Bioenerg Commun 2020.1 https://doi:10.26124/bec:2020-0001.v1

Consortium communication

Mitochondrial physiology

Gnaiger Erich et al ― MitoEAGLE Task Group*

Living Communication Source: MitoFit Preprint Arch: doi:10.26124/mitofit:190001.v6 Extended resource of Mitochondrial respiratory states and rates

Overview Internal and external respiration (mt) Mitochondrial catabolic respiration

JkO2 is the O2 consumption in the oxidation of fuel substrates (electron donors) and reduction of O2 catalysed by the electron transfer system ETS, which drives the

protonmotive force pmF. JkO2 excludes mitochondrial residual oxygen consumption, mt-Rox ().

(ce) Cell respiration JrO2 is internal cellular O2 consumption, taking into account all chemical reactions r that consume O2 in the cells. Catabolic cell respiration is the O2 consumption associated with catabolic pathways in the cell, including mitochondrial (mt) catabolism, and: mt-Rox (); non-mt O2 consumption by catabolic reactions, particularly peroxisomal oxidases and microsomal cytochrome P450 systems (); non-mt Rox by reactions unrelated to catabolism (). (ext) External respiration balances internal respiration at steady state, including extracellular Rox () and aerobic respiration by the microbiome (). External O2 is transported from the environment across the respiratory cascade by circulation between tissues and diffusion across cell membranes, to the intracellular compartment. The respiratory quotient RQ is the molar CO2/O2 exchange ratio; combined with the nitrogen quotient N/O2 (mol N given off per mol O2 consumed), the RQ reflects the proportion of carbohydrate, lipid and protein utilized in cell respiration during aerobically balanced steady states. Bicarbonate and CO2 are transported in reverse to the extracellular milieu and the organismic environment. Hemoglobin provides the molecular paradigm for the combined CO2/O2 exchange, as do lungs and gills on the morphological level, but CO2/O2 exchange across the skin and other surfaces is less interdependent, and highly independent in cell respiration. Respiratory states are defined in Table 1. Rates are illustrated in Figure 5. Consult Tables 4 and 8 for terms, symbols, and units.

Updates: https://www.bioenergetics-communications.org/index.php/BEC_2020.1_doi10.26124bec2020-0001.v1

Table of contents

Abstract – 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

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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

Abstract International Union of Pure and Applied Chemistry (IUPAC) on terminology in As the knowledge base and importance of physical chemistry, extended by mitochondrial physiology to evolution, considerations of open systems and health and disease expands, the necessity thermodynamics of irreversible for harmonizing the terminology processes. The concept-driven concerning mitochondrial respiratory constructive terminology incorporates states and rates has become increasingly the meaning of each quantity and aligns apparent. The chemiosmotic theory concepts and symbols with the establishes the mechanism of energy nomenclature of classical bioenergetics. transformation and coupling in oxidative We endeavour to provide a balanced view phosphorylation. The unifying concept of of mitochondrial respiratory control and the protonmotive force provides the a critical discussion on reporting data of framework for developing a consistent mitochondrial respiration in terms of theoretical foundation of mitochondrial metabolic flows and fluxes. Uniform physiology and bioenergetics. We follow standards for evaluation of respiratory the latest SI guidelines and those of the states and rates will ultimately contribute

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BEC 2020.1 doi:10.26124/bec:2020-0001.v1 to reproducibility between laboratories maintained between the mitochondrial and thus support the development of data matrix and intermembrane compartment or repositories of mitochondrial respiratory outer mitochondrial space. Compartmental function in species, tissues, and cells. coupling depends on ion translocation Clarity of concept and consistency of across a semipermeable membrane, which is nomenclature facilitate effective defined as vectorial metabolism and transdisciplinary communication, distinguishes OXPHOS from cytosolic education, and ultimately further fermentation as counterparts of cellular core discovery. energy metabolism (Overview). Cell respiration is thus distinguished from Keywords―MitoPedia: Respiratory states • SI fermentation: (1) Electron acceptors are - The International System of Units • IUPAC • supplied by external respiration for the Coupling control • Mitochondrial maintenance of redox balance, whereas preparations • Protonmotive force • fermentation is characterized by an internal Uncoupling • Oxidative phosphorylation • electron acceptor produced in intermediary Phosphorylation efficiency • Electron metabolism. In aerobic cell respiration, transfer pathway • LEAK respiration • redox balance is maintained by O2 as the Residual oxygen consumption • electron acceptor. (2) Compartmental Normalization of rate • Flow • Flux • Flux coupling in vectorial OXPHOS contrasts to control ratio • Mitochondrial marker • Cell scalar substrate-level phosphorylation in count • Oxygen fermentation.

2. When measuring mitochondrial metabolism, the contribution of Executive summary fermentation and other cytosolic interactions must be excluded from analysis In view of the broad implications for health by disrupting the barrier function of the care, mitochondrial researchers face an plasma membrane. Selective removal or increasing responsibility to disseminate permeabilization of the plasma membrane their fundamental knowledge and novel yields mitochondrial preparations— discoveries to a wide range of stakeholders including isolated mitochondria, tissue and and scientists beyond the group of cell preparations—with structural and specialists. This requires implementation of functional mitochondrial integrity. a commonly accepted terminology within Subsequently, extramitochondrial the discipline and standardization in the concentrations of oxidizable ‘fuel’ substrates, translational context. Authors, reviewers, as well as ADP, ATP, inorganic phosphate, journal editors, and lecturers are challenged and cations including H+ can be controlled to to collaborate with the aim to harmonize the determine mitochondrial function under a nomenclature in the growing field of set of conditions defined as respiratory mitochondrial physiology and bioenergetics, states. We strive to incorporate an easily from evolutionary biology and comparative recognized and understood concept-driven physiology to mitochondrial medicine. In the terminology of bioenergetics with explicit present communication we focus on the terms and symbols that define the nature of following concepts in mitochondrial respiratory states. physiology: 3. Mitochondrial coupling states are 1. Aerobic respiration is the O2 flux in defined according to the control of catabolic reactions coupled to respiratory oxygen flux by the protonmotive phosphorylation of ADP to ATP, and O2 flux force pmF, in an interaction of the electron in a variety of O2 consuming reactions apart transfer system generating the pmF and the from oxidative phosphorylation (OXPHOS). phosphorylation system utilizing the pmF. Coupling in OXPHOS is mediated by the Capacities of OXPHOS and electron transfer translocation of protons across the are measured at kinetically-saturating mitochondrial inner membrane (mtIM) concentrations of fuel substrates, ADP and through proton pumps generating or inorganic phosphate, and O2, or at optimal utilizing the protonmotive force that is uncoupler concentrations, respectively, in

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Consortium Communication the absence of Complex IV inhibitors such as NO, CO, or H2S. Respiratory capacity is a Box 1: In brief – Mitochondria and measure of the upper limit of the rate of bioblasts respiration; it depends on the fuel substrate type undergoing oxidation in a ‘For the physiologist, mitochondria mitochondrial pathway, and provides afforded the first opportunity for an reference values for the diagnosis of health experimental approach to structure- and disease. Evaluation of the impact of function relationships, in particular those evolutionary background, age, gender and involved in active transport, vectorial sex, lifestyle and environment represents a metabolism, and metabolic control major challenge for mitochondrial mechanisms on a subcellular level’ respiratory physiology and pathology. (Ernster and Schatz 1981) [38].

4. Incomplete tightness of coupling, i.e., Mitochondria are oxygen-consuming some degree of uncoupling relative to the electrochemical generators (Figure 1). They mitochondrial pathway-dependent coupling evolved from the endosymbiotic stoichiometry, is a characteristic of energy- alphaproteobacteria which became transformations across membranes. integrated into a host cell related to Asgard Uncoupling or dyscoupling are caused by Archaea [85; 72; 117]. Richard Altmann physiological, pathological, toxicological, described the ‘bioblasts’ in 1894 [1], which pharmacological and environmental include not only mitochondria as presently conditions that exert an influence not only on defined, but also symbiotic and free-living the proton leak and cation cycling, but also bacteria. The word ‘mitochondria’ (Greek on proton slip within the proton pumps and mitos: thread; chondros: granule) was the structural integrity of the mitochondria. introduced by Carl Benda in 1898 [4]. A more loosely coupled state is induced by Mitochondrion is singular and mitochondria stimulation of mitochondrial superoxide is plural. Abbreviation: mt, as generally used formation and the bypass of proton pumps. in mtDNA. In addition, the use of protonophores Contrary to past textbook dogma, which represents an experimental uncoupling describes mitochondria as individual intervention to assess the transition from a organelles, mitochondria form dynamic well-coupled to a noncoupled state of networks within eukaryotic cells. mitochondrial respiration. Mitochondrial movement is supported by 5. Respiratory oxygen consumption rates microtubules. Mitochondrial size and have to be carefully normalized to enable number can change in response to energy meta-analytic studies beyond the question of requirements of the cell via processes known a particular experiment. Therefore, all raw as fusion and fission; these interactions data on rates and variables for normalization allow mitochondria to communicate within a should be published in an open access data network [18]. Mitochondria can even repository. Normalization of rates for: (1) traverse cell boundaries in a process known the number of objects (cells, organisms); (2) as horizontal mitochondrial transfer [133]. the volume or mass of the experimental Another defining morphological sample; and (3) the concentration of characteristic of mitochondria is the double mitochondrial markers in the experimental membrane. The mitochondrial inner chamber are sample-specific normalizations, membrane, mtIM, forms dynamic tubular to which are distinguished from system- disk-shaped cristae that separate the specific normalization for the volume of the mitochondrial matrix, i.e., the negatively experimental chamber (the measuring charged internal mitochondrial system). compartment, from the intermembrane

6. The consistent use of terms and space; the latter being enclosed by the symbols facilitates transdisciplinary mitochondrial outer membrane, mtOM, and communication and will support the further positively charged with respect to the matrix. development of a collaborative database on Intracellular stress factors may cause bioenergetics and mitochondrial physiology. shrinking or swelling of the mitochondrial matrix that can ultimately result in

