Studying mitochondrial respiratory function is critical to understanding the plasticity of cellular energetics. Mitochondrial respiratory function is usually studied ex vivo and requires isolation of high-quality mitochondrial preparations. However, mitochondria are usually damaged by this process, and the degree of damage is typically assessed by measuring respiratory control ratios (RCRs) and the change in respiration rate mediated by exogenous cytochrome C addition to isolated mitochondrial preparations. The standard thresholds in the field for determining whether isolated mitochondria are “acceptable” are based on studies in common laboratory rodents and are entirely arbitrary. However, zoologists are increasingly employing nonstandard animal models in the study of mitochondrial respiratory function across a wide variety of fields and there is growing evidence that the canonical thresholds of acceptable RCRs and the cytochrome C response that have been used to identify healthy mitochondrial preparations may not be useful in other species. In this commentary, we discuss the limitations of using RCRs and cytochrome C-mediated changes in mitochondrial respiration in the study of nontraditional laboratory species and propose alternative means of evaluating mitochondrial integrity that may permit more accurate assessment of the quality of mitochondria isolated, and thus better support comparison of data across species and studies.

Variations in environmental oxygen availability can induce oxidative stress in mammalian cells; episodes of hypoxia/re-oxygenation can be deleterious to cardiac function. Subterranean-living African naked mole-rats putatively experience repeated bouts of hypoxia/re-oxygenation in their natural habitat and are among the most hypoxia-tolerant mammals. To mitigate the potentially deleterious effects of frequent hypoxia/re-oxygenation exposure, naked mole-rats possess cardiac mitochondrial adaptations that may limit oxidative damage caused by fluctuating environmental oxygen levels. These include low respiratory flux through complex II (succinate dehydrogenase) of the mitochondrial electron transport system and robust cardiac reactive oxygen species (ROS) scavenging capacities. We hypothesized that these specializations are common traits in all hypoxia-tolerant African mole-rats. To test this hypothesis, we evaluated mitochondrial respiration and both ROS efflux and scavenging capabilities from naked mole-rats, six additional hypoxia-tolerant African mole-rat species, and hypoxia-intolerant mice (Mus musculus), rats (Rattus norvegicus; non-subterranean), and star-nosed moles (Condylura cristata; subterranean). We found a clear and inverse relationship between complex II-fuelled respiration of isolated mitochondria and hypoxia-tolerance across all species tested. Conversely, we found no relationship between hypoxia-tolerance and rates of complex I-fuelled respiration, H2O2 efflux (with any substrate), or mitochondrial H2O2 scavenging capacity. Our results suggest that downregulating the catalytic capacity of cardiac mitochondrial complex II may be an adaptation that supports systemic hypoxia-tolerance in African mole-rats.

Hypoxemia from exposure to intermittent and/or acute environmental hypoxia (lower oxygen concentration) is a severe stressor for many animal species. The response to hypoxia of the hypothalamic-pituitary-adrenal axis (HPA-axis), which culminates in the release of glucocorticoids, has been well-studied in hypoxia-intolerant surface-dwelling mammals. Several group-living (social) subterranean species, including most African mole -rats, are hypoxia-tolerant, likely due to regular exposure to intermittent hypoxia in their underground bur-rows. Conversely, solitary mole-rat species, lack many adaptive mechanisms, making them less hypoxia-tolerant than the social genera. To date, the release of glucocorticoids in response to hypoxia has not been measured in hypoxia-tolerant mammalian species. Consequently, this study exposed three social African mole-rat species and two solitary mole-rat species to normoxia, or acute hypoxia and then measured their respective plasma gluco-corticoid (cortisol) concentrations. Social mole-rats had lower plasma cortisol concentrations under normoxia than the solitary genera. Furthermore, individuals of all three of the social mole-rat species exhibited signifi-cantly increased plasma cortisol concentrations after hypoxia, similar to those of hypoxia-intolerant surface -dwelling species. By contrast, individuals of the two solitary species had a reduced plasma cortisol response to acute hypoxia, possibly due to increased plasma cortisol under normoxia. If placed in perspective with other closely related surface-dwelling species, the regular exposure of the social African mole-rats to hypoxia may have reduced the basal levels of the components for the adaptive mechanisms associated with hypoxia exposure, including circulating cortisol levels. Similarly, the influence of body mass on plasma cortisol levels cannot be ignored. This study demonstrates that both hypoxia-tolerant rodents and hypoxia-intolerant terrestrial laboratory-bred rodents may possess similar HPA-axis responses from exposure to hypoxia. Further research is required to confirm the results from this pilot study and to further confirm how the cortisol concentrations may influence responses to hypoxia in African mole-rats.

