Measured O2 Consumption Research Articles

The influence of temperature on oxygen uptake of red alga Hildenbrandia rivularis – the next step of the indicatory potential revision

Hildenbrandia rivularis belongs to the freshwater red algae and is cosmopolitan. In some European countries, this species is protected, e.g., in Poland, where it mainly inhabits highly oxygenated, fast-flowing ecosystems. This alga is often considered both a bioindicator of oligotrophic waters and a relatively rare species in Europe. However, the expansion and ecological tolerance of H. rivularis have increased over the last decades; thus, there is an urgent call to retest its environmental optima and significance for bioindicative potential. In this paper, H. rivularis from Welna River (Poland) growing on hard substrates was tested. In addition to genetic, microscopic, and physicochemical analyses, we also delivered for the first time the relationship between the transient temperature changes (15 – 45 °C, with 5 °C intervals) and oxygen uptake of H. rivularis (based on ex situ measurements of O2 consumption by thalli). Interestingly, for the eurythermal H. rivularis, at the lowest temperature (15 °C) treatment, the O2 uptake was undetectable, but the respiratory rate reached maximal velocity at the two highest temperatures (40 and 45 °C). Importantly, the respiration of this alga was relatively stable across temperature gradient 20 – 35 °C. This observation could explain why this species has been disappearing from colder waters of uplands and mountains and started to prefer warmer lowland water ecosystems. The further increase in global warming can significantly accelerate this tendency, thus causing a significant change in the H. rivularis distribution pattern known from the previous literature. Finally, our research sheds new light on the bioindicative potential of H. rivularis.

A potent and selective activator of large-conductance Ca2+-activated K+ channels induces preservation of mitochondrial function after hypoxia and reoxygenation by handling of calcium and transmembrane potential.

Ischaemic heart disease remains a significant cause of mortality globally. A pharmacological agent that protects cardiac mitochondria against oxygen deprivation injuries is welcome in therapy against acute myocardial infarction. Here, we evaluate the effect of large-conductance Ca2+-activated K+ channels (BKCa) activator, Compound Z, in isolated mitochondria under hypoxia and reoxygenation. Mitochondria from mice hearts were obtained by differential centrifugation. The isolated mitochondria were incubated with a BKCa channel activator, Compound Z, and subjected to normoxia or hypoxia/reoxygenation. Mitochondrial function was evaluated by measurement of O2 consumption in the complexes I, II, and IV in the respiratory states 1, 2, 3, and by maximal uncoupled O2 uptake, ATP production, ROS production, transmembrane potential, and calcium retention capacity. Incubation of isolated mitochondria with Compound Z under normoxia conditions reduced the mitochondrial functions and induced the production of a significant amount of ROS. However, under hypoxia/reoxygenation, the Compound Z prevented a profound reduction in mitochondrial functions, including reducing ROS production over the hypoxia/reoxygenation group. Furthermore, hypoxia/reoxygenation induced a large mitochondria depolarization, which Compound Z incubation prevented, but, even so, Compound Z created a small depolarization. The mitochondrial calcium uptake was prevented by the BKCa activator, extruding the mitochondrial calcium present before Compound Z incubation. The Compound Z acts as a mitochondrial BKCa channel activator and can protect mitochondria function against hypoxia/reoxygenation injury, by handling mitochondrial calcium and transmembrane potential.

Exposure to a winter photoperiod to produce large Atlantic salmon smolts (Salmo salar) increases energetic costs and reduces hypoxia tolerance during seawater transfer