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BEC 2020.1 doi:10.26124/bec:2020-0001.v1 permeability transition mtPT [77]. The mtIM between mitochondria and other cellular contains the non-bilayer phospholipid components. For example, the crosstalk cardiolipin, which is also involved in the between mitochondria and the endoplasmic mtOM [47] but is not present in any other reticulum is involved in the regulation of eukaryotic cellular membrane. Cardiolipin calcium homeostasis, cell division, has many regulatory functions [101]; it autophagy, differentiation, and anti-viral promotes and stabilizes the formation of signaling [98]. Mitochondria contribute to supercomplexes (SCInIIInIVn) based on the formation of peroxisomes, which are dynamic interactions between specific hybrids of mitochondrial and ER-derived respiratory complexes [58; 80; 87], and it precursors [131]. Cellular mitochondrial supports proton transfer on the mtIM from homeostasis (mitostasis) is maintained the electron transfer system to F1FO-ATPase through regulation at transcriptional, post- (ATP synthase [144]). The mtIM is plastic translational and epigenetic levels [81; 82], and exerts an influence on the functional resulting in dynamic regulation of properties of incorporated proteins [135]. mitochondrial turnover by biogenesis of new Mitochondria constitute the structural mitochondria and removal of damaged and functional elementary components of mitochondria by fusion, fission and cell respiration. Aerobic respiration is the mitophagy [128]. Cell signalling modules reduction of molecular oxygen by electron contribute to homeostatic regulation transfer coupled to electrochemical proton throughout the cell cycle or even cell death translocation across the mtIM. In the process by activating proteostatic modules, e.g., the of OXPHOS, the catabolic reaction sequence ubiquitin-proteasome and autophagy- of oxygen consumption is electrochemically lysosome/vacuole pathways, specific coupled to the transformation of energy in proteases like LON, and genome stability the phosphorylation of ADP to adenosine modules in response to varying energy triphosphate, ATP [92; 93]. Mitochondria are demands and stress cues [109]. In addition, the powerhouses of the cell that contain the several post-translational modifications, machinery of the OXPHOS pathways, including acetylation and nitrosylation, are including transmembrane respiratory capable of influencing the bioenergetic complexes (proton pumps with FMN, Fe-S response, with clinically significant and cytochrome b, c, aa3 redox systems); implications for health and disease [17]. alternative dehydrogenases and oxidases; Mitochondria of higher eukaryotes the coenzyme ubiquinone, Q; F1FO-ATPase; typically maintain several copies of their the enzymes of the tricarboxylic acid cycle, own circular genome known as TCA, fatty acid and amino acid oxidation; mitochondrial DNA, mtDNA (hundred to transporters of ions, metabolites and co- thousands per cell [27]), which is maternally factors; iron/sulphur cluster synthesis; and inherited in many species. However, mitochondrial kinases related to catabolic biparental mitochondrial inheritance is pathways. TCA cycle intermediates are vital documented in some exceptional cases in precursors for macromolecule biosynthesis humans [83], is widespread in birds, fish, [30]. The mitochondrial proteome comprises reptiles and invertebrate groups, and is even over 1200 types of protein [13; 14], mostly the norm in some bivalve taxonomic groups encoded by nuclear DNA, nDNA, with a [9; 140]. variety of functions, many of which are The mitochondrial genome of the relatively well known, e.g., proteins angiosperm Amborella contains a record of regulating mitochondrial biogenesis or six mitochondrial genome equivalents apoptosis, while others are still under acquired by horizontal transfer of entire investigation, or need to be identified, e.g., genomes, two from angiosperms, three from mtPT pore and alanine transporter. The algae and one from mosses [114]. In mammalian mitochondrial proteome can be unicellular organisms, i.e., protists, the used to discover and characterize the genetic structural organization of mitochondrial basis of mitochondrial diseases [102; 142]. genomes is highly variable and includes Numerous cellular processes are circular and linear DNA [145]. While some of orchestrated by a constant crosstalk the free-living flagellates exhibit the largest

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Figure 1. Cell respiration and oxidative phosphorylation (OXPHOS) Mitochondrial respiration is the oxidation of fuel substrates (electron donors) with electron transfer to O2 as the electron acceptor. For explanation of symbols see also Overview. (a) Respiration of living cells: Extramitochondrial catabolism of macrofuels and uptake of small molecules by the cell provide the mitochondrial fuel substrates. Dashed arrows indicate the connection between the redox proton pumps (respiratory Complexes CI, CIII and CIV) and the transmembrane protonmotive force pmF. Coenzyme Q (Q) and the cytochromes b, c, and aa3 are redox systems of the mitochondrial inner membrane, mtIM. Glycerol-3-phosphate, Gp. (b) Respiration in mitochondrial preparations: The mitochondrial electron transfer system ETS is (1) fuelled by diffusion and transport of substrates across the mtOM and mtIM, and in addition consists of the (2) matrix-ETS, and (3) membrane-ETS. Electron transfer converges at the N-junction, and from CI, CII and electron transferring flavoprotein complex CETF at the Q- junction. Unlabeled arrows converging at the Q-junction indicate additional ETS-sections with electron entry into Q through glycerophosphate dehydrogenase, dihydroorotate dehydrogenase, proline dehydrogenase, choline dehydrogenase, and sulfide-ubiquinone oxidoreductase. The dotted arrow indicates the branched pathway of oxygen consumption by alternative quinol oxidase AOX. ET pathways are coupled to the phosphorylation pathway. H+pos/O2 shows the ratio of the outward proton flux from the matrix space to the positively (pos) charged vesicular compartment, divided by catabolic O2 flux in the NADH pathway. The H+neg/P» ratio is the inward proton flux from the inter-membrane space to the negatively (neg) charged matrix space, divided by the flux of phosphorylation of ADP to ATP. These stoichiometries are not fixed because of ion leaks and proton slip. Moreover, the H+neg/P» ratio is linked to the F1FO-ATPase c-ring stoichiometry, which is species-dependent and defines the bioenergetic cost of P». Modified from [78; 116]. + (c) OXPHOS-coupling: The H circuit couples O2 flux JkO2 through the catabolic ET pathway to flux JP» through the phosphorylation pathway converting ADP to ATP. (d) Phosphorylation pathway catalyzed by the proton pump F1FO•ATPase (ATP synthase), adenine nucleotide translocase ANT, and inorganic phosphate carrier PiC. The H+neg/P» stoichiometry is the sum of the coupling stoichiometry in the F1FO-ATPase reaction (•2.7 H+pos from the positive intermembrane space, 2.7 H+neg to the matrix, i.e., the negative compartment) and the proton balance in the translocation of ADP3•, ATP4- and Pi2- (negative for substrates). Modified from [54]. known gene coding capacity, e.g., jakobid tRNAs, and two ribosomal RNAs. Additional Andalucia godoyi mtDNA codes for 106 genes gene content has been suggested to include [12], some protist groups, e.g., alveolates, microRNAs, piRNA, smithRNAs, repeat possess mitochondrial genomes with only associated RNA, long noncoding RNAs, and three protein-coding genes and two rRNAs even additional proteins or peptides [23; 35; [42]. The complete loss of mitochondrial 74; 111]. The mitochondrial genome genome is observed in the highly reduced requires nuclear-encoded mitochondrially mitochondria of Cryptosporidium species targeted proteins, e.g., TFAM, for its [83]. Reaching the final extreme, the maintenance and expression [110]. The microbial eukaryote, oxymonad nuclear and the mitochondrial genomes Monocercomonoides, has no mitochondrion encode peptides of the membrane spanning whatsoever and lacks all typical nuclear- redox pumps (CI, CIII and CIV) and F1FO- encoded mitochondrial proteins, showing ATPase, leading to strong constraints in the that while in 99 % of organisms coevolution of both genomes [6]. mitochondria play a vital role, this organelle Given the multiple roles of mitochondria, is not indispensable [65]. it is perhaps not surprising that In vertebrates, but not all invertebrates, mitochondrial dysfunction is associated with mtDNA is compact (16.5 kB in humans) and a wide variety of genetic and degenerative encodes 13 protein subunits of the diseases [41]. Robust mitochondrial function transmembrane respiratory Complexes CI, is supported by physical exercise and caloric CIII, CIV and ATP synthase (F1FO-ATPase), 22 balance, and is central for sustained

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Figure 1.

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Consortium Communication metabolic health throughout life. Therefore, of experimental outcomes requires a more consistent set of definitions for harmonization of protocols between mitochondrial physiology will increase our research groups at different institutes. In understanding of the etiology of disease and addition to quality control, a conceptual improve the diagnostic repertoire of framework is also required to standardise mitochondrial medicine with a focus on and harmonise terminology and protective medicine, evolution, lifestyle, methodology. Vague or ambiguous jargon environment, and healthy aging. can lead to confusion and may convert valuable signals to wasteful noise [100]. For this reason, measured values must be 1. Introduction expressed in standard units for each parameter used to define mitochondrial Mitochondria are the powerhouses of the cell respiratory function. A consensus on with numerous morphological, fundamental nomenclature and conceptual physiological, molecular, and genetic coherence, however, is missing in the functions (Box 1). Every study of expanding field of mitochondrial physiology. mitochondrial health and disease faces To fill this gap, the present communication Evolution, Age, Gender and sex, Lifestyle, provides an in-depth review on and Environment (MitoEAGLE) as essential harmonization of nomenclature and background conditions intrinsic to the definition of technical terms, which are individual person or cohort, species, tissue essential to improve the awareness of the and to some extent even cell line. As a large intricate meaning of current and past and coordinated group of laboratories and scientific vocabulary. This is important for researchers, the mission of the global documentation and integration into data MitoEAGLE Network is to generate the repositories in general, and quantitative necessary scale, type, and quality of modelling in particular [3]. consistent data sets and conditions to In this review, we focus on coupling states address this intrinsic complexity. and fluxes through metabolic pathways of Harmonization of experimental protocols aerobic energy transformation in and implementation of a quality control and mitochondrial preparations in the attempt to data management system are required to establish a conceptually-oriented interrelate results gathered across a nomenclature in bioenergetics and spectrum of studies and to generate a mitochondrial physiology in a series of rigorously monitored database focused on communications, prepared in the frame of mitochondrial respiratory function. In this the EU COST Action MitoEAGLE open to way, researchers from a variety of global bottom-up input. disciplines can compare their findings using clearly defined and accepted international 2. Coupling states and rates in standards. mitochondrial preparations

With an emphasis on quality of research, ‘Every professional group develops its own published data can be useful far beyond the technical jargon for talking about matters specific question of a particular experiment. of critical concern ... People who know a For example, collaborative data sets support word can share that idea with other the development of open-access databases members of their group, and a shared such as those for National Institutes of vocabulary is part of the glue that holds Health sponsored research in genetics, people together and allows them to create proteomics, and metabolomics. Indeed, a shared culture’ (Miller 1991) [91]. enabling meta-analysis is the most economic way of providing robust answers to 2.1. Cellular and mitochondrial respiration biological questions [25]. However, the reproducibility of quantitative results 2.1.1. Aerobic and anaerobic catabolism depend on accurate measurements under and ATP turnover: In respiration, electron strictly-defined conditions. Likewise, transfer is coupled to the phosphorylation of meaningful interpretation and comparability ADP to ATP, with energy transformation

