Are reactive oxygen species always bad? Lessons from hypoxic ectotherms.

Oxygen (O2) is required for aerobic energy metabolism but can produce reactive oxygen species (ROS), which are a wide variety of oxidant molecules with a range of biological functions from causing cell damage (oxidative distress) to cell signalling (oxidative eustress). The balance between the rate and amount of ROS generated and the capacity for scavenging systems to remove them is affected by several biological and environmental factors, including oxygen availability. Ectotherms, and in particular hypoxia-tolerant ectotherms, are hypothesized to avoid oxidative damage caused by hypoxia, although it is unclear whether this translates to an increase in ecological fitness. In this Review, we highlight the differences between oxidative distress and eustress, the current mechanistic understanding of the two and how they may affect ectothermic physiology. We discuss the evidence of occurrence of oxidative damage with hypoxia in ectotherms, and that ectotherms may avoid oxidative damage through (1) high levels of antioxidant and scavenging systems and/or (2) low(ering) levels of ROS generation. We argue that the disagreements in the literature as to how hypoxia affects antioxidant enzyme activity and the variable metabolism of ectotherms makes the latter strategy more amenable to ectotherm physiology. Finally, we argue that observed changes in ROS production and oxidative status with hypoxia may be a signalling mechanism and an adaptive strategy for ectotherms encountering hypoxia.

Physiological and behavioural strategies of aquatic animals living in fluctuating environments.

Shallow or near-shore environments, such as ponds, estuaries and intertidal zones, are among the most physiologically challenging of all aquatic settings. Animals inhabiting these environments experience conditions that fluctuate markedly over relatively short temporal and spatial scales. Living in these habitats requires the ability to tolerate the physiological disturbances incurred by these environmental fluctuations. This tolerance is achieved through a suite of physiological and behavioural responses that allow animals to maintain homeostasis, including the ability to dynamically modulate their physiology through reversible phenotypic plasticity. However, maintaining the plasticity to adjust to some stresses in a dynamic environment may trade off with the capacity to deal with other stressors. This paper will explore studies on select fishes and invertebrates exposed to fluctuations in dissolved oxygen, salinity and pH. We assess the physiological mechanisms these species employ to achieve homeostasis, with a focus on the plasticity of their responses, and consider the resulting physiological trade-offs in function. Finally, we discuss additional factors that may influence organismal responses to fluctuating environments, such as the presence of multiple stressors, including parasites. We echo recent calls from experimental biologists to consider physiological responses to life in naturally fluctuating environments, not only because they are interesting in their own right but also because they can reveal mechanisms that may be crucial for living with increasing environmental instability as a consequence of climate change.

Rates of warming impact oxidative stress in zebrafish (Danio rerio).

Potentially negative effects of thermal variation on physiological functions may be modulated by compensatory responses, but their efficacy depends on the time scale of phenotypic adjustment relative to the rate of temperature change. Increasing temperatures in particular can affect mitochondrial bioenergetics and rates of reactive oxygen species (ROS) production. Our aim was to test whether different rates of temperature increase affect mitochondrial bioenergetics and modulate oxidative stress. We exposed zebrafish (Danio rerio) to warming from 20°C to 28°C over 3, 6, 24 or 48 h, and compared these with a control group that was kept at constant 20°C. Fish exposed to the fastest (3 h) and slowest (48 h) rates of warming had significantly higher rates of H2O2 production relative to the control treatment, and the proportion of O2 converted to H2O2 (H2O2/O2 ratio) was significantly greater in these groups. However, ROS production was not paralleled by differences in mitochondrial substrate oxidation rates, leak respiration rates or coupling (respiratory control ratios). Increased rates of ROS production did not lead to damage of proteins or membranes, which may be explained by a moderate increase in catalase activity at the fastest, but not the slowest, rate of warming. The increase in ROS production at the slowest rate of warming indicates that even seemingly benign environments may be stressful. Understanding how animals respond to different rates of temperature change is important, because the rate determines the time period for phenotypic adjustments and it also alters the environmental thermal signal that triggers compensatory pathways.