A goal of salmon aquaculture is to produce larger, physiologically robust smolts to improve performance at time of seawater transfer and reduce time spent in marine net pens. However, little is known about whether standard processes to induce smoltification provide benefits in the production of large smolts. In Atlantic salmon (Salmo salar) reared under continuous light (24 L:0D) we applied an artificial photoperiod treatment (8 weeks of 12 L:12D followed by 4 weeks of 24 L:0D before seawater transfer) at three sizes to produce fish of ∼150–200 g, ∼400–600 g, and ∼ 1200–1300 g at seawater transfer. We then measured O2 consumption (a proxy for metabolic rate) of photoperiod treated and control salmon (kept in constant 24 L:0D until time of seawater transfer) at three time points (in freshwater prior to seawater entry, 24 h after seawater entry, and 1 month after seawater entry) to assess impacts of photoperiod treatment and seawater transfer on performance. Consistent with known higher metabolic rates in smolts vs parr, photoperiod treatment (12 L:12D) increased minimum O2 consumption (ṀO2min) by ∼13% across all three sizes and at all time points measured. Increased ṀO2min was not solely a result of increased growth rates and was associated with lower hypoxia tolerance at time of seawater transfer in ∼500 g and ∼ 1200 g salmon. Transfer to seawater reduced maximum O2 consumption (ṀO2max) acutely in ∼200 g and ∼ 500 g salmon regardless of the photoperiod treatment (12 L:12D or 24 L:0D), consistent with potential ion and osmotic disturbances leading to an osmo-respiratory compromise 24 h after seawater transfer. For both sizes of ∼200 g and ∼ 500 g, ṀO2max recovered to freshwater levels 1 month after seawater transfer. In contrast, ∼1200 g salmon showed no significant changes in ṀO2max following seawater transfer. Overall, we showed little benefits of traditional photoperiod treatments (12 L:12D) on performance of large salmon following seawater transfer. In fact, photoperiod treatment (12 L:12D) may lead to negative impacts on large salmon by increasing ṀO2min and sensitivity to environmental stress such as hypoxia. Our results, combined with other recent studies, suggest that traditional photoperiod manipulations may be unnecessary for the development of larger Atlantic salmon smolts. However, further research is needed to fully understand impacts of photoperiod manipulations on physiological performance of larger salmon at time of seawater transfer.

Quantification of oxygen consumption in head and neck cancer using fluorescent sensor foil technology.

Head and neck squamous cell carcinoma (HNSCC) patients suffer from frequent local recurrences that negatively impact on prognosis. Hence, distinguishing tumor and normal tissue is of clinical importance as it may improve the detection of residual tumor tissue in surgical resection margins and during imaging-based surgery planning. Differences in O2 consumption (OC) can be used to this aim, as they provide options for improved surgical, image-guided approaches. In the present study, the potential of a fluorescent sensor foil-based technology to quantify OC in HNSCC was evaluated in an in vitro 3D model and in situ in patients. In vitro measurements of OC using hypopharyngeal and esophageal cell lines allowed a specific detection of tumor cell spheroids embedded together with cancer-associated fibroblasts in type I collagen extracellular matrix down to a diameter of 440 µm. Pre-surgery in situ measurements were conducted with a handheld recording device and sensor foils with an oxygen permeable membrane and immobilized O2-reactive fluorescent dyes. Lateral tongue carcinoma and carcinoma of the floor of the mouth were chosen for analysis owing to their facilitated accessibility. OC was evaluated over a time span of 60 seconds and was significantly higher in tumor tissue compared to healthy mucosa in the vicinity of the tumor. Hence, OC quantification using fluorescent sensor foil-based technology is a relevant parameter for the differentiation of tumor tissue of the head and neck region and may support surgery planning.

Bioprotection by non-Saccharomyces yeasts in oenology: Evaluation of O2 consumption and impact on acetic acid bacteria