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BEC 2020.1 doi:10.26124/bec:2020-0001.v1 mediated by the protonmotive force pmF the activity of solute carriers, e.g., the (Figure 2). Anabolic reactions are coupled to sodium-dependent dicarboxylate catabolism, both by ATP as the intermediary transporter SLC13A3 and the sodium- energy currency and by small organic dependent phosphate transporter SLC20A2, precursor molecules as building blocks for which transport specific metabolites across biosynthesis [30]. Glycolysis involves the plasma membrane of various cell types, substrate-level phosphorylation of ADP to and the availability of plasma membrane- ATP in fermentation without utilization of permeable succinate [37]. These limitations O2, studied mainly in living cells and are overcome by the use of mitochondrial organisms. Many cellular fuel substrates are preparations. catabolized to acetyl-CoA or to glutamate, and further electron transfer reduces nicotinamide adenine dinucleotide to NADH or flavin adenine dinucleotide to FADH2. Subsequent mitochondrial electron transfer to O2 is coupled to proton translocation for the control of the pmF and phosphorylation of ADP (Figure 1b and 1c). In contrast, extramitochondrial oxidation of odd chain Figure 2. Four-compartment model of fatty acids, very long chain fatty acids, and oxidative phosphorylation some amino acids proceeds partially in Respiratory states (ET, OXPHOS, LEAK; peroxisomes without coupling to ATP Table 1) and corresponding rates (E, P, L) production: acyl-CoA oxidase catalyzes the are connected by the protonmotive force oxidation of FADH with electron transfer to 2 pmF. (1) ET capacity E is partitioned into (2) O ; amino acid oxidases oxidize flavin 2 dissipative LEAK respiration L, when the mononucleotide FMNH or FADH (Figure 2 2 Gibbs energy change of catabolic O flux is 1a). 2 irreversibly lost, (3) net-OXPHOS capacity The plasma membrane separates the (P-L), with partial conservation of the intracellular compartment including the cytosol, nucleus, and organelles from the capacity to perform work, and (4) the ET- excess capacity (E-P). Modified from [54]. extracellular environment. Cell membranes include the plasma membrane and organellar membranes. The plasma 2.1.2. Specification of biochemical dose membrane consists of a lipid bilayer with and exposure: Substrates, uncouplers, embedded proteins and attached organic inhibitors, and other chemical reagents are molecules that collectively control the titrated to analyse cellular and selective permeability of ions, organic mitochondrial function. Nominal molecules, and particles across the cell concentrations of these substances are boundary. The intact plasma membrane usually reported as initial amount of -1 prevents the passage of many water-soluble substance concentration cB [mol∙L ] in the mitochondrial substrates and inorganic incubation medium. ions—such as succinate, adenosine Kinetically-saturating conditions are diphosphate (ADP) and inorganic phosphate evaluated by substrate kinetics to obtain the maximum reaction velocity or maximum (Pi) that must be precisely controlled at kinetically-saturating concentrations for the pathway flux, in contrast to solubility- analysis of mitochondrial respiratory saturated conditions. When aiming at the capacities (Figure 2). Respiratory capacities measurement of kinetically-saturated delineate―comparable to channel capacity processes—such as OXPHOS capacities—the in information theory [123]―the upper concentrations for substrates can be chosen according to half-saturating substrate boundary of the rate of O2 consumption measured in defined respiratory states. The concentrations c50, for metabolic pathways, intact plasma membrane limits the scope of or the Michaelis constant Km, for enzyme investigations into mitochondrial kinetics. In the case of hyperbolic kinetics, respiratory function in living cells, despite only 80 % of maximum respiratory capacity is obtained at a substrate concentration of

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Consortium Communication four times the c50, whereas substrate in vivo deviate from these experimentally concentrations of 5, 9, 19 and 49 times the c50 obtained states; this is because kinetically- are theoretically required for reaching 83, saturating concentrations, e.g., of ADP, 90, 95 or 98 % of the maximal rate [51]. oxygen (O2; dioxygen) or fuel substrates, Other reagents are chosen to inhibit or may not apply to physiological intracellular alter a particular process. The amount of conditions. Further information is obtained these chemicals in an experimental in studies of kinetic responses to variations incubation is selected to maximize effect, in fuel substrate concentrations, [ADP], or avoiding unacceptable off-target [O2] in the range between kinetically- consequences that would adversely affect saturating concentrations and anoxia [51]. the data being sought. Specifying the amount The cholesterol content of the plasma of substance in an incubation as nominal membrane is high compared to concentration in the aqueous incubation mitochondrial membranes [70]. Therefore, medium can be ambiguous [33], particularly mild detergents—such as digitonin and for cations (TPP+; fluorescent dyes such as saponin—can be applied to selectively safranin, TMRM [22]) and lipophilic permeabilize the plasma membrane via substances (oligomycin, uncouplers, interaction with cholesterol; this allows free permeabilization agents [32]), which exchange of organic molecules and inorganic accumulate in the mitochondrial matrix or ions between the cytosol and the immediate on biological membranes, respectively. cell environment, while maintaining the Generally, dose can be specified per unit of integrity and localization of organelles, biological sample, i.e., (nominal moles of cytoskeleton, and the nucleus. Application of xenobiotic)/(number of cells) [mol∙x•1] or, as permeabilization agents (mild detergents or appropriate, per mass of biological sample toxins) leads to washout of cytosolic marker [mol∙kg-1]. This approach to specification of enzymes—such as lactate dehydrogenase— dose provides a scalable parameter that can and results in the complete loss of cell be used to design experiments, help viability (tested by nuclear staining using interpret a wide variety of experimental plasma membrane-impermeable dyes), results, and provide absolute information while mitochondrial function remains intact that allows researchers worldwide to make (tested by cytochrome c stimulation of the most use of published data [33]. respiration). Exposure includes the additional dimension Digitonin concentrations have to be of time in contact with a particular dose. optimized according to cell type, particularly since mitochondria from cancer cells contain 2.2. Mitochondrial preparations significantly higher contents of cholesterol in both membranes [2]. For example, a dose of Mitochondrial preparations are defined as digitonin per cell of 8 fmol∙x•1 [10 pg∙x•1; 10 either isolated mitochondria or tissue and µg∙(106 x)•1] is optimal for permeabilization cell preparations in which the barrier of endothelial cells, and the concentration in function of the plasma membrane is the incubation medium has to be adjusted disrupted. Since this entails the loss of cell according to the cell-mass concentration viability, mitochondrial preparations are not [32]. Respiration of isolated mitochondria studied in vivo. In contrast to isolated remains unaltered after the addition of low mitochondria and tissue homogenate concentrations of digitonin or saponin. In preparations, mitochondria in addition to mechanical cell disruption during permeabilized tissues and cells are in situ homogenization of tissue, permeabilization relative to the plasma membrane. When agents may be applied to ensure studying mitochondrial preparations, permeabilization of all cells in tissue substrate-uncoupler-inhibitor-titration homogenates. (SUIT) protocols are used to establish Suspensions of cells permeabilized in the respiratory Coupling-Control States (CCS) respiration chamber and crude tissue and Pathway-Control States (PCS) that homogenates contain all components of the provide reference values for various output cell at highly dilute concentrations. All variables (Table 1). Physiological conditions

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Table 1. Coupling states and rates, and residual oxygen consumption in mitochondrial

preparations. Respiration- and phosphorylation flux, JkO2 and JP», are rates, characteristic of a state in conjunction with the protonmotive force pmF. Coupling states are established at kinetically-saturating concentrations of oxidizable ‘fuel’ substrates and O2.

State Rate JkO2 JP» pmF Inducing Limiting factors factors

LEAK L low, cation 0 max. back-flux of JP» = 0: (1) without ADP, leak- cations including L(n); (2) max. ATP/ADP dependent proton leak, ratio, L(T); or (3) inhibition respiration proton slip of the phosphorylation pathway, L(Omy), L(Cat)

OXPHOS P high, ADP- max. high kinetically- ET capacity limits JkO2, or stimulated saturating [ADP] phosphorylation-pathway respiration, and [Pi] capacity limits JP» and in turn OXPHOS JkO2 capacity

ET E max., 0 low optimal external JkO2 by ET capacity noncoupled uncoupler respiration, concentration for

ET capacity max. JO2,E

ROX Rox min., residual 0 0 JO2,Rox in non-ET inhibition of all ET O2 pathway pathways; or absence of fuel consumption oxidation substrates reactions mitochondria are retained in chemically- mtIM. In addition, the mitochondrial permeabilized mitochondrial preparations electron transfer system ETS consists of the and crude tissue homogenates. In the matrix-ETS and membrane-ETS (Figure 1b). preparation of isolated mitochondria, Upstream sections of ET pathways converge however, the mitochondria are separated at the NADH-junction (N-junction). NADH is from other cell fractions and purified by mainly generated in the TCA cycle and is differential centrifugation, entailing the loss oxidized by Complex I (CI), with further of mitochondria at typical recoveries ranging electron entry into the coenzyme Q-junction from 30 to 80 % of total mitochondrial (Q-junction). Similarly, succinate is formed content [71]. Using Percoll or sucrose in the TCA cycle and oxidized by CII to density gradients to maximize the purity of fumarate. CII is part of both the TCA cycle isolated mitochondria may compromise the and the ETS, and reduces FAD to FADH2 with mitochondrial yield or structural and further reduction of ubiquinone to ubiquinol functional integrity. Therefore, downstream of the TCA cycle in the Q- mitochondrial isolation protocols need to be junction. Thus FADH2 is not a substrate but is optimized according to each study. The term the product of CII, in contrast to erroneous mitochondrial preparation neither includes metabolic maps shown in many publications. living cells, nor submitochondrial particles β-oxidation of fatty acids FA supplies and further fractionated mitochondrial reducing equivalents via (1) FADH2 as the components. substrate of electron transferring flavoprotein complex CETF; (2) acetyl-CoA 2.3. Electron transfer pathways generated by chain shortening; and (3) NADH generated via 3-hydroxyacyl-CoA Mitochondrial electron transfer (ET) dehydrogenases. The ATP yield depends on pathways are fuelled by diffusion and whether acetyl-CoA enters the TCA cycle, or transport of substrates across the mtOM and is for example used in ketogenesis.