Temperature sensitivity differs between heart and red muscle mitochondria in mahi-mahi (Coryphaena hippurus).

Maintaining energy balance over a wide range of temperatures is critical for an active pelagic fish species such as the mahi-mahi (Coryphaena hippurus), which can experience rapid changes in temperature during vertical migrations. Due to the profound effect of temperature on mitochondrial function, this study was designed to investigate the effects of temperature on mitochondrial respiration in permeabilized heart and red skeletal muscle (RM) fibres isolated from mahi-mahi. As RM is thought to be more anatomically isolated from rapid ambient temperature changes compared to the myocardium, it was hypothesized that heart mitochondria would be more tolerant of temperature changes through a greater ability to match respiratory capacity to an increase in temperature and to maintain coupling, when compared to RM mitochondria. Results show that heart fibres were more temperature sensitive and increased respiration rate with temperature increases to a greater degree than RM. Respiratory coupling ratios at the three assay temperatures (20, 26, and 30 °C), revealed that heart mitochondria were less coupled at a lower temperature (26 °C) compared to RM mitochondria (30 °C). In response to an in vitro acute temperature challenge, both tissues showed irreversible effects, where both heart and RM increased uncoupling whether the assay temperature was acutely changed from 20 to 30 °C or 30 to 20 °C. The findings from this study indicate that mahi-mahi heart mitochondria were more temperature sensitive compared to those from RM.

Convergent evolution of conserved mitochondrial pathways underlies repeated adaptation to extreme environments.

Extreme environments test the limits of life; yet, some organisms thrive in harsh conditions. Extremophile lineages inspire questions about how organisms can tolerate physiochemical stressors and whether the repeated colonization of extreme environments is facilitated by predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying convergent evolution of tolerance to hydrogen sulfide (H2S)-a toxicant that impairs mitochondrial function-across evolutionarily independent lineages of a fish (Poecilia mexicana, Poeciliidae) from H2S-rich springs. Using comparative biochemical and physiological analyses, we found that mitochondrial function is maintained in the presence of H2S in sulfide spring P. mexicana but not ancestral lineages from nonsulfidic habitats due to convergent adaptations in the primary toxicity target and a major detoxification enzyme. Genome-wide local ancestry analyses indicated that convergent evolution of increased H2S tolerance in different populations is likely caused by a combination of selection on standing genetic variation and de novo mutations. On a macroevolutionary scale, H2S tolerance in 10 independent lineages of sulfide spring fishes across multiple genera of Poeciliidae is correlated with the convergent modification and expression changes in genes associated with H2S toxicity and detoxification. Our results demonstrate that the modification of highly conserved physiological pathways associated with essential mitochondrial processes mediates tolerance to physiochemical stress. In addition, the same pathways, genes, and-in some instances-codons are implicated in H2S adaptation in lineages that span 40 million years of evolution.

Species- and tissue-specific differences in ROS metabolism during exposure to hypoxia and hyperoxia plus recovery in marine sculpins.

Animals that inhabit environments that fluctuate in oxygen must not only contend with disruptions to aerobic metabolism, but also the potential effects of reactive oxygen species (ROS). The goal of this study was to compare aspects of ROS metabolism in response to O2 variability (6 h hypoxia or hyperoxia, with subsequent normoxic recovery) in two species of intertidal sculpin fishes (Cottidae, Actinopterygii) that can experience O2 fluctuations in their natural environment and differ in whole-animal hypoxia tolerance. To assess ROS metabolism, we measured the ratio of glutathione to glutathione disulfide as an indicator of tissue redox environment, MitoP/MitoB ratio to assess in vivo mitochondrial ROS generation, thiobarbituric acid reactive substances (TBARS) for lipid peroxidation, and total oxidative scavenging capacity (TOSC) in the liver, brain and gill. In the brain, the more hypoxia-tolerant Oligocottusmaculosus showed large increases in TBARS levels following hypoxia and hyperoxia exposure that were generally not associated with large changes in mitochondrial H2O2 In contrast, the less-tolerant Scorpaenichthysmarmoratus showed no significant changes in TBARS or mitochondrial H2O2 in the brain. More moderate increases were observed in the liver and gill of O. maculosus exposed to hypoxia and hyperoxia with normoxic recovery, whereas S. marmoratus had a greater response to O2 variability in these tissues compared with the brain. Our results show a species- and tissue-specific relationship between hypoxia tolerance and ROS metabolism.