Bioprotection by yeast addition is increasingly used in oenology as an alternative to sulfur dioxide (SO2). Recent studies have also shown that it is likely to consume dissolved O2. This ability could limit O2 for other microorganisms and the early oxidation of the grape must. However, the ability of yeasts to consume O2 in a context of bioprotection was poorly studied so far considering the high genetic diversity of non-Saccharomyces. The first aim of the present study was to perform an O2 consumption rate (OCR) screening of strains from a large multi species collection found in oenology. The results demonstrate significant inter and intra species diversity with regard to O2 consumption. In the must M. pulcherrima consumes O2 faster than Saccharomyces cerevisiae and then other studied non-Saccharomyces species. The O2 consumption was also evaluate in the context of a yeast mix used as industrial bioprotection (Metschnikowia pulcherrima and Torulaspora delbrueckii) in red must. These non-Saccharomyces yeasts were then showed to limit the growth of acetic acid bacteria, with a bioprotective effect comparable to that of the addition of sulfur dioxide. Laboratory experiment confirmed the negative impact of the non-Saccharomyces yeasts on Gluconobacter oxydans that may be related to O2 consumption. This study sheds new lights on the use of bioprotection as an alternative to SO2 and suggest the possibility to use O2 consumption measurements as a new criteria for non-Saccharomyces strain selection in a context of bioprotection application for the wine industry.

Measuring O2 Consumption in Drosophila Melanogaster using Coulometric Microrespirometry.

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable environment. A coulometric microrespirometer consists of an airtight chamber in which O2 is consumed and the CO2 produced by the organism is removed by an absorbent medium. The resulting pressure decrease triggers electrolytic O2 production, and the amount of O2 produced is measured by recording the amount of charge used to generate it. In the present study, the method has been adapted to Drosophila melanogaster tested in small groups, with the sensitivity of the apparatus and the environmental conditions optimized for high stability. The amount of O2 consumed by wildtype flies in this apparatus is consistent with that measured by previous studies. Mass-specific O2 consumption by CASK mutants, which are smaller and known to be less active, was not different from congenic controls. However, the small size of CASK mutants resulted in a significant reduction in O2 consumption on a per-fly basis. Therefore, the microrespirometer is capable of measuring O2 consumption in D. melanogaster, can distinguish modest differences between genotypes, and adds a versatile tool for measuring metabolic rates.

Measurement of 24-h continuous human CH4 release in a whole room indirect calorimeter.

We describe the technology and validation of a new whole room indirect calorimeter (WRIC) methodology to quantify volume of methane (VCH4) released from the human body over 24 h concurrently with the assessment of energy expenditure and substrate utilization. The new system extends the assessment of energy metabolism by adding CH4, a downstream product of microbiome fermentation that could contribute to energy balance. Our new system consists of an established WRIC combined with the addition of off-axis integrated-cavity output spectroscopy (OA-ICOS) to measure CH4 concentration ([CH4]). Development, validation, and reliability of the system included environmental experiments to measure the stability of the atmospheric [CH4], infusing CH4 into the WRIC and human cross-validation studies comparing [CH4] quantified by OA-ICOS and mid-infrared dual-comb spectroscopy (MIR DCS).Our infusion data indicated that the system measured 24-h [CH4] and VCH4 with high sensitivity, reliability, and validity. Cross-validation studies showed good agreement between OA-ICOS and MIR DCS technologies (r = 0.979, P < 0.0001). Human data revealed 24-h VCH4 was highly variable between subjects and within/between days. Finally, our method to quantify VCH4 released by breath or colon suggested that over 50% of the CH4 was eliminated through the breath. The method allows, for the first time, measurement of 24-h VCH4 (in kcal) and therefore the measurement of the proportion of human energy intake fermented to CH4 by the gut microbiome and released via breath or from the intestine; also, it allows us to track the effects of dietary, probiotic, bacterial, and fecal microbiota transplantation on VCH4.NEW & NOTEWORTHY This is the first time that continuous assessment of CH4 is reported in parallel with measurements of O2 consumption and CO2 production inside a whole room indirect calorimeter in humans and over 24 h. We provide a detailed description of the whole system and its parts. We carried out studies of reliability and validity of the whole system and its parts. CH4 is released in humans during daily activities.

