phosphoproteins was significantly altered in CAD patients, with 18 phosphoproteins showing altered expression in AVS patients. Inflammatory pathways were implicated in the changes in phosphoprotein expression in both groups. Inter­disease comparison for the same ventricular chamber at both timepoints revealed differences relating to inflammation and adrenergic and calcium signalling. In conclusion, the present study found that ischemic arrest and reperfusion trigger different changes in the proteomes and phosphoproteomes of LV and RV of CAD and AVS patients undergoing surgery, with markedly more changes in CAD patients despite a significantly shorter ischaemic period.

Pathology-related changes in cardiac energy metabolites, inflammatory response and reperfusion injury following cardioplegic arrest in patients undergoing open-heart surgery.

Introduction: Changes in cardiac metabolites in adult patients undergoing open-heart surgery using ischemic cardioplegic arrest have largely been reported for non-ventricular tissue or diseased left ventricular tissue, with few studies attempting to assess such changes in both ventricular chambers. It is also unknown whether such changes are altered in different pathologies or linked to the degree of reperfusion injury and inflammatory response. The aim of the present work was to address these issues by monitoring myocardial metabolites in both ventricles and to establish whether these changes are linked to reperfusion injury and inflammatory/stress response in patients undergoing surgery using cold blood cardioplegia for either coronary artery bypass graft (CABG, n = 25) or aortic valve replacement (AVR, n = 16). Methods: Ventricular biopsies from both left (LV) and right (RV) ventricles were collected before ischemic cardioplegic arrest and 20 min after reperfusion. The biopsies were processed for measuring selected metabolites (adenine nucleotides, purines, and amino acids) using HPLC. Blood markers of cardiac injury (Troponin I, cTnI), inflammation (IL- 6, IL-8, Il-10, and TNFα, measured using Multiplex) and oxidative stress (Myeloperoxidase, MPO) were measured pre- and up to 72 hours post-operatively. Results: The CABG group had a significantly shorter ischemic cardioplegic arrest time (38.6 ± 2.3 min) compared to AVR group (63.0 ± 4.9 min, p = 2 x 10-6). Cardiac injury (cTnI release) was similar for both CABG and AVR groups. The inflammatory markers IL-6 and Il-8 were significantly higher in CABG patients compared to AVR patients. Metabolic markers of cardiac ischemic stress were relatively and significantly more altered in the LV of CABG patients. Comparing diabetic and non-diabetic CABG patients shows that only the RV of diabetic patients sustained major ischemic stress during reperfusion and that diabetic patients had a significantly higher inflammatory response. Discussion: CABG patients sustain relatively more ischemic stress, systemic inflammatory response and similar injury and oxidative stress compared to AVR patients despite having significantly shorter cross-clamp time. The higher inflammatory response in CABG patients appears to be at least partly driven by a higher incidence of diabetes amongst CABG patients. In addition to pathology, the use of cold blood cardioplegic arrest may underlie these differences.

Corrigendum: Agrin-Mediated Cardiac Regeneration: Some Open Questions.

[This corrects the article on p. 594 in vol. 8, PMID: 32612983.].

Agrin-Mediated Cardiac Regeneration: Some Open Questions.

After cardiac injury, the mammalian adult heart has a very limited capacity to regenerate, due to the inability of fully differentiated cardiomyocytes (CMs) to efficiently proliferate. This has been directly linked to the extracellular matrix (ECM) surrounding and connecting cardiomyocytes, as its increasing rigidity during heart maturation has a crucial impact over the proliferative capacity of CMs. Very recent studies using mouse models have demonstrated how the ECM protein agrin might promote heart regeneration through CMs de-differentiation and proliferation. In maturing CMs, this proteoglycan would act as an inducer of a specific molecular pathway involving ECM receptor(s) within the transmembrane dystrophin-glycoprotein complex (DGC) as well as intracellular Yap, an effector of the Hippo pathway involved in the replication/regeneration program of CMs. According to the mechanism proposed, during mice heart development agrin gets progressively downregulated and ultimately replaced by other ECM proteins eventually leading to loss of proliferation/ regenerative capacity in mature CMs. Although the role played by the agrin-DGC-YAP axis during human heart development remains still largely to be defined, this scenario opens up fascinating and promising therapeutic avenues. Herein, we discuss the currently available relevant information on this system, with a view to explore how the fundamental understanding of the regenerative potential of this cellular program can be translated into therapeutic treatment of injured human hearts.

Hypertension Programmed in Adult Hens by Isolated Effects of Developmental Hypoxia In Ovo.

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% O2) or hypoxia (H; 13.5%-14% O2) 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.