Maternal hypoxia decreases capillary supply and increases metabolic inefficiency leading to divergence in myocardial oxygen supply and demand

Maternal hypoxia is associated with a decrease in left ventricular capillary density while cardiac performance is preserved, implying a mismatch between metabolism and diffusive exchange. We hypothesised this requires a switch in substrate metabolism to maximise efficiency of ATP production from limited oxygen availability. Rat pups from pregnant females exposed to hypoxia (FIO2=0.12) at days 10-20 of pregnancy were grown to adulthood and working hearts perfused ex vivo. 14 C-labelled glucose and 3 H-palmitate were provided as substrates and metabolism quantified from recovery of 14CO2 and 3 H2O, respectively. Hearts of male offspring subjected to Maternal Hypoxia showed a 20% decrease in cardiac output (P<0.05), despite recording a 2-fold increase in glucose oxidation (P<0.01) and 2.5-fold increase (P<0.01) in palmitate oxidation. Addition of insulin to Maternal Hypoxic hearts, further increased glucose oxidation (P<0.01) and suppressed palmitate oxidation (P<0.05), suggesting preservation in insulin signalling in the heart. In vitro enzyme activity measurements showed that Maternal Hypoxia increased both total and the active component of cardiac pyruvate dehydrogenase (both P<0.01), although pyruvate dehydrogenase sensitivity to insulin was lost (NS), while citrate synthase activity declined by 30% (P<0.001) and acetyl-CoA carboxylase activity was unchanged by Maternal Hypoxia, indicating realignment of the metabolic machinery to optimise oxygen utilisation. Capillary density was quantified and oxygen diffusion characteristics examined, with calculated capillary domain area increased by 30% (P<0.001). Calculated metabolic efficiency decreased 4-fold (P<0.01) for Maternal Hypoxia hearts. Paradoxically, the decline in citrate synthase activity and increased metabolism suggest that the scope of individual mitochondria had declined, rendering the myocardium potentially more sensitive to metabolic stress. However, decreasing citrate synthase may be essential to preserve local PO2, minimising regions of hypoxia and hence maximising the area of myocardium able to preserve cardiac output following maternal hypoxia

Prenatal hypoxia induces increased cardiac contractility on a background of decreased capillary density.

Background: Chronic hypoxia in utero (CHU) is one of the most common insults to fetal development and may be associated with poor cardiac recovery from ischaemia-reperfusion injury,yet the effects on normal cardiac mechanical performance are poorly understood. Methods: Pregnant female wistar rats were exposed to hypoxia (12% oxygen, balance nitrogen)for days 10–20 of pregnancy. Pups were born into normal room air and weaned normally. At 10 weeks of age, hearts were excised under anaesthesia and underwent retrograde 'Langendorff' perfusion. Mechanical performance was measured at constant filling pressure (100 cm H2O) with intraventricular balloon. Left ventricular free wall was dissected away and capillary density estimated following alkaline phosphatase staining. Expression of SERCA2a and Nitric Oxide Synthases (NOS) proteins were estimated by immunoblotting. Results: CHU significantly increased body mass (P < 0.001) compared with age-matched control rats but was without effect on relative cardiac mass. For incremental increases in left ventricular balloon volume, diastolic pressure was preserved. However, systolic pressure was significantly greater following CHU for balloon volume = 50 μl (P < 0.01) and up to 200 μl (P < 0.05). For higher balloon volumes systolic pressure was not significantly different from control. Developed pressures were correspondingly increased relative to controls for balloon volumes up to 250 μl (P < 0.05).Left ventricular free wall capillary density was significantly decreased in both epicardium (18%; P <0.05) and endocardium (11%; P < 0.05) despite preserved coronary flow. Western blot analysis revealed no change to the expression of SERCA2a or nNOS but immuno-detectable eNOS protein was significantly decreased (P < 0.001) in cardiac tissue following chronic hypoxia in utero. Conclusion: These data offer potential mechanisms for poor recovery following ischaemia, including decreased coronary flow reserve and impaired angiogenesis with subsequent detrimental effects of post-natal cardiac performance

Ephemeral detection of Bonamia exitiosa (Haplosporida) in adult and larval European flat oysters Ostrea edulis in the Solent, United Kingdo