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Selected mitochondrial catabolic Pathway-control states PCS and coupling- pathways of electron transfer from the control states CCS are complementary, since oxidation of fuel substrates to the reduction mitochondrial preparations depend on (1) of O2 are stimulated by addition of fuel an exogenous supply of pathway-specific substrates to the mitochondrial respiration fuel substrates and oxygen, and (2) medium after depletion of endogenous exogenous control of phosphorylation substrates (Figure 1b). Substrate (Figure 1). combinations and specific inhibitors of ET pathway enzymes are used to obtain defined 2.4.2. Phosphorylation P» and P»/O2 pathway-control states in mitochondrial ratio: Phosphorylation in the context of preparations [54]. OXPHOS is defined as phosphorylation of ADP by Pi to form ATP. On the other hand, the 2.4. Respiratory coupling control term phosphorylation is used generally in many contexts, e.g., protein phosphorylation. 2.4.1. Coupling: Coupling of electron transfer This provides the argument for introducing a (ET) to phosphorylation of ADP to ATP is symbol more discriminating and specific mediated by vectorial translocation of protons than P as used in the P/O ratio (phosphate to across the mtIM. Proton pumps generate or atomic oxygen ratio), where P indicates utilize the electrochemical pmF (Figure 1). phosphorylation of ADP to ATP or GDP to The pmF is the sum of two partial forces, the GTP (Figure 1): The symbol P» indicates the electric force (electric potential difference) and endergonic (uphill) direction of chemical force (proton chemical potential phosphorylation ADP→ATP, and likewise P« difference, related to ΔpH [92; 93]). The the corresponding exergonic (downhill) catabolic flux of scalar reactions is hydrolysis ATP→ADP. P» refers mainly to collectively measured as O2 flux JkO2. electrontransfer phosphorylation but may Thus mitochondria are elementary also involve substrate-level phosphorylation components of energy transformation. as part of the TCA cycle (succinyl-CoA ligase, Energy is a conserved quantity and cannot be phosphoglycerate kinase) and lost or produced in any internal process phosphorylation of ADP catalyzed by (First Law of Thermodynamics). Open and pyruvate kinase, and of GDP phosphorylated closed systems can gain or lose energy only by phosphoenolpyruvate carboxykinase. by external fluxes—by exchange with the Transphosphorylation is performed by environment. Therefore, energy can neither adenylate kinase, creatine kinase (mtCK), be produced by mitochondria, nor is there hexokinase and nucleoside diphosphate any internal process without energy kinase. In isolated mammalian mitochondria, conservation. Exergy or Gibbs energy (‘free ATP production catalyzed by adenylate energy’) is the part of energy that can kinase (2 ADP ↔ ATP + AMP) proceeds potentially be transformed into work under without fuel substrates in the presence of conditions of constant temperature and ADP [69]. Kinase cycles are involved in pressure. Coupling is the interaction of an intracellular energy transfer and signal exergonic process (spontaneous, negative transduction for regulation of energy flux. exergy change) with an endergonic process The P»/O2 ratio (P»/4 e-) is two times the (positive exergy change) in energy ‘P/O’ ratio (P»/2 e•). P»/O2 is a generalized transformations which conserve part of the symbol, not specific for reporting Pi exergy change. Exergy is not completely consumption (Pi/O2 flux ratio), ADP conserved, however, except at the limit of depletion (ADP/O2 flux ratio), or ATP 100 % efficiency of energy transformation in production (ATP/O2 flux ratio). The a coupled process [49]. The exergy or Gibbs mechanistic P»/O2 ratio—or P»/O2 energy change that is not conserved by stoichiometry—is calculated from the coupling is irreversibly dissipated, and is proton–to–O2 and proton–to– accounted for as the entropy change of the phosphorylation coupling stoichiometries surroundings and the system, multiplied by (Figure 1c):

+ the absolute temperature of the irreversible Hpos/O2 P»/O2 = + (1) process [50]. Hneg/P»

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+ The H pos/O2 coupling stoichiometry addition, YP»/O2 depends on several (referring to the full four electron reduction experimental conditions of flux control, of O2) depends on the relative involvement of increasing as a hyperbolic function of [ADP] the three coupling sites (respiratory to a maximum value [51]. Uncoupling of Complexes CI, CIII and CIV) in the catabolic mitochondrial respiration is a general term ET pathway from reduced fuel substrates comprising diverse mechanisms (Figure 3): (electron donors) to the reduction of O 2 1. Proton leak across the mtIM from the (electron acceptor). This varies with a positive to the negative compartment (H+ bypass of: (1) CI by single or multiple leak-uncoupled); electron input into the Q-junction; and (2) 2. Cycling of other cations, strongly CIV by involvement of alternative oxidases, stimulated by mtPT; comparable to the AOX. AOX are expressed in all plants, some use of protonophores, cation cycling is fungi, many protists, and several animal experimentally induced by valinomycin in phyla, but are not expressed in vertebrate the presence of K+; mitochondria [86]. 3. Decoupling by proton slip in the redox The H+ /O coupling stoichiometry pos 2 proton pumps (CI, CIII and CIV) when equals 12 in the ET pathways involving CIII protons are effectively not pumped in the and CIV as proton pumps, increasing to 20 ETS, or are not driving phosphorylation for the NADH pathway through CI (Figure (F1FO-ATPase); 1b). A general consensus on H+ /O pos 2 4. Loss of vesicular (compartmental) stoichiometries, however, remains to be integrity when electron transfer is reached [59; 122; 141]. The H+ /P» neg acoupled; coupling stoichiometry (3.7; Figure 1b) is 5. Electron leak in the loosely coupled the sum of 2.7 H+ required by the F F - neg 1 O univalent reduction of O to superoxide ATPase of vertebrate and most invertebrate 2 (O2•­; superoxide anion radical). species [138] and the proton balance in the translocation of ADP, ATP and Pi (Figure 1c). Differences of terms—uncoupled vs. Taken together, the mechanistic P»/O2 ratio noncoupled—are easily overlooked, is calculated at 5.4 and 3.3 for the N- and S although they relate to different meanings of pathway, respectively (Eq. 1). The uncoupling (Table 2 and Figure 3). corresponding classical P»/O ratios (referring to the 2 electron reduction of 0.5 2.5. Coupling states and respiratory rates O2) are 2.7 and 1.6 [138], in agreement with the measured P»/O ratio for succinate of To extend the classical nomenclature on 1.58 ± 0.02 [57]. mitochondrial respiratory states (Section 2.6) by a concept-driven terminology that 2.4.3. Uncoupling: The effective P»/O2 flux explicitly incorporates information on the meaning of respiratory states, the ratio (YP»/O2 = JP»/JkO2) is diminished relative terminology must be general and not to the mechanistic P»/O2 ratio by intrinsic and extrinsic uncoupling or dyscoupling restricted to any particular experimental (Figure 3). This is distinct from switching protocol or mitochondrial preparation [53]. between mitochondrial pathways that Diagnostically meaningful and reproducible involve fewer than three proton pumps conditions are defined for measuring (‘coupling sites’: Complexes CI, CIII and CIV), mitochondrial function and respiratory bypassing CI through multiple electron capacities of core energy metabolism. entries into the Q-junction, or bypassing CIII Standard respiratory coupling-control states and CIV through AOX (Figure 1b). are obtained while maintaining a defined ET- Reprogramming of mitochondrial pathways pathway state with constant fuel substrates leading to different types of substrates being and inhibitors of specific branches of the ET oxidized may be considered as a switch of pathway. Concept-driven nomenclature gears (changing the stoichiometry by aims at mapping the meaning and concept altering the substrate that is oxidized) rather behind the words and acronyms onto the than uncoupling (loosening the tightness of forms of words and acronyms [91]. The focus coupling relative to a fixed stoichiometry). In of concept-driven nomenclature is primarily

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Figure 3. Mechanisms of respiratory uncoupling An intact mitochondrial inner membrane, mtIM, is required for vectorial, compartmental coupling. Inducible uncoupling, e.g., by activation of UCP1, increases LEAK respiration; experimentally noncoupled respiration provides an estimate of ET capacity obtained by titration of protonophores stimulating respiration to maximum O2 flux. H+ leak-uncoupled, decoupled, and loosely coupled respiration are components of intrinsic uncoupling (Table 2). Pathological dysfunction may affect all types of uncoupling, including permeability transition mtPT, causing intrinsically dyscoupled respiration. Similarly, toxicological and environmental stress factors can cause extrinsically dyscoupled respiration. ‘Acoupled’ respiration is the consequence of structural disruption with catalytic activity of non-compartmental mitochondrial fragments. Reduced fuel substrates, red; oxidized products, ox. the conceptual why, along with clarification limitation of OXPHOS capacity mediated of the experimental how (Table 1). by the phosphorylation pathway. By application of external uncouplers, ET LEAK: The contribution of intrinsically capacity is measured as noncoupled uncoupled O2 consumption is studied by respiration.

preventing the stimulation of The three coupling states, LEAK, OXPHOS, phosphorylation either in the absence of and ET are shown schematically with the ADP or by inhibition of the corresponding respiratory rates, phosphorylation pathway. The abbreviated as L, P, and E, respectively corresponding states are collectively (Figure 2). We distinguish between classified as LEAK states when O2 metabolic pathways and metabolic states consumption compensates mainly for ion with the corresponding metabolic rates; for leaks, including the proton leak. example: ET pathways, ET states, and ET OXPHOS: The ET- and phosphorylation capacities E, respectively (Table 1). The pathways comprise coupled segments of protonmotive force pmF is maximum in the the OXPHOS-system and provide LEAK state of coupled mitochondria, driven reference values of respiratory capacities. by LEAK respiration at a minimum back-flux The OXPHOS capacity is measured at of cations to the matrix side, high in the kinetically-saturating concentrations of OXPHOS state when it drives ADP, Pi, fuel substrates and O2. phosphorylation, and very low in the ET state ET: Compared to OXPHOS capacity, the when uncouplers short-circuit the proton oxidative ET capacity reveals the cycle (Table 1).

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Table 2. Terms on respiratory coupling and uncoupling

Term JkO2 P»/O2 Notes

uncoupled L 0 non-phosphorylating LEAK respiration (Fig. 2) proton leak- 0 component of L, H+ diffusion across the mtIM (Fig.

uncoupled 2b-d)

inducibly 0 by UCP1 or cation (e.g., Ca2+) cycling, strongly uncoupled stimulated by permeability transition mtPT; experimentally induced by valinomycin in the presence of K+ decoupled 0 component of L, proton slip when protons are effectively not pumped in the redox proton pumps CI, CIII and CIV or are not driving phosphorylation (F1FO-ATPase [16]) (Fig. 2b-d) loosely 0 component of L, lower coupling due to superoxide coupled formation and bypass of proton pumps by electron

intrinsic, no protonophore added protonophore no intrinsic, leak with univalent reduction of O2 to superoxide (O2•–; superoxide anion radical) dyscoupled 0 mitochondrial dysfunction due to pathologically, toxicologically, environmentally increased uncoupling noncoupled E 0 ET capacity, non-phosphorylating respiration stimulated to maximum flux at optimum exogenous protonophore concentration (Fig. 2d) well-coupled P high OXPHOS capacity, phosphorylating respiration with an intrinsic LEAK component (Fig. 2c) fully coupled P – L max. OXPHOS capacity corrected for LEAK respiration (Fig. 2a) acoupled 0 electron transfer in mitochondrial fragments without vectorial proton translocation upon loss of vesicular (compartmental) integrity

2.5.1. LEAK state (Figure 4a): The LEAK Adjustment of the nominal concentration of state is defined as a state of mitochondrial these inhibitors to the concentration of respiration when O2 flux mainly biological sample applied can minimize or compensates for ion leaks in the absence of avoid inhibitory side-effects exerted on ET ATP synthesis, at kinetically-saturating capacity or even some dyscoupling. The concentrations of O2 and respiratory fuel chelator EGTA is added to mt-respiration substrates. LEAK respiration is measured to media to bind free Ca2+, thus limiting cation obtain an estimate of intrinsic uncoupling cycling. The LEAK rate is a function of without addition of an experimental respiratory state, hence it depends on (1) the uncoupler: (1) in the absence of adenylates, barrier function of the mtIM (‘leakiness’), (2) i.e., AMP, ADP and ATP; (2) after depletion of the electrochemical potential differences ADP at a maximum ATP/ADP ratio; or (3) and concentration differences across the after inhibition of the phosphorylation mtIM, and (3) the H+/O2 ratio of the ET pathway by inhibitors of F1FO-ATPase pathway (Figure 1b).

