Blood flow restricted resistance exercise and reductions in oxygen tension attenuate mitochondrial H2 O2 emission rates in human skeletal muscle.

KEY POINTS: Blood flow restricted resistance exercise (BFR-RE) is capable of inducing comparable adaptations to traditional resistance exercise (RE), despite a lower total exercise volume. It has been suggested that an increase in reactive oxygen species (ROS) production may be involved in this response; however, oxygen partial pressure ( PO2 ) is reduced during BFR-RE, and the influence of PO2 on mitochondrial redox balance remains poorly understood. In human skeletal muscle tissue, we demonstrate that both maximal and submaximal mitochondrial ROS emission rates are acutely decreased 2 h following BFR-RE, but not RE, occurring along with a reduction in tissue oxygenation during BFR-RE. We further suggest that PO2 is involved in this response because an in vitro analysis revealed that reducing PO2 dramatically decreased mitochondrial ROS emissions and electron leak to ROS. Altogether, these data indicate that mitochondrial ROS emission rates are attenuated following BFR-RE, and such a response is likely influenced by reductions in PO2 . ABSTRACT: Low-load blood flow restricted resistance exercise (BFR-RE) training has been proposed to induce comparable adaptations to traditional resistance exercise (RE) training, however, the acute signalling events remain unknown. Although a suggested mechanism of BFR-RE is an increase in reactive oxygen species (ROS) production, oxygen partial pressure ( PO2 ) is reduced during BFR-RE, and the influence of O2 tension on mitochondrial redox balance remains ambiguous. We therefore aimed to determine whether skeletal muscle mitochondrial bioenergetics were altered following an acute bout of BFR-RE or RE, and to further examine the role of PO2 in this response. Accordingly, muscle biopsies were obtained from 10 males at rest and 2 h after performing three sets of single-leg squats (RE or BFR-RE) to failure at 30% one-repetition maximum. We determined that mitochondrial respiratory capacity and ADP sensitivity were not altered in response to RE or BFR-RE. Although maximal (succinate) and submaximal (non-saturating ADP) mitochondrial ROS emission rates were unchanged following RE, BFR-RE attenuated these responses by ∼30% compared to pre-exercise, occurring along with a reduction in skeletal muscle tissue oxygenation during BFR-RE (P < 0.01 vs. RE). In a separate cohort of participants, evaluation of mitochondrial bioenergetics in vitro revealed that mild O2 restriction (50 µm) dramatically attenuated maximal (∼4-fold) and submaximal (∼50-fold) mitochondrial ROS emission rates and the fraction of electron leak to ROS compared to room air (200 µm). Combined, these data demonstrate that mitochondrial ROS emissions are attenuated following BFR-RE, a response which may be mediated by a reduction in skeletal muscle PO2 .

Effect of skeletal muscle mitochondrial phenotype on H2O2 emission.

Reactive oxygen species (ROS) are a key output of the skeletal muscle mitochondrial information processing system both at rest and during exercise. In skeletal muscle, mitochondrial ROS release depends on multiple factors; however, fiber-type specific differences remain ambiguous in part owing to the use of mitochondria from mammalian muscle that consist of mixed fibers. To elucidate fiber-type specific differences, we used mitochondria isolated from rainbow trout (Oncorhynchus mykiss) red and white skeletal muscles that consist of spatially distinct essentially pure red and white fibers. We first characterized the assay conditions for measuring ROS production (as H2O2) in isolated fish red and white skeletal muscle mitochondria (RMM and WMM) and thereafter compared the rates of emission during oxidation of different substrates and the responses to mitochondrial electron transport system (ETS) pharmacological modulators. Our results showed that H2O2 emission rates by RMM and WMM can be quantified using the same protein concentration and composition of the Amplex UltraRed-horseradish peroxidase (AUR-HRP) detection system. For both RMM and WMM, protein normalized H2O2 emission rates were highest at the lowest protein concentration tested and decreased exponentially thereafter. However, the absolute values of H2O2 emission rates depended on the calibration curves used to convert fluorescent signals to H2O2 while the trends depended on the normalization strategy. We found substantial qualitative and quantitative differences between RMM and WMM in the H2O2 emission rates depending on the substrates being oxidized and their concentrations. Similarly, pharmacological modulators of the ETS altered the magnitudes and trends of the H2O2 emission differently in RMM and WMM. While comparable concentrations of substrates elicited maximal albeit quantitively different emission rates in RMM and WMM, different concentrations of pharmacological ETS modulators may be required for maximal H2O2 emission rates depending on muscle fiber-type. Taken together, our study suggests that biochemical differences exist in RMM compared with WMM that alter substrate oxidation and responses to ETS modulators resulting in fiber-type specific mitochondrial H2O2 emission rates.