The comparative efficacy of two different doses of fentanyl on hemodynamic response to laryngoscopy and tracheal intubation: Prospective, randomized control trail

General anaesthesia with muscle relaxants using controlled ventilation involves laryngoscopy and tracheal intubation, which is associated with haemodynamic changes in the form of tachycardia and hypertension due to increased sympathoadrenal activity and are probably of no consequence in healthy individuals, but they may be hazardous to those with Myocardial Insufficiency and cerebrovascular disease. The objective of the present study was to compare the effect of two different doses of fentanyl with etomidate as an induction agent in attenuating haemodynamic stress response during laryngoscopy and endotracheal intubation. A randomised control trial was carried out on 60 adult patients (ASA I, II, III) undergoing elective surgery under general anaesthesia requiring endotracheal intubation. The patients were randomly allocated into two groups of 30 each i.e. group F2.5 and group F5 receiving fentanyl 2.5μg/kg and 5 μg/kg intravenously five minutes before intubation respectively. The pulse rate, systolic blood pressure, diastolic blood pressure, mean arterial pressure and rate pressure product were recorded at various time intervals up to ten minutes after intubation. The study showed that both the doses were equally effective in blunting the pulse rate response, but the 5μg /kg proved significantly effective in blunting the blood pressure response. The rate pressure product, a measure of cardiac O2 consumption was found to be significantly lower in fentanyl 5μg/kg compared to fentanyl 2.5μg/kg. Hence, we conclude that fentanyl in 5 mcg/kg dose is more effective in attenuating hemodynamic responses to intubation as compared to 2.5 mcg/kg.

Discovery of Mitochondrial Complex I Inhibitors as Anticancer and Radiosensitizer Drugs Based on Compensatory Stimulation of Lactate Release

Cancer cells may stimulate glycolytic flux when O2 becomes insufficient. Increase in L-lactate release therefore appears as an escape mechanism to drugs targeting mitochondrial respiration but also represents a response that may be exploited to screen for compounds blocking either mitochondrial carriers of oxidizable substrates or the electron transport chain. Here, we developed a screening procedure based on the capacity of cancer cells to release L-lactate to gain insights on the development of mitochondrial complex I inhibitors. For this purpose, we synthesized derivatives of carboxyamidotriazole, a compound previously described as a potential OXPHOS inhibitor. Two series of derivatives were generated by cycloaddition between benzylazide and either cyanoacetamides or alkynes. A primary assay measuring L-lactate release as a compensatory mechanism upon OXPHOS inhibition led us to identify 15 hits among 28 derivatives. A secondary assay measuring O2 consumption in permeabilized cancer cells confirmed that 12 compounds among the hits exhibited reversible complex I inhibitory activity. Anticancer effects of a short list of 5 compounds identified to induce more L-lactate release than reference compound were then evaluated on cancer cells and tumor-mimicking 3D spheroids. Human and mouse cancer cell monolayers exhibiting high level of respiration in basal conditions were up to 3-fold more sensitive than less oxidative cancer cells. 3D tumor spheroids further revealed potency differences between selected compounds in terms of cytotoxicity but also radiosensitizing activity resulting from local reoxygenation. In conclusion, this study documents the feasibility to efficiently screen in 96-well plate format for mitochondrial complex I inhibitors based on the capacity of drug candidates to induce L-lactate release.

Differentiating between fresh and frozen-thawed fish fillets by mitochondrial permeability measurement

In this study, we investigated the properties of mitochondria to discriminate fresh from frozen-thawed fish fillets. Mitochondria were isolated from gilthead seabream fillets and the impact of freezing was evaluated by measuring the permeability of mitochondrial membranes. Freezing led to permeabilization of mitochondrial inner membranes to reduced nicotinamide adenine dinucleotide (NADH). The increase in permeability related to freezing shock was compared to the physiological permeabilization of mitochondria isolated from gilthead seabream fillets stored at 4 °C. Two approaches were chosen to measure the increase in permeability: a spectrophotometric method to measure the consumption of NADH by complex I, and an oxygraphic method to measure O2 consumption by respiratory chains after exposure of mitochondria to NADH. Mitochondria isolated from frozen-thawed fillets were highly permeable to NADH and were no longer sensitive to a membrane permeabilizing agent: alamethicin. Altogether, our scientific approach allowed us to discriminate mitochondria isolated from fillets that have been exposed or not to a freezing shock (−80 °C) and thus to discriminate between fresh and frozen-thawed fish fillets.