The haplosporidian parasite Bonamia exitiosa was detected using PCR in four adult and six larval brood samples of the European flat oyster Ostrea edulis from the Solent, UK. This represents the second reported detection of this parasite along the south coast of England. Adult oysters were collected and preserved from seabed populations or restoration broodstock cages between 2015 and 2018. The larvae within brooding adults sampled during 2017 and 2018 were also preserved. Molecular analysis of all samples was performed in 2019. The DNA of B. exitiosa was confirmed to be present within the gill tissue of one oyster within the Portsmouth wild fishery seabed population (n = 48), sampled in November 2015; the congeneric parasite Bonamia ostreae was not detected in this individual. This is the earliest record of B. exitiosa in the Solent. Concurrent presence of both B. ostreae and B. exitiosa, determined by DNA presence, was confirmed in the gill and heart tissue of three mature individuals from broodstock cages sampled in October 2017 (n = 99), two from a location on the River Hamble and one from the Camber Dock in Portsmouth Harbour. B. exitiosa was not detected in the November 2018 broodstock populations. A total of six larval broods were positive for B. exitiosa, with five also positive for B. ostreae. None of the brooding adults were positive for B. exitiosa suggesting that horizontal transmission from the surrounding environment to the brooding larvae is occurring. Further sampling of broodstock populations conducted by the Fish Health Inspectorate at the Centre for Environment, Fisheries and Aquaculture Science in June 2019 did not detect infection of O. edulis by B. exitiosa. These findings together suggest that the pathogen has not currently established in the area

Metabolomics using anion-exchange chromatography mass spectrometry for the analysis of cells, tissues and biofluids

The direct coupling of ion-exchange chromatography with mass spectrometry using electrochemical ion suppression creates a hyphenated technique with selectivity and specificity for the analysis of highly polar and ionic compounds. The technique has enabled new applications in environmental chemistry, food chemistry, forensics, cell biology and, more recently, metabolomics. Robust, reproducible and quantitative methods for the analysis of highly polar and ionic metabolites help meet a longstanding analytical need in metabolomics. Here, we provide step-by-step instructions for both untargeted and semi-targeted metabolite analysis from cell, tissue or biofluid samples by using anion-exchange chromatography–high-resolution tandem mass spectrometry (AEC-MS/MS). The method requires minimal sample preparation and is robust, sensitive and selective. It provides comprehensive coverage of hundreds of metabolites found in primary and secondary metabolic pathways, including glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, purine and pyrimidine metabolism, amino acid degradation and redox metabolism. An inline electrolytic ion suppressor is used to quantitatively neutralize OH− ions in the eluent stream, after chromatographic separation, enabling AEC to be directly coupled with MS. Counter ions are also removed during this process, creating a neutral pH, aqueous eluent with a simplified matrix optimal for negative ion MS analysis. Sample preparation through to data analysis and interpretation is described in the protocol, including a guide to which metabolites and metabolic pathways are suitable for analysis by using AEC-MS/MS

Energy Metabolism in the Failing Right Ventricle: Limitations of Oxygen Delivery and the Creatine Kinase System

Pulmonary arterial hypertension (PAH) results in hypertrophic remodeling of the right ventricle (RV) to overcome increased pulmonary pressure. This increases the O₂ consumption of the myocardium, and without a concomitant increase in energy generation, a mismatch with demand may occur. Eventually, RV function can no longer be sustained, and RV failure occurs. Beta-adrenergic blockers (BB) are thought to improve survival in left heart failure, in part by reducing energy expenditure and hypertrophy, however they are not currently a therapy for PAH. The monocrotaline (MCT) rat model of PAH was used to investigate the consequence of RV failure on myocardial oxygenation and mitochondrial function. A second group of MCT rats was treated daily with the beta-1 blocker metoprolol (MCT + BB). Histology confirmed reduced capillary density and increased capillary supply area without indications of capillary rarefaction in MCT rats. A computer model of O₂ flux was applied to the experimentally recorded capillary locations and predicted a reduction in mean tissue Po₂ in MCT rats. The fraction of hypoxic tissue (defined as Po₂ < 0.5 mmHg) was reduced following beta-1 blocker (BB) treatment. The functionality of the creatine kinase (CK) energy shuttle was measured in permeabilized RV myocytes by sequential ADP titrations in the presence and absence of creatine. Creatine significantly decreased the KmADP in cells from saline-injected control (CON) rats, but not MCT rats. The difference in KmADP with or without creatine was not different in MCT + BB cells compared to CON or MCT cells. Improved myocardial energetics could contribute to improved survival of PAH with chronic BB treatment

Glycogen metabolism protects against metabolic insult to preserve carotid body function during glucose deprivation