Interactions of binary mixtures of metals on rainbow trout (Oncorhynchus mykiss) heart mitochondrial H2O2 homeodynamics.

For continuous pumping of blood, the heart needs a constant supply of energy (ATP) that is primarily met via oxidative phosphorylation in the mitochondria of cardiomyocytes. However, sustained high rates of electron transport for energy conversion redox reactions predisposes the heart to the production of reactive oxygen species (ROS) and oxidative stress. Mitochondrial ROS are fundamental drivers of responses to environmental stressors including metals but knowledge of how combinations of metals alter mitochondrial ROS homeodynamics remains sparse. We explored the effects and interactions of binary mixtures of copper (Cu), cadmium (Cd), and zinc (Zn), metals that are common contaminants of aquatic systems, on ROS (hydrogen peroxide, H2O2) homeodynamics in rainbow trout (Oncorhynchus mykiss) heart mitochondria. Isolated mitochondria were energized with glutamate-malate or succinate and exposed to a range of concentrations of the metals singly and in equimolar binary concentrations. Speciation analysis revealed that Cu was highly complexed by glutamate or Tris resulting in Cu2+ concentrations in the picomolar to nanomolar range. The concentration of Cd2+ was 7.2-7.5 % of the total while Zn2+ was 15 % and 21 % of the total during glutamate-malate and succinate oxidation, respectively. The concentration-effect relationships for Cu and Cd on mitochondrial H2O2 emission depended on the substrate while those for Zn were similar during glutamate-malate and succinate oxidation. Cu + Zn and Cu + Cd mixtures exhibited antagonistic interactions wherein Cu reduced the effects of both Cd and Zn, suggesting that Cu can mitigate oxidative distress caused by Cd or Zn. Binary combinations of the metals acted additively to reduce the rate constant and increase the half-life of H2O2 consumption while concomitantly suppressing thioredoxin reductase and stimulating glutathione peroxidase activities. Collectively, our study indicates that binary mixtures of Cu, Zn, and Cd act additively or antagonistically to modulate H2O2 homeodynamics in heart mitochondria.

Exhaustive exercise alters native and site-specific H2O2 emission in red and white skeletal muscle mitochondria.

Mitochondrial reactive oxygen species (ROS) homeostasis is intricately linked to energy conversion reactions and entails regulation of the mechanisms of ROS production and removal. However, there is limited understanding of how energy demand modulates ROS balance. Skeletal muscle experiences a wide range of energy requirements depending on the intensity and duration of exercise and therefore is an excellent model to probe the effect of altered energy demand on mitochondrial ROS production. Because in most fish skeletal muscle exists essentially as pure spatially distinct slow-twitch red oxidative and fast-twitch white glycolytic fibers, it provides a natural system for investigating how functional specialization affects ROS homeostasis. We tested the hypothesis that acute increase in energy demand imposed by exhaustive exercise will increase mitochondrial H2O2 emission to a greater extent in red muscle mitochondria (RMM) compared with white muscle mitochondria (WMM). We found that native H2O2 emission rates varied by up to 6-fold depending on the substrate being oxidized and muscle fiber type, with RMM emitting at higher rates with glutamate-malate and palmitoylcarnitine while WMM emitted at higher rates with succinate and glyceral-3-phosphate. Exhaustive exercise increased the native and site-specific H2O2 emission rates; however, the maximal emission rates depended on the substrate, fiber type and redox site. The H2O2 consumption capacity and activities of individual antioxidant enzymes including the glutathione- and thioredoxin-dependent peroxidases as well as catalase were higher in RMM compared with WMM indicating that the activity of antioxidant defense system does not explain the differences in H2O2 emission rates in RMM and WMM. Overall, our study suggests that substrate selection and oxidation may be the key factors determining the rates of ROS production in RMM and WMM following exhaustive exercise.

Factors affecting liver mitochondrial hydrogen peroxide emission.