A greater capacity of endogenous matrix antioxidants has recently been hypothesized to characterize mitochondria of long-lived species, curbing bursts of reactive oxygen species (ROS) generated in this organelle. Evidence for this has been obtained from studies comparing the long-lived naked mole rat to laboratory mice. We tested this hypothesis by comparing the longest-lived metazoan, the marine bivalve Arctica islandica (MLSP = 507 y), with shorter-lived and evolutionarily related species. We used a recently developed fluorescent technique to assess mantle and gill tissue mitochondria's capacity to consume hydrogen peroxide (H2O2) in multiple physiological states ex vivo. Depending on the type of respiratory substrate provided, mitochondria of Arctica islandica could consume between 3 and 14 times more H2O2 than shorter-lived species. These findings support the contention that a greater capacity for the elimination of ROS characterizes long-lived species, a novel property of mitochondria thus far demonstrated in two key biogerontological models from distant evolutionary lineages.

Star-nosed moles (Condylura cristata) have an impressive diving performance and burrowing lifestyle, yet no ventilatory data are available for this or any other talpid mole species. We predicted that, like many other semi-aquatic and fossorial small mammals, star-nosed moles would exhibit: (i) a blunted (i.e. delayed or reduced) hypoxic ventilatory response, (ii) a reduced metabolic rate and (iii) a lowered body temperature (Tb) in hypoxia. We thus non-invasively measured these variables from wild-caught star-nosed moles exposed to normoxia (21% O-2) or acute graded hypoxia (21-6% O-2). Surprisingly, star-nosed moles did not exhibit a blunted HVR or decreased Tb in hypoxia, and only manifested a significant, albeit small (<8%), depression of metabolic rate at 6% O-2 relative to normoxic controls. Unlike small rodents inhabiting similar niches, star-nosed moles are thus intolerant to hypoxia, which may reflect an evolutionary trade-off favouring the extreme sensory biology of this unusual insectivore.

Oxidative phosphorylation is compromised in hypoxia, but many organisms live and exercise in low oxygen environments. Hypoxia-driven adaptations at the mitochondrial level are common and may enhance energetic efficiency or minimize deleterious reactive oxygen species (ROS) generation. Mitochondria from various hypoxia-tolerant animals exhibit robust functional changes following in vivo hypoxia and we hypothesized that similar plasticity would occur in naked mole-rat skeletal muscle. To test this, we exposed adult subordinate naked mole-rats to normoxia (21% O-2) or acute (4 h, 7% O-2) or chronic hypoxia (4-6 weeks, 11% O-2) and then isolated skeletal muscle mitochondria. Using high-resolution respirometry and a fluorescent indicator of ROS production, we then probed for changes in: i) lipid- (palmitoylcarnitine-malate), ii) carbohydrate- (pyruvatemalate), and iii) succinate-fueled metabolism, and also iv) complex IV electron transfer capacity, and v) H2O2 production. Compared to normoxic values, a) lipid-fueled uncoupled respiration was reduced similar to 15% during acute and chronic hypoxia, b) complex I-II capacity and the rate of ROS efflux were both unaffected, and c) complex II and IV uncoupled respiration were supressed similar to 16% following acute hypoxia. Notably, complex II-linked H2O2 efflux was 33% lower after acute hypoxia, which may reduce deleterious ROS bursts during reoxygenation. These mild changes in lipid- and carbohydrate-fueled respiratory capacity may reflect the need for this animal to exercise regularly in highly variable and intermittently hypoxic environments in which more robust plasticity may be energetically expensive.