(oligomycin, Omy) or adenine nucleotide • Proton leak and uncoupled translocase (carboxyatractyloside, Cat). respiration: The intrinsic proton leak is

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the uncoupled leak current of protons in superoxide by cytochrome c, whereby which protons diffuse across the mtIM in electrons may re-enter the ETS with the dissipative direction of the downhill proton translocation by CIV. pmF without coupling to phosphorylation • Dyscoupled respiration: Mitochondrial (Figure 4a). The proton leak flux depends injuries may lead to dyscoupling as a non-linearly on the electric membrane pathological or toxicological cause of potential difference [31; 45], which is a uncoupled respiration. Dyscoupling may temperature-dependent property of the involve any type of uncoupling mtIM and may be enhanced due to mechanism, e.g., opening the mtPT pore. possible contamination by free fatty acids. Dyscoupled respiration is distinguished Inducible uncoupling mediated by from experimentally induced noncoupled uncoupling protein 1 (UCP1) is respiration in the ET state (Table 2). physiologically controlled, e.g., in brown • Protonophore titration and non- adipose tissue. UCP1 is a member of the coupled respiration: Protonophores are mitochondrial carrier family that is uncouplers which are titrated to obtain involved in the translocation of protons maximum noncoupled respiration as a across the mtIM [64]. Consequently, this measure of ET capacity. short-circuit lowers the pmF and • Loss of compartmental integrity and stimulates electron transfer, respiration, acoupled respiration: Electron transfer and heat dissipation in the absence of and catabolic O2 flux proceed without phosphorylation of ADP. compartmental proton translocation in • Cation cycling: There can be other cation disrupted mitochondrial fragments. Such contributors to leak current including fragments are an artefact of Ca2+ and probably magnesium. Ca2+ influx mitochondrial isolation, and may not fully is balanced by mitochondrial Na+/Ca2+ or fuse to re-establish structurally intact H+/Ca2+ exchange, which is balanced by mitochondria. Loss of mtIM integrity, Na+/H+ or K+/H+ exchanges. This is therefore, is the cause of acoupled another effective uncoupling mechanism respiration, which is a nonvectorial different from proton leak (Table 2). dissipative process without control by the • Proton slip and decoupled respiration: pmF. Proton slip is the decoupled process in which protons are only partially 2.5.2. OXPHOS state (Figure 4b): The translocated by a redox proton pump of OXPHOS state is defined as the respiratory the ET pathways and slip back to the state with kinetically-saturating original vesicular compartment. The concentrations of ADP and Pi proton leak is the dominant contributor (phosphorylation substrates), respiratory to the overall leak current in mammalian fuel substrates and O2, in the absence of mitochondria incubated under exogenous uncoupler, to estimate the physiological conditions at 37 °C, whereas maximal respiratory capacity in the OXPHOS proton slip increases at lower state for any given ET-pathway state. experimental temperature [16]. Proton Respiratory capacities at kinetically- slip can also happen in association with saturating substrate concentrations provide the F1FO-ATPase, in which the proton slips reference values or upper limits of downhill across the pump to the matrix performance, aiming at the generation of without contributing to ATP synthesis. In data sets for comparative purposes. each case, proton slip is a property of the Physiological activities and effects of proton pump and increases with the substrate kinetics can be evaluated relative pump turnover rate. to the OXPHOS capacity. • Electron leak and loosely coupled As discussed previously, 0.2 mM ADP respiration: Superoxide production by does not kinetically-saturate flux in isolated the ETS leads to a bypass of redox proton mitochondria [51; 107]; greater [ADP] is pumps and correspondingly lower P»/O2 required, particularly in permeabilized ratio. This depends on the actual site of muscle fibers and cardiomyocytes, to electron leak and the scavenging of overcome limitations by intracellular

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Figure 4. Respiratory coupling states (a) LEAK state and rate L: Oxidation only, since phosphorylation is arrested, JP» = 0, and catabolic O2 flux JkO2,L is controlled mainly by the proton leak and slip JmH+neg (motive, subscript m), at maximum protonmotive force (Figure 2). ATP may be hydrolyzed by ATPases, JP«; then phosphorylation must be blocked. (b) OXPHOS state and rate P:

Oxidation JkO2,P coupled to phosphorylation JP»,P, which is stimulated by kinetically- saturating [ADP] and [Pi]. A high protonmotive force is maintained by pumping of protons JmH+pos to the positive

compartment. O2 flux JkO2,P is well-coupled -1 at a P»/O2 flux ratio of JP»,P∙JkO2,P . Extramitochondrial ATPases may recycle ATP to ADP, JP«. (c) ET state and rate E: Oxidation only, since phosphorylation is zero, JP» = 0, at optimum exogenous uncoupler concentration when noncoupled

respiration JkO2,E is maximum. The F1FO- ATPase may hydrolyze extramitochondrial ATP translocated into the matrix. Modified from [54]. diffusion and by the reduced conductance of estimate of ET capacity. The ET state is the mtOM [61; 63; 127], either through defined as the noncoupled state with interaction with tubulin [118] or other optimum exogenous uncoupler intracellular structures [5]. In addition, concentration for maximum O2 flux at kinetically-saturating ADP concentrations kinetically-saturating concentrations of need to be evaluated under different respiratory fuel substrates and O2. experimental conditions such as Uncouplers are weak lipid-soluble acids temperature [78] and with different animal which function as protonophores. These models [7]. In permeabilized muscle fiber overcome the mtIM barrier function and bundles of high respiratory capacity, the thus short-circuit the protonmotive system, apparent Km for ADP increases up to 0.5 mM functioning like a clutch in a mechanical [120], consistent with experimental system. As a consequence of the nearly evidence that >90 % kinetic saturation is collapsed protonmotive force, the driving reached only at >5 mM ADP [104]. Similar force is insufficient for phosphorylation, and ADP concentrations are also required for JP» = 0. The most frequently used uncouplers accurate determination of OXPHOS capacity are carbonyl cyanide m-chloro phenyl in human clinical cancer samples and hydrazone (CCCP), carbonyl cyanide p- permeabilized cells [67; 68]. 2.5 to 5 mM trifluoromethoxyphenylhydrazone (FCCP), ADP is sufficient to obtain the actual OXPHOS or dinitrophenol (DNP). Stepwise titrations capacity in many types of permeabilized of uncouplers stimulate respiration up to or tissue and cell preparations, but above the level of O2 consumption rates in experimental validation is required in each the OXPHOS state; respiration is inhibited, specific case. however, above optimum uncoupler concentrations [93]. Data obtained with a 2.5.3. Electron transfer state (Figure 4c): single dose of uncoupler must be evaluated O2 flux determined in the ET state yields an with caution, particularly when a fixed

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Consortium Communication uncoupler concentration is used in studies enzyme activities, availability of specific exploring a treatment or disease that may substrates, O2 concentration, and electron alter the mitochondrial content or leakage leading to the formation of reactive mitochondrial sensitivity to inhibition by oxygen species. uncouplers. There is a need for new protonophoric uncouplers that drive 2.5.5. Quantitative relations: E may exceed maximal respiration across a broad dosing or be equal to P. E > P is observed in many range and do not inhibit respiration at high types of mitochondria, varying between concentrations [66]. The effect on ET species, tissues and cell types [53]. E-P is the capacity of the reversed function of F1FO- ET-excess capacity pushing the ATPase (JP«; Figure 4c) can be evaluated in phosphorylation-flux to the limit of its the presence and absence of capacity for utilizing the pmF (Figure 2). In extramitochondrial ATP, Omy, or Cat. addition, the magnitude of E-P depends on the tightness of respiratory coupling or 2.5.4. ROX state: The state of residual O2 degree of uncoupling, since an increase of L consumption ROX, is not a coupling state, but causes P to increase towards the limit of E is relevant to assess respiratory function [79]. The ET-excess capacity E-P, therefore, (Overview). The rate of residual oxygen provides a sensitive diagnostic indicator of consumption Rox is defined as O2 specific injuries of the phosphorylation consumption due to oxidative reactions pathway, under conditions when E remains measured after inhibition of ET with constant but P declines relative to controls. antimycin A alone, or in combination with Substrate cocktails supporting simultaneous rotenone and malonic acid. Cyanide and convergent electron transfer to the Q- azide not only inhibit CIV, but also catalase junction for reconstitution of TCA cycle and several peroxidases involved in Rox, function establish pathway-control states whereas AOX is not inhibited (Figure 1b). with high ET capacity, and consequently High concentrations of antimycin A, but not increase the sensitivity of the E-P assay. rotenone or cyanide, inhibit peroxisomal Theoretically E cannot be lower than P. acyl-CoA oxidase and D-amino acid oxidase E