Detection of changes in mitochondrial hydrogen sulfide i n vivo in the fish model Poecilia mexicana (Poeciliidae).

In this paper, we outline the use of a mitochondria-targeted ratiometric mass spectrometry probe, MitoA, to detect in vivo changes in mitochondrial hydrogen sulfide (H2S) in Poecilia mexicana (family Poeciliidae). MitoA is introduced via intraperitoneal injection into the animal and is taken up by mitochondria, where it reacts with H2S to form the product MitoN. The MitoN/MitoA ratio can be used to assess relative changes in the amounts of mitochondrial H2S produced over time. We describe the use of MitoA in the fish species P. mexicana to illustrate the steps for adopting the use of MitoA in a new organism, including extraction and purification of MitoA and MitoN from tissues followed by tandem mass spectrometry. In this proof-of-concept study we exposed H2S tolerant P. mexicana to 59 µM free H2S for 5 h, which resulted in increased MitoN/MitoA in brain and gills, but not in liver or muscle, demonstrating increased mitochondrial H2S levels in select tissues following whole-animal H2S exposure. This is the first time that accumulation of H2S has been observed in vivo during whole-animal exposure to free H2S using MitoA. This article has an associated First Person interview with the first author of the paper.

Interspecific variation in brain mitochondrial complex I and II capacity and ROS emission in marine sculpins.

Environmental hypoxia presents a metabolic challenge for animals because it inhibits mitochondrial respiration and can lead to the generation of reactive oxygen species (ROS). We investigated the interplay between O2 use for aerobic respiration and ROS generation among sculpin fishes (Cottidae, Actinopterygii) that are known to vary in whole-animal hypoxia tolerance. We hypothesized that mitochondria from hypoxia-tolerant sculpins would show more efficient O2 use with a higher phosphorylation efficiency and lower ROS emission. We showed that brain mitochondria from more hypoxia-tolerant sculpins had lower complex I and higher complex II flux capacities compared with less hypoxia-tolerant sculpins, but these differences were not related to variation in phosphorylation efficiency (ADP/O) or mitochondrial coupling (respiratory control ratio). The hypoxia-tolerant sculpins had higher mitochondrial H2O2 emission per O2 consumed (H2O2/O2) under oligomycin-induced state 4 conditions compared with less hypoxia-tolerant sculpins. An in vitro redox challenge experiment revealed species differences in how well mitochondria defend their glutathione redox status when challenged with high levels of reduced glutathione, but the redox challenge elicited the same H2O2/O2 in all species. Furthermore, in vitro anoxia recovery lowered absolute H2O2 emission (H2O2 per mg mitochondrial protein) in all species and negatively impacted state 3 respiration rates in some species, but the responses were not related to hypoxia tolerance. Overall, we clearly demonstrate a relationship between hypoxia tolerance and complex I and II flux capacities in sculpins, but the differences in complex flux capacity do not appear to be directly related to variation in ROS metabolism.

Effects of cold on murine brain mitochondrial function.

Therapeutic hypothermia is a strategy that reduces metabolic rate and brain damage during clinically-relevant hypoxic events. Mitochondrial respiration is compromised by hypoxia, with deleterious consequences for the mammalian brain; however, little is known about the effects of reduced temperature on mitochondrial metabolism. Therefore, we examined how mitochondrial function is impacted by temperature using high resolution respirometry to assess electron transport system (ETS) function in saponin-permeabilized mouse brain at 28 and 37°C. Respirometric analysis revealed that, at the colder temperature, ETS respiratory flux was ~ 40-75% lower relative to the physiological temperature in all respiratory states and for all fuel substrates tested. In whole brain tissue, the enzyme maximum respiratory rates for complexes I-V were similarly reduced by between 37-88%. Complexes II and V were particularly temperature-sensitive; a temperature-mediated decrease in complex II activity may support a switch to complex I mediated ATP-production, which is considerably more oxygen-efficient. Finally, the mitochondrial H+-gradient was more tightly coupled, indicating that mitochondrial respiration is more efficient at the colder temperature. Taken together, our results suggest that improvements in mitochondrial function with colder temperatures may contribute to energy conservation and enhance cellular viability in hypoxic brain.