Mitochondria generate aerobic cellular energy (i.e., ATP) through the reduction of oxygen to water via oxidative phosphorylation. The efficiency of this pathway can be measured by the phosphate/oxygen (ATP/O) ratio, which is the amount of ATP produced per oxygen atom reduced. This ratio thus provides a measure of the efficiency of mitochondrial respiration that can be readily compared between species. The magnesium green (MgGr) fluorometric method permits easy measurement of ATP/O ratios from isolated mitochondria but the standard analysis approach employs an endpoint method to calculate ATP/O ratios. Here, we present a modified method of ATP/O calculation that permits dynamic observation of fluorescent measurements of the consumption of O2 (JO2) and the production of ATP (JATP). Specifically, by substituting the slope of a straight line within a given period of time (seconds to minutes) with the slope of a tangent to each time point (per second), it is possible to evaluate JO2, JATP and the ATP/O ratio in a dynamic manner.•Provides second-by-second visualization of ATP/O ratios throughout experiments vs. a single measurement.•Dynamic visualization allows for easy identification of outlying data and more accurate calculation of mean ATP/O ratios.

Executive summary – Box 1: In brief: Mitochondria and bioblasts .......................................... 2 1. Introduction ............................................................................................................................................................................. 8 2. Coupling states and rates in mitochondrial preparations ................................................................................... 8 2.1. Cellular and mitochondrial respiration ................................................................................................................. 8 2.1.1. Aerobic and anaerobic catabolism and ATP turnover Consortium Communication 2 of 44 Gnaiger E et al ― MitoEAGLE Task Group (2020) Bioenerg Commun 2020.1 2.1.2. Specification of biochemical dose and exposure 2.2. Mitochondrial preparations .................................................................................................................................... 10 2.3. Electron transfer pathways ..................................................................................................................................... 11 2.4. Respiratory coupling control .................................................................................................................................. 12 2.4.1. Coupling 2.4.2. Phosphorylation P» and P»/O2 ratio 2.4.3. Uncoupling 2.5. Coupling states and respiratory rates ................................................................................................................. 13 2.5.1. LEAK state 2.5.2. OXPHOS state 2.5.3. Electron transfer state 2.5.4. ROX state 2.5.5. Quantitative relations 2.5.6. The steady state 2.6. Classical terminology for isolated mitochondria ............................................................................................ 19 2.6.1. – 2.6.5. State 1 – State 5 2.7. Control and regulation .............................................................................................................................................. 21 3. What is a rate? – Box 2: Metabolic flows and fluxes: vectoral, vectorial, and scalar ............................. 21 4. Normalization of rate per sample................................................................................................................................. 23 4.1. Flow: per object ............................................................................................................................................................ 23 4.1.1. Count concentration 4.1.2. Flow per single object 4.2. Size-specific flux: per sample size .......................................................................................................................... 25 4.2.1. Mass concentration 4.2.2. Size-specific flux 4.3. Marker-specific flux: per mitochondrial content ............................................................................................. 26 4.3.1. Mitochondrial concentration and mitochondrial density 4.3.2. mt-Marker-specific flux 5. Normalization of rate per system ................................................................................................................................ 28 5.1. Flow: per chamber ...................................................................................................................................................... 28 5.2. Flux: per chamber volume ....................................................................................................................................... 28 5.2.1. System-specific flux 5.2.2. Advancement per volume 6. Conversion of units ............................................................................................................................................................ 30 7. Conclusions – Box 3: Recommendations for studies with mitochondrial preparations ...................... 31 References ............................................................................................................................................................................. 36 Authors (MitoEAGLE Task Group) – Author contributions .............................................................................. 41 Acknowledgements – Competing financial interests – Correspondence