Effects of calcium salts of palm oil inclusion and ad libitum feeding regimen on growth performance, carcass characteristics, and plasma glucose-dependent insulinotropic polypeptide concentration of feedlot steers.

Sixty Angus × SimAngus-crossbred steers (body weight [BW] 279 ± 16 kg) were used to evaluate the effect of calcium salts of palm oil inclusion (CPO) and the amount of feed offered (AFO) on plasma glucose-dependent insulinotropic polypeptide (GIP) concentration and its association with energy metabolism and marbling score (MS) in feedlot steers. Steers were blocked by BW and gain to feed (G:F) and randomly assigned to individual feedlot pens. Treatments (2 × 2 factorial) consisted of ad libitum-fed steers without (ANF) or with (AWF) the inclusion of CPO or restricted-fed steers (85% of the ad libitum intake of ANF) without (RNF) or with the inclusion of CPO (RWF). After weaning, steers were adapted to individual pens and fed a corn silage-based diet for 30 d and subsequently placed in a ground corn (GC)-based diet. Diets were given ad libitum or at 85% of the ANF intake and with or without CPO. After 59 d on the finishing diet, all steers had ad libitum access to the finishing diet until harvest. Measurements of CO2 emission and O2 consumption to estimate respiratory quotient (RQ) were taken (n = 9/treatment). Correlations between plasma GIP and insulin concentrations and RQ were analyzed. A linear regression was performed to evaluate the association of plasma GIP and MS. All data were analyzed using the PROC MIXED procedure of SAS. During the first 103 d of the trial, there were AFO × CPO interactions (P ≤ 0.01) for BW, dry matter intake (DMI), average daily gain (ADG), and net energy for maintenance (NEm) intake. Ad libitum-fed steers without CPO presented the greatest DMI among dietary treatments and had greater BW and ADG compared with steers in the RWF and RNF treatments. After all steers had ad libitum access to dietary treatments, steers that were previously restricted showed a 30% and 19% increase (P ≤ 0.01) in ADG and G:F, respectively. There was a three-way interaction time × CPO × AFO (P = 0.04) for plasma GIP concentration. There was no correlation (P = 0.96) of GIP with RQ, whereas insulin demonstrated marginal significance for a positive (P = 0.07) and negative (P = 0.08) correlation with plasma GIP and RQ, respectively. There was no association (P = 0.30) between GIP and MS. These data indicate that GIP secretion results from an interaction between CPO and energy intake depending on the time relative to feed intake that GIP might indirectly regulate energy metabolism through insulin secretion, and that GIP does not appear to be associated with MS.

Single Cell Assessment of Mitochondrial Function

Cellular mitochondrial function can be assessed using high resolution respirometry that measures O2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDHmax) in isolated human airway smooth muscle (hASM) cells using 1‐methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDHmax, the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil‐immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDHmax was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP‐mediated disruption of the mitochondrial proton gradient does not affect SDHmax, while SDHmax is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDHmax was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDHmax can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.

Measurement of oxygen consumption in Tenebrio molitor using a sensitive, inexpensive, sensor-based coulometric microrespirometer.

Coulometric respirometry is a highly sensitive method for measuring O2 consumption in small organisms but it is not in widespread use among physiologists. Here, we describe a coulometric microrespirometer based on a digital environmental sensor inside a sealed glass chamber and controlled by an Arduino™ microcontroller. As O2 is consumed, exhaled CO2 is removed, causing pressure to decrease in the chamber. The sensor detects the decreased pressure, and the controller activates electrolytic production of O2, returning pressure to the initial value. O2 consumption is calculated from electrolytic charge transfer. The effects of developmental stage, body mass and temperature on O2 consumption of Tenebrio molitor beetles were easily measured by the apparatus. This straightforward design is a significant innovation in that it provides continuous data regarding environmental conditions inside the experimental chamber, can be fabricated easily, and is adaptable to a wide range of uses.