Naked mole rat brain mitochondria electron transport system flux and H+ leak are reduced during acute hypoxia.

Mitochondrial respiration and ATP production are compromised by hypoxia. Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals and reduce metabolic rate in hypoxic environments; however, little is known regarding mitochondrial function during in vivo hypoxia exposure in this species. To address this knowledge gap, we asked whether the function of NMR brain mitochondria exhibits metabolic plasticity during acute hypoxia. Respirometry was utilized to assess whole-animal oxygen consumption rates and high-resolution respirometry was utilized to assess electron transport system (ETS) function in saponin-permeabilized NMR brain. We found that NMR whole-animal oxygen consumption rate reversibly decreased by ∼85% in acute hypoxia (4 h at 3% O2). Similarly, relative to untreated controls, permeabilized brain respiratory flux through the ETS was decreased by ∼90% in acutely hypoxic animals. Relative to carbonyl cyanide p-trifluoro-methoxyphenylhydrazone-uncoupled total ETS flux, this functional decrease was observed equally across all components of the ETS except for complex IV (cytochrome c oxidase), at which flux was further reduced, supporting a regulatory role for this enzyme during acute hypoxia. The maximum enzymatic capacities of ETS complexes I-V were not altered by acute hypoxia; however, the mitochondrial H+ gradient decreased in step with the decrease in ETS respiration. Taken together, our results indicate that NMR brain ETS flux and H+ leak are reduced in a balanced and regulated fashion during acute hypoxia. Changes in NMR mitochondrial metabolic plasticity mirror whole-animal metabolic responses to hypoxia.

Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.

Nitrate (NO3-) and nitrite (NO2-) are known to be cardioprotective and to alter energy metabolism in vivo NO3- action results from its conversion to NO2- by salivary bacteria, but the mechanism(s) by which NO2- affects metabolism remains obscure. NO2- may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2--dependent S-nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2- under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2- in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2- on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2-, combined with the lack of S-nitrosation during anoxia alone or by NO2- during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2- exposure.

Evolution of Cytochrome c Oxidase in Hypoxia Tolerant Sculpins (Cottidae, Actinopterygii).

Vertebrate hypoxia tolerance can emerge from modifications to the oxygen (O2) transport cascade, but whether there is adaptive variation to O2 binding at the terminus of this cascade, mitochondrial cytochrome c oxidase (COX), is not known. In order to address the hypothesis that hypoxia tolerance is associated with enhanced O2 binding by mitochondria we undertook a comparative analysis of COX O2 kinetics across species of intertidal sculpins (Cottidae, Actinopterygii) that vary in hypoxia tolerance. Our analysis revealed a significant relationship between hypoxia tolerance (critical O2 tension of O2 consumption rate; Pcrit), mitochondrial O2 binding affinity (O2 tension at which mitochondrial respiration was half maximal; P50), and COX O2-binding affinity (apparent Michaelis-Menten constant for O2 binding to COX; Km,app O2). The more hypoxia tolerant species had both a lower mitochondrial P50 and lower COX Km,app O2, facilitating the maintenance of mitochondrial function to a lower O2 tension than in hypoxia intolerant species. Additionally, hypoxia tolerant species had a lower overall COX Vmax but higher mitochondrial COX respiration rate when expressed relative to maximal electron transport system respiration rate. In silico analyses of the COX3 subunit postulated as the entry point for O2 into the COX protein catalytic core, points to variation in COX3 protein stability (estimated as free energy of unfolding) contributing to the variation in COX Km,app O2. We propose that interactions between COX3 and cardiolipin at four amino acid positions along the same alpha-helix forming the COX3 v-cleft represent likely determinants of interspecific differences in COX Km,app O2.