The view that the carotid body (CB) type I cells are direct physiological sensors of hypoglycaemia is challenged by the finding that the basal sensory neuronal outflow from the whole organ is unchanged in response to low glucose. The reason for this difference in viewpoint and how the whole CB maintains its metabolic integrity when exposed to low glucose is unknown. Here we show that, in the intact superfused rat CB, basal sensory neuronal activity was sustained during glucose deprivation for 29.1 ± 1.2 min, before irreversible failure following a brief period of excitation. Graded increases in the basal discharge induced by reducing the superfusate PO2 led to proportional decreases in the time to the pre-failure excitation during glucose deprivation which was dependent on a complete run-down in glycolysis and a fall in cellular energy status. A similar ability to withstand prolonged glucose deprivation was observed in isolated type I cells. Electron micrographs and immunofluorescence staining of rat CB sections revealed the presence of glycogen granules and the glycogen conversion enzymes glycogen synthase I and glycogen phosphorylase BB, dispersed throughout the type I cell cytoplasm. Furthermore, pharmacological attenuation of glycogenolysis and functional depletion of glycogen both significantly reduced the time to glycolytic run-down by ∼33 and 65%, respectively. These findings suggest that type I cell glycogen metabolism allows for the continuation of glycolysis and the maintenance of CB sensory neuronal output in periods of restricted glucose delivery and this may act as a key protective mechanism for the organ during hypoglycaemia. The ability, or otherwise, to preserve energetic status may thus account for variation in the reported capacity of the CB to sense physiological glucose concentrations and may even underlie its function during pathological states associated with augmented CB discharge.</p

On the interdependence of ketone body oxidation,glycogen content, glycolysis and energy metabolism in the heart

In heart, glucose and glycolysis are important for anaplerosis and potentially therefore for d-β-hydroxybutyrate (βHB) oxidation. As a glucose store, glycogen may also furnish anaplerosis. We determined the effects of glycogen content on βHB oxidation and glycolytic rates, and their downstream effects on energetics, in the isolated rat heart. High glycogen (HG) and low glycogen (LG) containing hearts were perfused with 11 mM [5-3H]glucose and/or 4 mM [14C]βHB to measure glycolytic rates or βHB oxidation, respectively, then freeze-clamped for glycogen and metabolomic analyses. Free cytosolic [NAD+]/[NADH] and mitochondrial [Q+]/[QH2] ratios were estimated using the lactate dehydrogenase and succinate dehydrogenase reaction, respectively. Phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations were measured using 31P-nuclear magnetic resonance spectroscopy. Rates of βHB oxidation in LG hearts were half that in HG hearts, with βHB oxidation directly proportional to glycogen content. βHB oxidation decreased glycolysis in all hearts. Glycogenolysis in glycogen-replete hearts perfused with βHB alone was twice that of hearts perfused with βHB and glucose, which had significantly higher levels of the glycolytic intermediates fructose 1,6-bisphosphate and 3-phosphoglycerate, and higher free cytosolic [NAD+]/[NADH]. βHB oxidation increased the Krebs cycle intermediates citrate, 2-oxoglutarate and succinate, the total NADP/H pool, reduced mitochondrial [Q+]/[QH2], and increased the calculated free energy of ATP hydrolysis (∆GATP). Although βHB oxidation inhibited glycolysis, glycolytic intermediates were not depleted, and cytosolic free NAD remained oxidised. βHB oxidation alone increased Krebs cycle intermediates, reduced mitochondrial Q and increased ∆GATP. We conclude that glycogen facilitates cardiac βHB oxidation by anaplerosis

L-Carnitine Stimulates In Vivo Carbohydrate Metabolism in the Type 1 Diabetic Heart as Demonstrated by Hyperpolarized MRI.

Funder: Danish Council for Strategic Research; Grant(s): “LIFE-DNP: hyperpolarized magnetic resonance for in vivo quantification of lipid, sugar and amino acid metabolism in lifestyle related diseases”The diabetic heart is energetically and metabolically abnormal, with increased fatty acid oxidation and decreased glucose oxidation. One factor contributing to the metabolic dysfunction in diabetes may be abnormal handling of acetyl and acyl groups by the mitochondria. L-carnitine is responsible for their transfer across the mitochondrial membrane, therefore, supplementation with L-carnitine may provide a route to improve the metabolic state of the diabetic heart. The primary aim of this study was to use hyperpolarized magnetic resonance imaging (MRI) to investigate the effects of L-carnitine supplementation on the in vivo metabolism of [1-13C]pyruvate in diabetes. Male Wistar rats were injected with either vehicle or streptozotocin (55 mg/kg) to induce type-1 diabetes. Three weeks of daily i.p. treatment with either saline or L-carnitine (3 g/kg/day) was subsequently undertaken. In vivo cardiac function and metabolism were assessed with CINE and hyperpolarized MRI, respectively. L-carnitine supplementation prevented the progression of hyperglycemia, which was observed in untreated streptozotocin injected animals and led to reductions in plasma triglyceride and ß-hydroxybutyrate concentrations. Hyperpolarized MRI revealed that L-carnitine treatment elevated pyruvate dehydrogenase flux by 3-fold in the diabetic animals, potentially through increased buffering of excess acetyl-CoA units in the mitochondria. Improved functional recovery following ischemia was also observed in the L-carnitine treated diabetic animals

Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation

Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with β-alanine buildup. Raising β-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function