Coenzyme Q10 and related quinones oxidize H2S to polysulfides and thiosulfate

In the canonical pathway for mitochondrial H2S oxidation electrons are transferred from sulfide:quinone oxidoreductase (SQR) to complex III via ubiquinone (CoQ10). We previously observed that a number of quinones directly oxidize H2S and we hypothesize that CoQ10 may have similar properties. Here we examine H2S oxidation by CoQ10 and more hydrophilic, truncated forms, CoQ1 and CoQ0, in buffer using H2S and polysulfide fluorophores (AzMC and SSP4), silver nanoparticles to measure thiosulfate (H2S2O3), mass spectrometry to identify polysulfides and O2-sensitive optodes to measure O2 consumption. We show that all three quinones concentration-dependently catalyze the oxidization of H2S to polysulfides and thiosulfate in buffer with the potency CoQ0>CoQ1>CoQ10 and that CoQ0 specifically oxidizes H2S to per-polysulfides, H2S2,3,4. These reactions consume and require oxygen and are augmented by addition of SOD suggesting that the quinones, not superoxide, oxidize H2S. Related quinones, MitoQ, menadione and idebenone, oxidize H2S in similar reactions. Exogenous CoQ0 decreases cellular H2S and increases polysulfides and thiosulfate production and this is also O2-dependent, suggesting that the quinone has similar effects on sulfur metabolism in cells. Collectively, these results suggest an additional endogenous mechanism for H2S metabolism and a potential therapeutic approach in H2S-related metabolic disorders.

Characterizing the Electron Transport Chain: Functional Approach Using Extracellular Flux Analyzer on Mouse Tissue Samples.

The Seahorse Extracellular Flux Analyzer enables the high-throughput characterization of oxidative phosphorylation capacity based on the electron transport chain organization and regulation with relatively small amount of material. This development over the traditional polarographic Clark-type electrode approaches make it possible to analyze the respiratory features of mitochondria isolated from tissue samples of particular animal models. Here we provide a description of an optimized approach to carry out multi-well measurement of O2 consumption, with the Agilent Seahorse XFe96 analyzer on mouse brain and muscles to determine the tissue-specific oxidative phosphorylation properties. Protocols include the preparation of the tissue samples, isolation of mitochondria, and analysis of their function; in particular, the preparation and optimization of the reagents and samples.

The effect of age on mechanisms of exercise tolerance: Reduced arteriovenous oxygen difference causes lower oxygen consumption in older people.

To assess the effect of age on mechanisms of exercise tolerance. Prospective observational study recruited 71 healthy individuals divided into two groups according to their age i.e. younger (≤40years of age, N=43); and older (≥55years of age, N=28). All participants underwent maximal graded cardiopulmonary exercise stress testing using cycle ergometer with simultaneous non-invasive gas-exchange and central haemodynamic measurements. Using the Fick equation, arteriovenous O2 difference was calculated as the ratio between measured O2 consumption and cardiac output. The mean age of younger and older participants was 26.0±5.7years, and 65.1±6.6years respectively. Peak O2 consumption was significantly lower in older compared to the younger age group (18.8±5.2 vs 34.4±9.8mL/kg/min, p<0.01). Peak exercise cardiac output and cardiac index were not significantly different between the younger and older age groups (22.7±5.0 vs 22.1±3.9L/min, p=0.59; and 12.4±2.9 vs 11.8±1.9L/min/m2, p=0.29). Despite demonstrating significantly lower peak heart rate by 33 beats/min (129±18.3 vs 162±19.9, p<0.01), older participants demonstrated significantly higher stroke volume and stroke volume index compared to the younger age group (173±41.5 vs 142±34.9mL/min, p<0.01; and 92.1±18.1 vs 78.3±19.5mL/m2, p<0.01). Arteriovenous O2 difference was significantly lower in older compared to younger age group participants (9.01±3.0 vs 15.8±4.3 mlO2/100mL blood, p<0.01). Ability of skeletal muscles to extract delivered oxygen represented by reduced arteriovenous O2 difference at peak exercise appears to be the key determinant of exercise tolerance in healthy older individuals.