Mitochondria are key cellular sources of reactive oxygen species (ROS) and contain at least 12 known sites on multiple enzymes that convert molecular oxygen to superoxide and hydrogen peroxide (H2O2). Quantitation of site-specific ROS emission is critical to understand the relative contribution of different sites and the pathophysiologic importance of mitochondrial ROS. However, factors that affect mitochondrial ROS emission are not well understood. We characterized and optimized conditions for maximal total and site-specific H2O2 emission during oxidation of standard substrates and probed the source of the high H2O2 emission in unenergized rainbow trout liver mitochondria. We found that mitochondrial H2O2 emission capacity depended on the substrate being oxidized, mitochondrial protein concentration, and composition of the ROS detection system. Contrary to our expectation, addition of exogenous superoxide dismutase reduced H2O2 emission. Titration of conventional mitochondrial electron transfer system (ETS) inhibitors over a range of conditions revealed that one size does not fit all; inhibitor concentrations evoking maximal responses varied with substrate and were moderated by the presence of other inhibitors. Moreover, the efficacy of suppressors of electron leak (S1QEL1.1 and S3QEL2) was low and depended on the substrate being oxidized. We found that H2O2 emission in unenergized rainbow trout liver mitochondria was suppressed by GKT136901 suggesting that it is associated with NADPH oxidase activity. We conclude that optimization of assay conditions is critical for quantitation of maximal H2O2 emission and would facilitate more valid comparisons of mitochondrial total and site-specific H2O2 emission capacities between studies, tissues, and species.

Anoxia-reoxygenation modulates cadmium-induced liver mitochondrial reactive oxygen species emission during oxidation of glycerol 3-phosphate.

Aquatic organisms are frequently exposed to multiple stressors including low dissolved oxygen (O2) and metals such as cadmium (Cd). Reduced O2 concentration and Cd exposure alter cellular function in part by impairing energy metabolism and dysregulating reactive oxygen species (ROS) homeostasis. However, little is known about the role of mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) in ROS homeostasis in fish and its response to environmental stress. In this study, mGPDH activity and the effects of anoxia-reoxygenation (A-RO) and Cd on ROS (as hydrogen peroxide, H2O2) emission in rainbow trout liver mitochondria during oxidation of glycerol 3-phosphate (G3P) were probed. Trout liver mitochondria exhibited low mGPDH activity that supported a low respiratory rate but substantial H2O2 emission rate. Cd evoked a low concentration stimulatory-high concentration inhibitory H2O2 emission pattern that was blunted by A-RO. At specific redox centers, Cd suppressed H2O2 emission from site IQ, but stimulated emission from sites IIIQo and GQ. In contrast, A-RO stimulated H2O2 emission from site IQ following 15 min exposure and augmented Cd-stimulated emission from site IIF after 30 min exposure but did not alter the rate of H2O2 emission from sites IIIQo and GQ. Additionally, Cd neither altered the activities of catalase, glutathione peroxidase, or thioredoxin reductase nor the concentrations of total glutathione, reduced glutathione, or oxidized glutathione. Overall, this study indicates that oxidation of G3P drives ROS production from mGPDH and complexes I, II and III, whereas Cd directly modulates redox sites but not antioxidant defense systems to alter mitochondrial H2O2 emission.

Anoxia-reoxygenation alters H2O2 efflux and sensitivity of redox centers to copper in heart mitochondria.

Mitochondrial reactive oxygen species (ROS) have been implicated in organ damage caused by environmental stressors, prompting studies on the effect of oxygen deprivation and metal exposure on ROS metabolism. However, how anoxia and copper (Cu) jointly influence heart mitochondrial ROS metabolism is not understood. We used rainbow trout heart mitochondria to probe the effects of anoxia-reoxygenation and Cu on hydrogen peroxide (H2O2) emission during oxidation of palmitoylcarnitine (PC), succinate, or glutamate-malate. In addition, we examined the influence of anoxia-reoxygenation and Cu on site-specific H2O2 emission capacities and key antioxidant enzymes, glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Results showed that anoxia-reoxygenation suppressed H2O2 emission regardless of substrate type or duration of anoxia. Anoxia-reoxygenation reduced mitochondrial sensitivity to Cu during oxidation of succinate or glutamate-malate whereas high Cu concentration additively stimulated H2O2 emission in mitochondria oxidizing PC. Prolonged anoxia-reoxygenation stimulated H2O2 emission from sites OF and IF, inhibited emission from sites IQ, IIF and IIIQo, and disparately altered the sensitivity of the sites to Cu. Interestingly, anoxia-reoxygenation increased GPx and TrxR activities, more prominently when reoxygenation followed a short duration of anoxia. Cu did not alter GPx but reduced TrxR activity in normoxic and anoxic-reoxygenated mitochondria. Overall, our study revealed potential mechanisms that may reduce oxidative damage associated with anoxia-reoxygenation and Cu exposure in heart mitochondria. The increased and decreased H2O2 emission from NADH/NAD+ and QH2/Q isopotential sites, respectively, may represent a balance between H2O2 required for oxygen deprivation-induced signaling and prevention of ROS burst associated with anoxia-reoxygenation.