Subfunctionalization of COX4 paralogs in fish.

Cytochrome c oxidase (COX) subunit 4 has two paralogs in most vertebrates. The mammalian COX4-2 gene is hypoxia responsive, and the protein has a disrupted ATP-binding site that confers kinetic properties on COX that distinguish it from COX4-1. The structure-function of COX4-2 orthologs in other vertebrates remains uncertain. Phylogenetic analyses suggest the two paralogs arose in basal vertebrates, but COX4-2 orthologs diverged faster than COX4-1 orthologs. COX4-1/4-2 protein levels in tilapia tracked mRNA levels across tissues, and did not change in hypoxia, arguing against a role for differential post-translational regulation of paralogs. The heart, and to a lesser extent the brain, showed a size-dependent shift from COX4-1 to COX4-2 (transcript and protein). ATP allosterically inhibited both velocity and affinity for oxygen in COX assayed from both muscle (predominantly COX4-2) and gill (predominantly COX4-1). We saw some evidence of cellular and subcellular discrimination of COX4 paralogs in heart. In cardiac ventricle, some non-cardiomyocyte cells were COX positive but lacked detectible COX4-2. Within heart, the two proteins partitioned to different mitochondrial subpopulations. Cardiac subsarcolemmal mitochondria had mostly COX4-1 and intermyofibrillar mitochondria had mostly COX4-2. Collectively, these data argue that, despite common evolutionary origins, COX4-2 orthologs of fish show unique patterns of subfunctionalization with respect to transcriptional and posttranslation regulation relative to the rodents and primates that have been studied to date.

Beating oxygen: chronic anoxia exposure reduces mitochondrial F1FO-ATPase activity in turtle (Trachemys scripta) heart.

The freshwater turtle Trachemys scripta can survive in the complete absence of O2 (anoxia) for periods lasting several months. In mammals, anoxia leads to mitochondrial dysfunction, which culminates in cellular necrosis and apoptosis. Despite the obvious clinical benefits of understanding anoxia tolerance, little is known about the effects of chronic oxygen deprivation on the function of turtle mitochondria. In this study, we compared mitochondrial function in hearts of T. scripta exposed to either normoxia or 2 weeks of complete anoxia at 5°C and during simulated acute anoxia/reoxygenation. Mitochondrial respiration, electron transport chain activities, enzyme activities, proton conductance and membrane potential were measured in permeabilised cardiac fibres and isolated mitochondria. Two weeks of anoxia exposure at 5°C resulted in an increase in lactate, and decreases in ATP, glycogen, pH and phosphocreatine in the heart. Mitochondrial proton conductance and membrane potential were similar between experimental groups, while aerobic capacity was dramatically reduced. The reduced aerobic capacity was the result of a severe downregulation of the F1FO-ATPase (Complex V), which we assessed as a decrease in enzyme activity. Furthermore, in stark contrast to mammalian paradigms, isolated turtle heart mitochondria endured 20 min of anoxia followed by reoxygenation without any impact on subsequent ADP-stimulated O2 consumption (State III respiration) or State IV respiration. Results from this study demonstrate that turtle mitochondria remodel in response to chronic anoxia exposure and a reduction in Complex V activity is a fundamental component of mitochondrial and cellular anoxia survival.

AMP-activated protein kinase plays a role in initiating metabolic rate suppression in goldfish hepatocytes.