Peripheral and pulmonary effects of inorganic nitrite during exercise in heart failure with preserved ejection fraction.

To determine whether inorganic nitrite improves peripheral and pulmonary oxygen (O2 ) transport during exercise in heart failure with preserved ejection fraction (HFpEF). Data from two invasive, randomized, double-blind, placebo-controlled trials with matched workload exercise of inhaled and intravenous sodium nitrite were pooled for this analysis (n= 51). Directly measured O2 consumption (VO2 ) and blood gas data were used to evaluate the effect of nitrite on skeletal muscle O2 conductance (Dm), VO2 kinetics, alveolar capillary membrane O2 conductance (DL ), and O2 utilization during submaximal exercise. As compared to placebo, treatment with nitrite resulted in an improvement in Dm (+4.9± 6.5 vs. -0.9± 4.3mL/mmHg*min, P= 0.0008) as well as VO2 kinetics measured by mean response time (-5.0± 6.9 vs. -0.6± 6.0s, P= 0.03), with preserved O2 utilization despite increased convective O2 delivery through cardiac output (+0.4± 0.7 vs. -0.3± 0.9L/min, P= 0.02). Nitrite improved DL (+2.5± 6.3 vs. -2.0± 9.0mL/mmHg*min, P= 0.05) with exercise, which was associated with lower pulmonary capillary pressures (r=-0.34, P= 0.02), and reduced pulmonary dead space ventilation fraction (-0.01 ± 0.05 vs. +0.02 ± 0.05, P= 0.02). Sodium nitrite enhances skeletal muscle Dm during exercise as well as pulmonary O2 diffusion, optimizing O2 kinetics in tandem with increased convective O2 delivery through cardiac output augmentation. The favourable combined pulmonary, cardiac and peripheral effects of nitrite may improve exercise tolerance in people with HFpEF and requires further investigation. ClinicalTrials.gov ID NCT01932606 and NCT02262078.

Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress.

Background/Aims:The role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mouse embryonic fibroblasts (MEFs), the roles of VDAC1 knockout (VDAC1−/−) in apoptotic cell death are contradictory. The contribution and underlying mechanism of VDAC1−/− in oxidative stress-induced cell death in cardiac cells has not been established. We hypothesized that VDAC1 is an essential regulator of oxidative stress-induced cell death in H9c2 cells.Methods:We knocked out VDAC1 in this rat cardiomyoblast cell line with CRISPR-Cas9 genome editing technique to produce VDAC1−/− H9c2 cells, and determined if VDAC1 is critical in promoting cell death via oxidative stress induced by tert-butylhydroper-oxide (tBHP), an organic peroxide, or rotenone (ROT), an inhibitor of mitochondrial complex I by measuring cell viability with MTT assay, cell death with TUNEL stain and LDH release. The mitochondrial and glycolytic stress were examined by measuring O2 consumption rate (OCR) and extracellular acidification rate (ECAR) with a Seahorse XFp analyzer.Results:We found that under control conditions, VDAC1−/− did not affect H9c2 cell proliferation or mitochondrial respiration. However, compared to the wildtype (WT) cells, exposure to either tBHP or ROT enhanced the production of ROS, ECAR, and the proton (H+) production rate (PPR) from glycolysis, as well as promoted apoptotic cell death in VDAC1−/− H9c2 cells. VDAC1−/− H9c2 cells also exhibited markedly reduced mitochondria-bound hexokinase II (HKII) and Bax. Restoration of VDAC1 in VDAC1−/− H9c2 cells reinstated mitochondria-bound HKII and concomitantly decreased tBHP and ROT-induced ROS production and cell death. Interestingly, mitochondrial respiration remained the same after tBHP treatment in VDAC1−/− and WT H9c2 cells.Conclusion:Our results suggest that VDAC1−/− in H9c2 cells enhances oxidative stress-mediated cell apoptosis that is directly linked to the reduction of mitochondria-bound HKII and concomitantly associated with enhanced ROS production, ECAR, and PPR.