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Table 3. Metabolic states of mitochondria (Chance and Williams, 1956; Table V). ADP Substrate Respiration Rate-limiting State [O2] level level rate substance 1 >0 low low slow ADP 2 >0 high ~0 slow substrate 3 >0 high high fast respiratory chain 4 >0 low high slow ADP 5 0 high high 0 oxygen however, are underestimated under these in non-equilibrium states of biochemical conditions [46]. In general, it is energy transformation. State variables inappropriate to use the term ATP (redox states; pmF) and metabolic rates production or ATP turnover for the difference (fluxes) are measured in defined of O2 fluxes measured in the OXPHOS- and mitochondrial respiratory states. Steady LEAK states. P-L is the upper limit of OXPHOS states can be obtained only in open systems, capacity that is freely available for ATP in which changes by internal production (corrected for LEAK respiration) transformations, e.g., O2 consumption, are and is fully coupled to phosphorylation with instantaneously compensated for by a maximum mechanistic stoichiometry external fluxes across the system boundary, (Figure 2). e.g., O2 supply, thus preventing a change of O2 LEAK respiration and OXPHOS capacity concentration in the system [50]. depend on (1) the tightness of coupling Mitochondrial respiratory states monitored under the influence of the respiratory in closed systems satisfy the criteria of uncoupling mechanisms (Figure 3), and (2) pseudo-steady states for limited periods of the coupling stoichiometry, which varies as a time, when changes in the system function of the substrate type undergoing (concentrations of O2, fuel substrates, ADP, oxidation in ET pathways with either two or Pi, H+) do not exert significant effects on three coupling sites (Figure 1b). When metabolic fluxes (respiration, substrate cocktails are used supporting the phosphorylation). Such pseudo-steady states convergent NADH- and succinate pathways require respiratory media with sufficient simultaneously, the relative contribution of buffering capacity and substrates ET pathways with three or two coupling sites maintained at kinetically-saturating cannot be controlled experimentally, is concentrations, and thus depend on the difficult to determine, and may shift in kinetics of the processes under investigation. transitions between LEAK-, OXPHOS- and ET states [54]. Under these experimental 2.6. Classical terminology for isolated conditions, we cannot separate the tightness mitochondria of coupling versus coupling stoichiometry as ‘When a code is familiar enough, it ceases the mechanisms of respiratory control in a appearing like a code; one forgets that shift of L/P ratios. The tightness of coupling there is a decoding mechanism. The and fully coupled O2 flux (P-L; Table 2), message is identical with its meaning’ therefore, are obtained from measurements (Hofstadter 1979) [60]. of coupling control of LEAK respiration, OXPHOS- and ET capacities in well-defined Chance and Williams [20; 21] introduced the pathway states, using either pyruvate and five classical mitochondrial respiratory and malate as substrates or the classical cytochrome redox states. Table 3 shows a succinate and rotenone substrate-inhibitor protocol with isolated mitochondria in a combination (Figure 1b). closed respirometric chamber, defining a sequence of respiratory states. States and 2.5.6. The steady state: Mitochondria rates are not distinguished in this represent a thermodynamically open system nomenclature.

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2.6.1. State 1 is obtained after addition of isolated mitochondria. The abbreviation isolated mitochondria to air-saturated ‘State 3u’ is occasionally used in isoosmotic/isotonic respiration medium bioenergetics, to indicate the state of containing Pi, but no mitochondrial fuel respiration after titration of an uncoupler, substrates and no adenylates. without sufficient emphasis on the fundamental difference between OXPHOS 2.6.2. State 2 is induced by addition of a capacity (well-coupled with an endogenous ‘high’ concentration of ADP (typically 100 to uncoupled component) and ET capacity 300 µM), which stimulates respiration (noncoupled). transiently on the basis of endogenous fuel substrates and phosphorylates only a small 2.6.4. State 4 is a LEAK state that is obtained portion of the added ADP. State 2 is then only if the mitochondrial preparation is obtained at a low respiratory activity limited intact and well-coupled. Depletion of ADP by by exhausted endogenous fuel substrate phosphorylation to ATP causes a decline of availability (Table 3). If addition of specific O2 flux in the transition from State 3 to State inhibitors of respiratory complexes such as 4. Under the conditions of State 4, a rotenone does not cause a further decline of maximum protonmotive force and high O2 flux, State 2 is equivalent to the ROX state ATP/ADP ratio are maintained. The gradual

(Table 1). Undefined endogenous fuel decline of YP»/O2 towards diminishing [ADP] substrates are a confounding factor of at State 4 must be taken into account for pathway control, contributing to the effect of calculation of P»/O2 ratios [51]. State 4 subsequently added external substrates and respiration L(T) (Table 1), reflects intrinsic inhibitors. In an alternative sequence of proton leak and ATP hydrolysis activity. O2 titration steps, the second state (not flux in State 4 is an overestimation of LEAK introduced as State 2) is induced by addition respiration if any contaminating ATP of fuel substrate without ADP or ATP [20, hydrolysis activity, JP«, recycles some ATP to 39]. In contrast to the original State 2 defined ADP and thus stimulates respiration coupled in Table 1 as a ROX state, the alternative to phosphorylation, JP» > 0. Some degree of ‘State 2’ is a LEAK state, L(n). Some mechanical disruption and loss of researchers have called this condition as mitochondrial integrity allows the exposed ‘pseudostate 4’. mitochondrial F1FO-ATPases to hydrolyze the ATP synthesized by the fraction of 2.6.3. State 3 is the state stimulated by coupled mitochondria. This can be tested by addition of fuel substrates while the ADP inhibition of the phosphorylation pathway concentration in the original State 2 is still using oligomycin, ensuring that JP» = 0 (State high (Table 3) and supports coupled energy 4o). On the other hand, the State 4 transformation. 'High ADP' is a respiration reached after exhaustion of concentration of ADP specifically selected to added ADP is a more physiological condition, allow the measurement of State 3 to State 4 i.e., presence of ATP, ADP and even AMP. transitions of isolated mitochondria in a Sequential ADP titrations re-establish State closed respirometric chamber. Repeated 3, followed by State 3 to State 4 transitions ADP titration re-establishes State 3 at ‘high while sufficient O2 is available. Anoxia may ADP’. Starting at O2 concentrations near air- be reached, however, before exhaustion of saturation (193 or 238 µM O2 at 37 °C or 25 ADP (State 5). °C and sea level at 1 atm or 101.32 kPa, and an O2 solubility of respiration medium at 2.6.5. State 5 ‘may be obtained by antimycin 0.92 times that of pure water [44]), the total A treatment or by anaerobiosis’ [20]. These ADP concentration added must be low definitions give State 5 two different enough (typically 100 to 300 µM) to allow meanings: ROX or anoxia. Anoxia is obtained phosphorylation to ATP at a coupled O2 flux after exhaustion of O2 in a closed that does not lead to O2 depletion during the respirometric chamber. Diffusion of O2 from transition to State 4. In contrast, kinetically- the surroundings into the aqueous solution saturating ADP concentrations usually are may be a confounding factor preventing 10-fold higher than 'high ADP', e.g., 2.5 mM in complete anoxia [51].

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In Table 3, only States 3 and 4 are exercise and nutrition; and environmental coupling-control states, with the restriction issues including thermal, atmospheric, toxic that rates in State 3 may be limited and pharmacological factors, exert an kinetically by non-saturating ADP influence on all control mechanisms listed concentrations. above. For reviews, see [10; 49; 53; 54; 97; 103]. 2.7. Control and regulation Lack of control by a metabolic pathway, e.g., phosphorylation pathway, means that The terms metabolic control and regulation there will be no response to a variable are frequently used synonymously, but are activating it, e.g., [ADP]. The reverse, distinguished in metabolic control analysis: however, is not true as the absence of a ‘We could understand the regulation as the response to [ADP] does not exclude the mechanism that occurs when a system phosphorylation pathway from having some maintains some variable constant over time, degree of control. The degree of control of a in spite of fluctuations in external conditions component of the OXPHOS pathway on an (homeostasis of the internal state). On the output variable, such as O2 flux, will in other hand, metabolic control is the power to general be different from the degree of change the state of the metabolism in control on other outputs, such as response to an external signal’ [43]. phosphorylation-flux or proton leak flux. Respiratory control may be induced by Therefore, it is necessary to be specific as to experimental control signals that exert an which input and output are under influence on: (1) ATP demand and ADP consideration [43]. phosphorylation-rate; (2) fuel substrate Respiratory control refers to the ability of composition, pathway competition; (3) mitochondria to adjust O2 flux in response to available amounts of substrates and O2, e.g., external control signals by engaging various starvation and hypoxia; (4) the mechanisms of control and regulation. protonmotive force, redox states, flux–force Respiratory control is monitored in a relationships, coupling and efficiency; (5) mitochondrial preparation under conditions Ca2+ and other ions including H+; (6) defined as respiratory states, preferentially inhibitors, e.g., nitric oxide or intermediary under near-physiological conditions of metabolites such as oxaloacetate; (7) temperature, pH, and medium ionic signalling pathways and regulatory proteins, composition, to generate data of higher e.g., insulin resistance, transcription factor biological relevance. When phosphorylation hypoxia inducible factor 1. of ADP to ATP is stimulated or depressed, an Mechanisms of respiratory control and increase or decrease is observed in electron regulation include adjustments of: (1) transfer measured as O2 flux in respiratory enzyme activities by allosteric mechanisms coupling states of intact mitochondria and phosphorylation; (2) enzyme content, (‘controlled states’ in the classical concentrations of cofactors and conserved terminology of bioenergetics). Alternatively, moieties such as adenylates, nicotinamide coupling of electron transfer with adenine dinucleotide [NAD+/NADH], phosphorylation is diminished by coenzyme Q, cytochrome c; (3) metabolic uncouplers. The corresponding coupling- channeling by supercomplexes; and (4) control state is characterized by a high mitochondrial density and morphology respiratory rate without control by P» (membrane area, cristae folding, fission and (noncoupled or ‘uncontrolled state’). fusion). Mitochondria are targeted directly by hormones, e.g., progesterone and 3. What is a rate? glucocorticoids, which affect their energy metabolism [48; 74; 96; 106; 128]. ‘Before stating the result of a measurement, it Evolutionary or acquired differences in the is essential that the quantity being presented genetic and epigenetic basis of is adequately described‘ [11]. The term rate is mitochondrial function (or dysfunction) not adequately defined to be useful for between individuals; age; biological sex, and reporting data. Normalization of rates leads hormone concentrations; life style including to a diversity of formats expressed in various

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Consortium Communication units. The second [s] is the SI unit for the • Intensive quantities: In contrast to size- base quantity time. It is also the standard specific properties, forces are intensive time-unit used in solution chemical kinetics quantities defined as the change of an (catalytic activity, unit catal [kat = mol∙s-1]). extensive quantity per advancement of an The inconsistency of the meanings of rate energy transformation [50]. becomes apparent when considering Galileo • Formats: Mass mX can be measured on Galilei’s famous principle, that ‘bodies of samples of any type of X, but a number of different weight all fall at the same rate (have objects NX and a molar amount nB can be a constant acceleration)’ [26]. A rate may be defined in samples of countable objects an extensive quantity [24], which is a flow I, only. The molar format is preferred for when expressed per single object (per metabolites including O2. As of 2019 May elementary entity) or per experimental 20, the definition of the SI unit mole [mol] chamber (system). ‘System’ is defined as the is based on a natural constant, namely the open or closed experimental chamber in the Avogadro constant: one mole contains analytical instrument including a sample s. exactly 6.02214076∙1023 elementary Alternatively, a rate is a flux J, when entities, in contrast to the former expressed as a size-specific quantity [50] definition in terms of the number of (Figure 5a; Box 2). Importantly, a rate can molaratoms in the mass of 0.012 kilogram be a nondimensional flux control ratio FCR. of carbon 12 [11]. Metabolic O2 flow and