Acute erythropoietin injection increases muscle mitochondrial respiratory capacity in young men: a double-blinded randomized crossover trial.

The aim was to investigate if acute recombinant human erythropoietin (rHuEPO) injection had an effect on mitochondrial function and if exercise would have an additive effect. Furthermore, to investigate if in vitro incubation with rHuEPO had an effect on muscle mitochondrial respiratory capacity. Eight healthy young men were recruited for this double-blinded randomized placebo-controlled crossover study. rHuEPO (400 IU/kg body wt) or saline injection was given intravenously, before an acute bout of exercise. Resting metabolic rate and fat oxidation were measured. Biopsies were obtained at baseline, 120 min after injection, and right after the acute exercise bout. Mitochondrial function (mitochondrial respiration and H2O2 emission) was measured in permeabilized skeletal muscle using high-resolution respirometry and fluorometry. Specific gene expression and enzyme activity were measured. Skeletal muscle mitochondrial respiratory capacity was measured with and without incubation with rHuEPO. Fat oxidation at rest increased after rHuEPO injection, but no difference was found in fat oxidation during exercise. Mitochondrial respiratory capacity was increased after rHuEPO injection when pyruvate was in the assay, which was not the case when saline was injected. No changes were seen in H2O2 emission after rHuEPO injection or acute exercise. Incubation of skeletal muscle fibers in vitro with rHuEPO increased mitochondrial respiratory capacity. Acute rHuEPO injection increased mitochondrial respiratory capacity when pyruvate was used in the assay. No statistical difference was found in H2O2 emission capacity, although a numerical increase was seen after rHuEPO injection. In vitro incubation of the skeletal muscle sample with rHuEPO increases mitochondrial respiratory capacity.NEW & NOTEWORTHY The effect of an acute rHuEPO injection on skeletal muscle mitochondrial function was investigated in young healthy male subjects. rHuEPO has an acute effect on skeletal muscle mitochondrial respiratory capacity in humans, where an increased mitochondrial respiratory capacity was seen. This could be the first step leading to increased mitochondrial biogenesis.

High-fat load: mechanism(s) of insulin resistance in skeletal muscle.

Skeletal muscle from sedentary obese patients is characterized by depressed electron transport activity, reduced expression of genes required for oxidative metabolism, altered mitochondrial morphology and lower overall mitochondrial content. These findings imply that obesity, or more likely the metabolic imbalance that causes obesity, leads to a progressive decline in mitochondrial function, eventually culminating in mitochondrial dissolution or mitoptosis. A decrease in the sensitivity of skeletal muscle to insulin represents one of the earliest maladies associated with high dietary fat intake and weight gain. Considerable evidence has accumulated to suggest that the cytosolic ectopic accumulation of fatty acid metabolites, including diacylglycerol and ceramides, underlies the development of insulin resistance in skeletal muscle. However, an alternative mechanism has recently been evolving, which places the etiology of insulin resistance in the context of cellular/mitochondrial bioenergetics and redox systems biology. Overnutrition, particularly from high-fat diets, generates fuel overload within the mitochondria, resulting in the accumulation of partially oxidized acylcarnitines, increased mitochondrial hydrogen peroxide (H2O2) emission and a shift to a more oxidized intracellular redox environment. Blocking H2O2 emission prevents the shift in redox environment and preserves insulin sensitivity, providing evidence that the mitochondrial respiratory system is able to sense and respond to cellular metabolic imbalance. Mitochondrial H2O2 emission is a major regulator of protein redox state, as well as the overall cellular redox environment, raising the intriguing possibility that elevated H2O2 emission from nutrient overload may represent the underlying basis for the development of insulin resistance due to disruption of normal redox control mechanisms regulating protein function, including the insulin signaling and glucose transport processes.