Metabolic and Hematological Responses to Endotoxin-Induced Inflammation in Chicks Experiencing Embryonic 2,3,7,8-Tetrachlorodibenzodioxin Exposure.

Dioxin exposure during bird embryonic development disrupts immunity as well as mechanisms involved in energy metabolism, potentially affecting negatively acute-phase responses to pathogens. Thus, we hypothesized that embryonic exposure to 2,3,7,8-tetrachlorodibenzodioxin (TCDD) changes the metabolism and blood physiology of domestic chicks, affecting their physiological competence for responding to immune challenges. To test this hypothesis, we injected doses of 0, 1.5, and 3 ng TCDD/egg (based on survival experiments) on embryonic day 4 and then measured O2 consumption and CO2 production for metabolic rate, ventilation, and body temperature (TB ) in 5-d-old chicks. Then, chicks were injected with lipopolysaccharide (LPS, endotoxin) or saline prior to repeating the physiological measurements. A second chick group exposed to identical TCDD and LPS treatments had blood partial pressure of oxygen, partial pressure of carbon dioxide, pH, bicarbonate concentration, lactate concentration, osmolality, hemoglobin concentration, red blood cell concentration, and hematocrit, as well as TB , analyzed at 1 and 5 h after LPS injection. Metabolism in chicks embryonically exposed to 1.5 and 3 ng TCDD/egg was up to 37% higher, whereas body mass of chicks exposed to 3 ng TCDD/egg was approximately 6% lower. Chicks embryonically exposed to 3 ng TCDD/egg challenged with LPS showed a relative persistent hypometabolism accompanied by elimination of the normal hematological and osmotic responses to LPS. We conclude that embryonic exposure to TCDD affects posthatching metabolism as well as impairs metabolic, hematological, and osmotic responses to LPS. Environ Toxicol Chem 2020;39:2208-2220. © 2020 SETAC.

Open-flow respirometry under field conditions: How does the airflow through the nest influence our results?

Open-flow respirometry is a common method to measure oxygen-uptake as a proxy of energy expenditure of organisms in real-time. Although most often used in the laboratory it has seen increasing application under field conditions. Air is drawn or pushed through a metabolic chamber or the nest with the animal, and the O2 depletion and/or CO2 accumulation in the air is analysed to calculate metabolic rate and energy expenditure. Under field conditions, animals are often measured within the microclimate of their nest and in contrast to laboratory work, the temperature of the air entering the nest cannot be controlled. Thus, the aim of our study was to determine the explanatory power of respirometry in a set-up mimicking field conditions. We measured O2 consumption of 14 laboratory mice (Mus musculus) using three different flow rates [50L*h-1 (834mL*min-1), 60L*h-1 (1000mL*min-1) and 70L*h-1 (1167mL*min-1)] and two different temperatures of the inflowing air; either the same as the temperature inside the metabolic chamber (no temperature differential; 20°C), or cooler (temperature differential of 10°C). Our results show that the energy expenditure of the mice did not change significantly in relation to a cooler airflow, nor was it affected by different flow rates, despite a slight, but significant decrease of about 1.5°C in chamber temperature with the cooler airflow. Our study emphasises the validity of the results obtained by open-flow respirometry when investigating energy budgets and physiological responses of animals to ambient conditions. Nevertheless, subtle changes in chamber temperature in response to changes in the temperature and flow rate of the air pulled or pushed through the system were detectable. Thus, constant airflow during open-flow respirometry and consequent changes in nest/chamber temperature should be measured.