In the present study, we test the hypothesis that AMP-activated protein kinase (AMPK) initiates metabolic rate suppression in isolated goldfish hepatocytes. To accomplish this, we attempted to pharmacologically activate AMPK in goldfish hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the thienopyridone, A769662, to examine the effects of AMPK activation on eukaryotic elongation factor-2 (eEF2), protein synthesis, and cellular oxygen consumption rate ([Formula: see text]). Goldfish hepatocytes treated with 1 mM AICAR under normoxic conditions (>200 µM O(2)) showed a modest but significant 1.1-fold increase in AMPK phosphorylation, a 7.5-fold increase in AMPK activity, a 1.4-fold increase in eEF2 phosphorylation, and a 24% decrease in [Formula: see text]. At physiologically relevant [O(2)] (<40 µM O(2)), the addition of 1 mM AICAR resulted in only a 13% decrease in cellular [Formula: see text] with no change in sensitivity to [O(2)] as assessed by estimates of cellular P(50) and P(90) values. The addition of compound C, a general protein kinase inhibitor, after AICAR incubation did not reverse the effects of AICAR on [Formula: see text] in normoxia. Treatment of hepatocytes with ≤200 µM A769662 did not affect AMPK activity, AMPK phosphorylation, eEF2 phosphorylation, or cellular [Formula: see text]. These data suggest that A769662 is not an activator of AMPK in goldfish hepatocytes. Although our study provides support for the hypothesis that AMPK plays a role in initiating metabolic rate suppression in goldfish hepatocytes, this support must be viewed cautiously because of the known off-target effects of the pharmacological agents used.

Effects of environmental hypoxia on cardiac energy metabolism and performance in tilapia.

The ability of an animal to depress ATP turnover while maintaining metabolic energy balance is important for survival during hypoxia. In the present study, we investigated the responses of cardiac energy metabolism and performance in the hypoxia-tolerant tilapia (Oreochromis hybrid sp.) during exposure to environmental hypoxia. Exposure to graded hypoxia (> or =92% to 2.5% air saturation over 3.6 +/- 0.2 h) followed by exposure to 5% air saturation for 8 h caused a depression of whole animal oxygen consumption rate that was accompanied by parallel decreases in heart rate, cardiac output, and cardiac power output (CPO, analogous to ATP demand of the heart). These cardiac parameters remained depressed by 50-60% compared with normoxic values throughout the 8-h exposure. During a 24-h exposure to 5% air saturation, cardiac ATP concentration was unchanged compared with normoxia and anaerobic glycolysis contributed to ATP supply as evidenced by considerable accumulation of lactate in the heart and plasma. Reductions in the provision of aerobic substrates were apparent from a large and rapid (in <1 h) decrease in plasma nonesterified fatty acids concentration and a modest decrease in activity of pyruvate dehydrogenase. Depression of cardiac ATP demand via bradycardia and an associated decrease in CPO appears to be an integral component of hypoxia-induced metabolic rate depression in tilapia and likely contributes to hypoxic survival.

Metabolic recovery in goldfish: A comparison of recovery from severe hypoxia exposure and exhaustive exercise.

Severe hypoxia exposure and exhaustive exercise in goldfish both elicit a strong activation of substrate-level phosphorylation with the majority of the metabolic perturbations occurring in the white muscle. Approximately half of the muscle glycogen breakdown observed during severe hypoxia exposure was accounted for by ethanol production and loss to the environment, which limited the extent of muscle glycogen recovery when animals were returned to normoxic conditions. Ethanol production in goldfish is not solely a response to anoxia/hypoxia exposure however, as a transient increase in ethanol production was observed during the early stages of recovery from exhaustive exercise. These data suggest that ethanol production is a ubiquitous "anaerobic" end product, which accumulates whenever metabolic demands exceed mitochondrial oxidative potential. Exhaustive exercise and hypoxia exposure both caused a 7 to 8 micromol g(-1) wet mass increase in muscle [lactate] and the rates of recovery following these perturbations were similar. The rates of muscle PCr and pHi recovery after hypoxia exposure and exhaustive exercise were similar with levels returning to controls values within 0.5 h. Surprisingly, liver [glycogen] was not depleted during exposure to severe hypoxia, however, during recovery from both hypoxia and exercise dramatically different responses in liver [glycogen] were noted. During the early stages of recovery, liver [glycogen] transiently increased to high levels after exhaustive exercise, while during recovery from hypoxia there was a transient decrease in liver glycogen over the same time frame. Overall, this points to the liver playing a dramatically different role in facilitating recovery from exercise compared with hypoxia exposure.