Skeletal muscle, a significant contributor to resting energy expenditure and reactive oxygen species, may play a critical role in body-weight regulation and aging processes. Methionine restriction (MR) is a dietary intervention which extends lifespan, lowers body-weight and enhances energy expenditure in rodents, all of which have been linked to mitochondrial function in various tissues including liver, kidney, heart and brown adipose tissue; however, mitochondrial responses to MR in skeletal muscle is largely unknown. Given the importance of skeletal muscle on energy metabolism and aging-related processes, we investigated if there are changes in skeletal muscle mitochondrial energetics in response to MR. Although MR lowers body-weight in rats, neither respiration, proton leak nor hydrogen peroxide metabolism were altered in isolated skeletal muscle mitochondria. This suggests that mitochondrial function in skeletal muscle remains conserved while MR alters metabolism in other tissues.

The mitochondrial oxidative theory of aging has been repeatedly investigated over the past 30 years by comparing the efflux of hydrogen peroxide (H2 O2 ) from isolated mitochondria of long- and short-lived species using horseradish peroxidase-based assays. However, a clear consensus regarding the relationship between H2 O2 production rates and longevity has not emerged. Concomitantly, novel insights into the mechanisms of reactive oxygen species (ROS) handling by mitochondria themselves should have raised concerns about the validity of this experimental approach. Here, we review pitfalls of the horseradish peroxidase/amplex red detection system for the measurement of mitochondrial ROS formation rates, with an emphasis on longevity studies. Importantly, antioxidant systems in the mitochondrial matrix are often capable of scavenging H2 O2 faster than mitochondria produce it. As a consequence, as much as 84% of the H2 O2 produced by mitochondria may be consumed before it diffuses into the reaction medium, where it can be detected by the horseradish peroxidase/amplex red system, this proportion is likely not consistent across species. Furthermore, previous studies often used substrates that elicit H2 O2 formation at a much higher rate than in physiological conditions and at sites of secondary importance in vivo. Recent evidence suggests that the activity of matrix antioxidants may correlate with longevity instead of the rate of H2 O2 formation. We conclude that past studies have been methodologically insufficient to address the putative relationship between longevity and mitochondrial ROS. Thus, novel methodological approaches are required that more accurately encompass mitochondrial ROS metabolism.

Naked mole-rats (NMRs) are mouse-sized mammals that exhibit an exceptionally long lifespan (>30 vs. <4 years for mice), and resist aging-related pathologies such as cardiovascular and pulmonary diseases, cancer, and neurodegeneration. However, the mechanisms underlying this exceptional longevity and disease resistance remain poorly understood. The oxidative stress theory of aging posits that (a) senescence results from the accumulation of oxidative damage inflicted by reactive oxygen species (ROS) of mitochondrial origin, and (b) mitochondria of long-lived species produce less ROS than do mitochondria of short-lived species. However, comparative studies over the past 28 years have produced equivocal results supporting this latter prediction. We hypothesized that, rather than differences in ROS generation, the capacity of mitochondria to consume ROS might distinguish long-lived species from short-lived species. To test this hypothesis, we compared mitochondrial production and consumption of hydrogen peroxide (H2 O2 ; as a proxy of overall ROS metabolism) between NMR and mouse skeletal muscle and heart. We found that the two species had comparable rates of mitochondrial H2 O2 generation in both tissues; however, the capacity of mitochondria to consume ROS was markedly greater in NMRs. Specifically, maximal observed consumption rates were approximately two and fivefold greater in NMRs than in mice, for skeletal muscle and heart, respectively. Our results indicate that differences in matrix ROS detoxification capacity between species may contribute to their divergence in lifespan.