• Extensive quantities: An extensive flux are expressed in molar units [mol] in quantity increases proportionally with biochemistry, but as volume [L] in the size of the object or a system. For ergometry. When necessary, these example, mass and volume of a sample or formats can be distinguished as JnO2/m and system are extensive quantities. Flow is JVO2/m, respectively, indicating the an extensive quantity. The magnitude of different formats of O2 in subscripts (n, V) an extensive quantity is completely with the symbols of the quantities in additive for non-interacting subsystems underlined italic font. In many cases it is [24]. more practical, however, to use simpler • Size-specific quantities: ‘The adjective symbols and provide the required specific before the name of an extensive definitions in the text and explicitly quantity is often used to mean divided by written units (Table 4 and Figure 5). mass’ [24]. The term specific, however, has different meanings in three particular Box 2: Metabolic flows and fluxes: contexts: (1) In the system-paradigm, (a) vectoral, vectorial, and scalar mass-specific flux is flow divided by mass of the system (the mass of the entire Flow is an extensive quantity (I; of the contents in the experimental chamber or system), distinguished from the size-specific reactor). (b) Rates are frequently quantity flux (J; per system size). Flows Itr are expressed as volume-specific flux (liquid defined for transformations tr as extensive volume of the experimental chamber). A quantities. This is a generalization derived mass-specific or volume-specific quantity from electrical terms: Electric charge per is independent of the extent of non- unit time is electric flow or current, Iel = interacting homogenous subsystems. (2) dQel∙dt-1 [A≡ C∙s•1]. When dividing Iel by size In the context of sample size, tissue- of the system (cross-sectional area of a specific quantities are related to the mass ‘wire’), we obtain flux as a size-specific or volume of the sample in contrast to the quantity; this is the current density (surface- mass or volume of the system (e.g., muscle density of flow) perpendicular to the mass-specific or cell volume-specific direction of flux, Jel = Iel∙A-1 [A∙m•2] [24]. normalization; Figure 5). (3) An entirely Fluxes with spatial geometric direction and different meaning of ‘specific’ is implied magnitude are vectors. Vector and scalar in the context of sample type, e.g., muscle- fluxes are related to flows as Jtr = Itr∙A-1 specific compared to brain-specific [mol∙s•1∙m•2] and Jtr = Itr∙V-1 [mol∙s•1∙m•3], properties. expressing flux as an area-specific vector or volume-specific vectorial or scalar quantity,

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Figure 5. Normalization of rate. (a) Normalization forms: Left (physiological): Rate can be expressed as

extensive flow IO2/X, if the sample of X can be quantified as a count NX (X: cell, organism). Rate is a size-specific flux, JO2/mX or JO2/VX, when expressed per mass or volume of sample of X in the chamber, mX or VX. Normalization per mitochondrial elementary marker mtE relies on determination of mtE expressed in a mt- elementary unit [mtEU]. A reference rate can be defined as an internal functional mtE, to obtain nondimensional flux control ratios that are independent of sample quantification and even chamber volume. Right (methodological): Flow in

experimental chamber IO2, or flux per chamber volume JV,O2. (b) O2 flow per cell IO2/ce: using CS activity as mtE, IO2/ce is the product of mt- -1 specific flux JO2/CS, mt-density DCS=CS∙mce , -1 and mass per cell, MUce=mce∙Nce . Then performance is the product of mass-specific •1 •1 flux ( JO2/mce=JO2/CS∙Dce [mol∙s ∙kg ]) and size -1 (mass per cell MUce [kg∙x ]), or the product of mitochondrial function (mt-specific flux

JO2/CS) and structure (CS per cell, CSUce =CS∙Nce-1). Modified from [54]. See Tab. 4. respectively [50]. We use the meter– normalization. Normalization is guided by kilogram–second–ampere (MKSA) physicochemical principles, methodological International System of Units (SI) for general considerations, and conceptual strategies cases ([m], [kg], [s] and [A]), with decimal SI (Table 4). Normalization per sample prefixes and [L = dm3] for specific concentration is routinely required to report applications (Table 4). respiratory data (Figure 5). We suggest defining: (1) vectoral fluxes, which are translocations as functions of 4.1. Flow: per object gradients with direction in geometric space in continuous systems; (2) vectorial fluxes, 4.1.1. Count concentration CX: The count which describe translocations in concentration of objects X is CX. 'Count' NX is discontinuous systems and are restricted to defined as the 'number of elementary information on compartmental differences entities' [11]. In the case of animals, NX is the (transmembrane proton flux); and (3) scalar number of organisms with concentration CX fluxes, which are localized transformations = NX∙V-1 [x∙L-1]. Similarly, the number of cells without translocation, such as chemical per chamber volume is the cell reactions or reaction sequences in a concentration, where the cell count Nce is the homogenous system (catabolic O2 flux JkO2). number of cells in the chamber (Table 4).

4. Normalization of rate per sample 4.1.2. Flow per single object IO2/X: O2 flow per cell is calculated from volume-specific O2 •1 •1 The challenges of measuring mitochondrial flux JV,O2 [nmol∙s ∙L ] (per V [L] of the respiratory rate are matched by those of experimental system), divided by the

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Table 4. Sample concentrations and normalization of flux. SI refers to ref. [11].

Expression Symbol Definition Unit Notes Sample X sample- or entity-type 1

elementary entity of X; unit X UX single countable object x SI; 1, 2

count of X; number of UX NX NX = N∙UX x SI; 2

mass of sample (entity-type X) mX kg SI; 3 -1 •1 mass per X MUX MUX = mX∙NX kg∙x 1, 3 Mitochondria mt mt-elementary entity mtE mtEU Concentration and density

molar concentration of X or B cX, cB cB = nB∙V-1 mol∙L•1 SI; 2

count concentration of X CX CX = NX∙V-1 x∙L•1 SI; 4 -1 •1 mass concentration of sample CmX CmX = mX∙V kg∙L 4

mitochondrial concentration CmtE CmtE = mtE∙V-1 mtEU∙L•1 5 -1 •1 mitochondrial density per VX DmtE/VX DmtE/VX = mtE∙VX mtEU∙L 6 -1 •1 mitochondrial density per mX DmtE/mX DmtE/mX = mtE∙mX mtEU∙kg 6 -1 •1 mitochondrial content per UX mtEUX mtEUX = mtE∙NX mtEU∙x 7

O2 flow and flux 8 -1 flow, system IO2 internal flow mol∙s 9 -1 -1 -1 volume-specific flux JV,O2 JV,O2 = IO2∙V mol∙s ∙L 1, 10 -1 -1 -1 flow per X IO2/X IO2/X = JV,O2∙CX mol∙s ∙x 11 -1 -1 -1 sample mass-specific flux JO2/mX JO2/mX = JV,O2∙CmX mol∙s ∙kg 1 -1 -1 -1 mt-marker-specific flux JO2/mtE JO2/mtE = JV,O2∙CmtE mol∙s ∙mtEU 12 1 A sample or subsample is one or more parts taken from a study group. A sample of X may contain countable objects. Quantities (NX, mX, VX) that relate to a sample in a mixture are distinguished from quantities for the thermodynamic system (m or V; mass or volume of the total contents in the experimental chamber). See Table 5 for entity-types X. 2 Entity-type X is indicated by a subscript (Nce; mce; NB; VO2). The elementary entity UX is count NX divided by the pure number N. The elementary unit [x] as the unit of count is not in the SI [95]. NX [x] and nX [mol] are count and amount of entity-type X; mX and VX are count-independent mass and volume of sample-type X, not quantities per UX (Table 5). 3 Units are given in the MKSA system and 1 L=10-3 m3 (Box 2). The prefix k is used for the SI base unit of mass (1 kg = 1000 g). Various SI prefixes are used to make numbers easily readable, e.g., 1 mg tissue, cell or mitochondrial mass -1 -1 instead of 0.000 001 kg. The units [kg∙x ] and [kg] distinguish mass per object, MUX [kg∙x ] (per single cell Uce) from the mass mce [kg] of a sample of cells that contains any number of cells. For MUX or M(UX), the SI uses m(X). -1 -1 4 IUPAC [24] term: ‘number concentration’ for CB. For X = ce, the cell-count concentration is Cce = Nce∙V . CmX = mX∙V for -1 -1 sample of X in a mixture. The SI quantity mass density ρ relates to a pure sample S, ρ = mS∙VS (≈1 kg∙L wet biomass). 5 mt-concentration is the experimental variable of sample concentration in the chamber: (1) CmtE = mtE∙V•1; (2) CmtE = mtEUX∙CX; (3) CmtE = DmtE/mX∙CmX; (4) CmtE = DmtE/VX∙CVX 6 For mtE expressed as mt-volume, DmtE/VX is the mt-volume fraction Φ in sample X(wet). VX ≈ mX, hence DmtE/VX ≈ DmtE/mX. -1 -1 7 mt-content mtEUce per single cell equals mtE per cell count Nce, mtEUce = mtE∙Nce = CmtE∙Cce . 8 O2 can be replaced by other chemicals to study different reactions, e.g., ATP, H2O2, or vesicular compartmental translocations, e.g., Ca2+. 9 IO2 and V are defined for the experimental system of constant volume (and typically constant temperature), which may

be closed or open (Figure 5). IO2 is abbreviated for IrO2, i.e., the metabolic internal O2 flow of the chemical reaction -1 sequence r in the sample by which O2 is consumed, with negative stoichiometric number νO2 = •1: IrO2 = drnO2/dt∙νO2 .

If r includes all reactions in which O2 participates, then drnO2 = dnO2 – denO2, where dnO2 is the change in the amount of O2 in the experimental chamber and denO2 is the amount of O2 added externally to the system. At steady state in oxystat mode, by definition dnO2 = 0, hence drnO2 = –denO2. Note that in this context ‘external’, e, refers to the experimental system, whereas in respiratory physiology ‘external’, ext, refers to the organism (See Overview). 10 Experimental quanity JV,O2, expressed per volume V of the experimental chamber, equal to VS for pure samples S only. -1 -1 11 Flow IO2/X per elementary entity UX [mol∙s ∙x ] is a physiological variable normalized for count. It depends on the size -1 (MUX, VUX) of UX. Flow IO2 [mol∙s ] (experimental system) is an experimental variable (Note 9; Figure 5). 12 There are many ways to normalize for a mitochondrial marker, that are used in different experimental approaches: -1 -1 -1 •1 •1 •1 -1 (1) JO2/mtE = JV,O2∙CmtE ; (2) JO2/mtE = JV,O2∙CmX ∙DmtE/mX = JO2/mX∙DmtE/mX ; (3) JO2/mtE = JV,O2∙CX ∙mtEUX = IO2/X∙mtEUX ; (4) -1 JO2/mtE = IO2∙mtE . The mt-elementary unit [mtEU] varies depending on the mt-marker mtE.