As the knowledge base and importance of mitochondrial physiology to human health expands, the necessity for harmonizing the terminologyconcerning mitochondrial respiratory states and rates has become increasingly apparent. Thechemiosmotic theoryestablishes the mechanism of energy transformationandcoupling in oxidative phosphorylation. Theunifying concept of the protonmotive force providestheframeworkfordeveloping a consistent theoretical foundation ofmitochondrial physiology and bioenergetics.We followguidelines of the International Union of Pure and Applied Chemistry(IUPAC)onterminology inphysical chemistry, extended by considerationsofopen systems and thermodynamicsof irreversible processes.Theconcept-driven constructive terminology incorporates the meaning of each quantity and alignsconcepts and symbols withthe nomenclature of classicalbioenergetics. We endeavour to provide a balanced view ofmitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes.Uniform standards for evaluation of respiratory states and rates will ultimatelycontribute to reproducibility between laboratories and thussupport the development of databases of mitochondrial respiratory function in species, tissues, and cells.Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery.

Mitochondrial electron transfer for oxidative ATP regeneration is linked to reactive oxygen species (ROS) production in aerobic eukaryotic cells. Because they can contribute to signaling as well as oxidative damage in cells, these ROS have profound impact for the physiology and survival of the organism. Although mitochondria have been recognized as a potential source for ROS for about 50 years, the mechanistic understanding on molecular sites and processes has advanced recently. Most experimental approaches neglect thermal variability among species although temperature impacts mitochondrial processes significantly. Here we delineate the importance of temperature by comparing muscle mitochondrial ROS formation across species. Measuring the thermal sensitivity of respiration, electron leak rate (ROS formation), and the antioxidant capacity (measured as H2O2 consumption) in intact mitochondria of representative ectothermic and endothermic vertebrate species, our results suggest that using a common assay temperature is inappropriate for comparisons of organisms with differing body temperatures. Moreover, we propose that measuring electron leak relative to the mitochondrial antioxidant capacity (the oxidant ratio) may be superior to normalizing relative to respiration rates or mitochondrial protein for comparisons of mitochondrial metabolism of ROS across species of varying mitochondrial respiratory capacities.

Mitochondria are widely recognized as a source of reactive oxygen species (ROS) in animal cells, where it is assumed that over-production of ROS leads to an overwhelmed antioxidant system and oxidative stress. In this Commentary, we describe a more nuanced model of mitochondrial ROS metabolism, where integration of ROS production with consumption by the mitochondrial antioxidant pathways may lead to the regulation of ROS levels. Superoxide and hydrogen peroxide (H2O2) are the main ROS formed by mitochondria. However, superoxide, a free radical, is converted to the non-radical, membrane-permeant H2O2; consequently, ROS may readily cross cellular compartments. By combining measurements of production and consumption of H2O2, it can be shown that isolated mitochondria can intrinsically approach a steady-state concentration of H2O2 in the medium. The central hypothesis here is that mitochondria regulate the concentration of H2O2 to a value set by the balance between production and consumption. In this context, the consumers of ROS are not simply a passive safeguard against oxidative stress; instead, they control the established steady-state concentration of H2O2 By considering the response of rat skeletal muscle mitochondria to high levels of ADP, we demonstrate that H2O2 production by mitochondria is far more sensitive to changes in mitochondrial energetics than is H2O2 consumption; this concept is further extended to evaluate how the muscle mitochondrial H2O2 balance should respond to changes in aerobic work load. We conclude by considering how differences in the ROS consumption pathways may lead to important distinctions amongst tissues, along with briefly examining implications for differing levels of activity, temperature change and metabolic depression.

Delineating the physiological and biochemical causes of aging process in the animal kingdom is a highly active area of research not only because of potential benefits for human health but also because aging process is related to life history strategies (growth and reproduction) and to responses of organisms to environmental conditions and stress. In this synthesis, we advocate studying bivalve species as models for revealing the determinants of species divergences in maximal longevity. This taxonomic group includes the longest living metazoan on earth (Arctica islandica), which insures the widest range of maximum life span when shorter living species are also included in the comparative model. This model can also be useful for uncovering factors modulating the pace of aging in given species by taking advantages of the wide disparity of lifespan among different populations of the same species. For example, maximal lifespan in different populations of A islandica range from approximately 36 years to over 500 years. In the last 15 years, research has revealed that either regulation or tolerance to oxidative stress is tightly correlated to longevity in this group which support further investigations on this taxon to unveil putative mechanistic links between Reactive Oxygen Species and aging process.

The most common methods of measuring mitochondrial hydrogen peroxide production are based on the extramitochondrial oxidation of a fluorescent probe such as amplex ultra red (AUR) by horseradish peroxidase (HRP). These traditional HRP-based assays only detect H2O2 that has escaped the matrix, raising the potential for substantial underestimation of production if H2O2 is consumed by matrix antioxidant pathways. To measure this underestimation, we characterized matrix consumers of H2O2 in rat skeletal muscle mitochondria, and developed specific means to inhibit these consumers. Mitochondria removed exogenously added H2O2 (2.5µM) at rates of 4.7 and 5.0nmol min−1 mg protein−1 when respiring on glutamate+malate and succinate+rotenone, respectively. In the absence of respiratory substrate, or after disrupting membranes by cycles of freeze-thaw, rates of H2O2 consumption were negligible. We concluded that matrix consumers are respiration-dependent (requiring respiratory substrates), suggesting the involvement of either the thioredoxin (Trx) and/or glutathione (GSH)-dependent enzymatic pathways. The Trx-reductase inhibitor auranofin (2µM), and a pre-treatment of mitochondria with 35µM of 1-chloro-2,4-dintrobenzene (CDNB) to deplete GSH specifically compromise these two consumption pathways. These inhibition approaches presented no undesirable “off-target” effects during extensive preliminary tests. These inhibition approaches independently and additively decreased the rate of consumption of H2O2 exogenously added to the medium (2.5µM). During traditional HRP-based H2O2 efflux assays, these inhibition approaches independently and additively increased apparent efflux rates. When used in combination (double inhibition), these inhibition approaches allowed accumulation of (endogenously produced) H2O2 in the medium at a comparable rate whether it was measured with an end point assay where 2.5µM H2O2 is initially added to the medium or with traditional HRP-based efflux assays. This finding confirms that a high degree of inhibition of all matrix consumers is attained with the double inhibition. Importantly, this double inhibition of the matrix consumers allowed revealing that a large part of the H2O2 produced in muscle mitochondria is consumed before escaping the matrix during traditional HRP-based efflux assays. The degree of this underestimation was substrate dependent, reaching >80% with malate, which complicates comparisons of substrates for their capacity to generate H2O2 in normal conditions i.e. when matrix consumers are active. Our results also urge caution in interpreting changes in H2O2 efflux in response to a treatment; when HRP-based assays are used, large changes in apparent H2O2 efflux may come from altered capacity of the matrix consumers.

Mitochondria are often regarded as a major source of reactive oxygen species (ROS) in animal cells, with H2O2 being the predominant ROS released from mitochondria; however, it has been recently demonstrated that energized brain mitochondria may act as stabilizers of H2O2 concentration (Starkov et al. [1]) based on the balance between production and the consumption of H2O2, the later of which is a function of [H2O2] and follows first order kinetics. Here we test the hypothesis that isolated skeletal muscle mitochondria, from the rat, are able to modulate [H2O2] based upon the interaction between the production of ROS, as superoxide/H2O2, and the H2O2 decomposition capacity. The compartmentalization of detection systems for H2O2 and the intramitochondrial metabolism of H2O2 leads to spacial separation between these two components of the assay system. This results in an underestimation of rates when relying solely on extramitochondrial H2O2 detection. We find that differentiating between these apparent rates found when using extramitochondrial H2O2 detection and the actual rates of metabolism is important to determining the rate constant for H2O2 consumption by mitochondria in kinetic experiments. Using the high rate of ROS production by mitochondria respiring on succinate, we demonstrate that net H2O2 metabolism by mitochondria can approach a stable steady-state of extramitochondrial [H2O2]. Importantly, the rate constant determined by extrapolation of kinetic experiments is similar to the rate constant determined as the [H2O2] approaches a steady state.