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Table 5. Sample preparations and elementary entities, count, mass, and volume

a b b Identity of sample or entity X Unit X Count Mass MUX Volume VUX mitochondrial preparation UX [x] NX [x] mX [kg] [kg∙x-1] VX [L] [L∙x-1]

isolated mitochondria imt Uimt Nimt mimt MUimt Vimt VUimt tissue homogenate thom mthom permeabilized tissue pti mpti permeabilized muscle fibers pfi mpfi permeabilized cells pce Upce Npce mpce MUpce Vpce VUpce c living cells ce Uce Nce mce MUce Vce VUce viable cells vce Uvce Nvce mvce MUvce Vvce VUvce dead cells dce Udce Ndce mdce MUdce Vdce VUdce organisms org Uorg Norg morg MUorg Vorg VUorg d molecules B UB NB mB MUB VB VUB a A sample of X may consist of elementary entities UX, which are countable objects identified as the defining units UX. b -1 -1 -1 -1 mX [kg] and VX [L] are mass and volume of the sample of X; MUX = mX∙NX [kg∙x ] and VUX = VX∙NX [L∙x ] are quantities per elementary entity UX (Table 4). Wet mass mw, dry mass md, or ash-free dry mass maf, have to be specified [56]. c Total cell count in a living cell population, which consists of viable and dead cells, Nce = Nvce + Ndce, without experimental permeabilization of the plasma membrane. Living cells have been called ‘intact’ cells in contrast to permeabilized cells; but injured cells have lost the property of being intact, yet may still be living. d IUPAC uses for MUB the term ‘mass of entity (molecule, formula unit)’ with symbol mf [24], but it should be ‘mass per -1 -1 entity’, MUB=mB∙NB . VB is the volume of molecules B, and the molecular volume is VUB=VB∙NB (compare molar volume). count concentration of cells, Cce = Nce∙V-1. The 4.2.2. Size-specific flux: Cellular O2 flow total cell count is the sum of viable and dead can be compared between cells of identical cells, Nce = Nvce+Ndce (Table 5). The cell size. To take into account changes and viability index, VIce = Nvce∙Nce•1, is the ratio of differences in cell size, normalization is the number of viable cells Nvce, per count of required to obtain cell size-specific or all cells in the population. After experimental mitochondrial marker-specific O2 flux [113] permeabilization, all cells are permeabilized, (Figure 5).

Npce = Nce. The cell viability index can be used • Sample mass-specific flux J to normalize respiration for the number of O2/mX [mol∙s•1∙kg•1]: Sample mass-specific flux is cells that have been viable before the expression of respiration per mass m experimental permeabilization, I = X O2/vce of a sample of X [mg]. Divide chamber •1 IO2/ce∙VI , considering that mitochondrial volume-specific flux JV,O by mass respiratory dysfunction in dead cells might 2 concentration of sample in the chamber, be a confounding factor. J = J ∙C -1. Cell mass-specific flux is O2/mX V,O2 mX obtained by dividing flow per unit cell by 4.2. Size-specific flux: per sample size mass per unit cell, J = I ∙M -1. O2/mX O2/X UX •1 • Cell volume-specific flux JO2/VX 4.2.1. Mass concentration CmX [kg∙L ]: •1 •1 Considering permeabilized tissue, [mol∙s ∙L ]: Sample volume-specific flux homogenate or cells as the sample of X, the is obtained by expressing respiration per volume of sample. sample mass mX [mg] is frequently measured as wet or dry mass, mw or md [mg], If size-specific O2 flux is constant and respectively, or as mass of protein mProtein. independent of mX or VX, then there is no The sample-mass concentration is the mass interaction between the subsystems. For of the (sub)sample per volume of the example, 1.5 mg and 3.0 mg sub-samples of -1 -1 experimental system, CmX = mX∙V [g∙L = muscle tissue respire at identical mass- mg∙mL•1]. Sample-types X are isolated specific flux. If mass-specific O2 flux, mitochondria, tissue homogenate, however, changes as a function of the mass of permeabilized muscle fibers or cells (Table a tissue sample, cells or isolated 4). mce [mg] is the total mass of all cells X=ce mitochondria in the experimental chamber, in the experimental system, whereas MUce = then the nature of the interaction becomes mce∙Nce-1 [mg∙x-1] is the average mass per an issue. Therefore, cell concentration must single or unit cell Uce (Table 5 and Figure 5). be optimized, particularly in experiments

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Consortium Communication carried out in wells, considering the the stock suspension of isolated confluency of the cell monolayer or clumps of mitochondria, CmtE,stock, and crude tissue cells [121]. homogenate, CmtE,thom, which together The complexity changes when provide information on the mitochondrial considering the scaling law of respiratory density DmtE in the sample (Table 4). physiology. Strong interactions are revealed When discussing concepts of between O2 flow and body mass M of an normalization, it is essential to consider the individual organism: basal metabolic rate question posed by the study. If the study (flow) does not increase linearly with body aims at comparing tissue performance— mass. Maximum mass-specific O2 flux, 푉̇O2max such as the effects of a treatment on a specific or 푉̇O2peak, depends less strongly on tissue, then normalization for tissue mass or individual body mass compared to basal protein content is appropriate. However, if metabolic flux [139]. Individuals, breeds and the aim is to find differences in species deviate substantially from the mitochondrial function independent of mitochondrial density (Table 4), then common scaling relationship. 푉̇O2peak of human endurance athletes is 60 to 80 mL normalization to a mitochondrial marker is imperative (Figure 5). One cannot assume O2·min•1·kg•1 body mass, converted to that quantitative changes in various JO peak/M = IO peak/org∙M-1 of 45 to 60 nmol·s•1·g•1 2 2 markers—such as mitochondrial proteins— [54] (Table 6). necessarily occur in parallel with one

another. It should be established that the 4.3. Marker-specific flux: per mitochondrial marker chosen is not selectively altered by content the performed treatment. In conclusion, the

normalization must reflect the question Tissues can contain multiple cell populations under investigation to reach a satisfying that may have distinct mitochondrial answer. On the other hand, the goal of subtypes. Mitochondria undergo dynamic comparing results across projects and fission and fusion cycles, and can exist in institutions requires standardization on multiple stages and sizes that may be altered normalization for entry into a databank. by a range of factors. The isolation of mitochondria (often achieved through 4.3.1. Mitochondrial concentration C differential centrifugation) can therefore mtE and mitochondrial density D : yield a subsample of the mitochondrial types mtE Mitochondrial organelles compose a present in a tissue, depending on the dynamic cellular reticulum in various states isolation protocols utilized. This possible of fusion and fission. Hence, the definition of bias should be taken into account when a ‘number’ of mitochondria is often planning experiments using isolated misconceived: mitochondria cannot be mitochondria. Different sizes of counted reliably as a number of occurring mitochondria are enriched at specific elementary particles. Therefore, centrifugation speeds, which can be used quantification of mitochondrial strategically for isolation of mitochondrial concentration and density depends on the subpopulations. measurement of chosen mitochondrial Part of the mitochondrial content of a markers. ‘Mitochondria are the structural tissue is lost during preparation of isolated and functional elementary units of cell mitochondria. The fraction of isolated respiration’ [54]. The quantity of a mitochondria obtained from a tissue sample mitochondrial marker can reflect the total of is expressed as mitochondrial recovery. At a mitochondrial elementary entities mtE, high mitochondrial recovery, the fraction of expressed in various mitochondrial isolated mitochondria is more elementary units [mtEU] specific for each representative of the total mitochondrial measured mt-marker (Table 4). However, population than in preparations since mitochondrial quality may change in characterized by low recovery. response to stimuli—particularly in Determination of the mitochondrial recovery mitochondrial dysfunction [15], exercise and yield is based on measurement of the training [105], and aging [29]—some concentration of a mitochondrial marker in

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BEC 2020.1 doi:10.26124/bec:2020-0001.v1 markers can vary while others are (Figure 5). Obviously, the numerical results unchanged: (1) Mitochondrial volume and and variability of JO2/mtE vary with the type of membrane area are structural markers, mitochondrial marker chosen for whereas mitochondrial protein mass is measurement of mtE and CmtE = mtE∙V-1 commonly used as a marker for isolated [mtEU∙L-1]. Different methods for the mitochondria. (2) Molecular and enzymatic quantification of mitochondrial markers mitochondrial markers (amounts or have different strengths and weaknesses. activities) can be selected as matrix markers, Some problems are common for all e.g., citrate synthase activity, mtDNA; mtIM- mitochondrial markers mtE: markers, e.g., cytochrome c oxidase activity, (1) Accuracy of measurement is crucial, aa3 content, cardiolipin; or mtOM-markers, since even a highly accurate and e.g., the voltage-dependent anion channel reproducible measurement of chamber (VDAC), TOM20. (3) Extending the volume-specific O2 flux results in an measurement of mitochondrial marker inaccurate and noisy expression if enzyme activity to mitochondrial pathway normalized by a biased and noisy capacity, ET- or OXPHOS capacity can be measurement of a mitochondrial marker. considered as an integrative functional This problem is acute in mitochondrial mitochondrial marker. respiration because the denominators used Depending on the type of mitochondrial (the mitochondrial markers) are often small marker, the mitochondrial elementary entity moieties of which accurate and precise mtE is expressed in marker-specific units. determination is difficult. In contrast, an Mitochondrial concentration CmtE in the internal marker is used when O2 fluxes experimental chamber and mitochondrial measured in substrate-uncoupler-inhibitor density DmtE in the tissue of origin are titration protocols are normalized for flux in quantified as (1) the quantity CmtE for a defined respiratory reference state within normalization in functional analyses, and (2) the assay, which yields flux control ratios the physiological output DmtE that is the FCR. FCRs are independent of externally result of mitochondrial biogenesis and measured markers and, therefore, are degradation, respectively (Table 4). It is statistically robust, considering the recommended, therefore, to distinguish limitations of ratios in general [62]. FCRs experimental mitochondrial concentration indicate qualitative changes of CmtE in the chamber, and physiological mitochondrial respiratory control, with mitochondrial density DmtE in the biological highest quantitative resolution, separating sample. The biological variable DmtE is the effect of mitochondrial density on JO2/mX expressed as mitochondrial elementary and IO2/X from that of function per entities per volume VX of cells or mass mX of mitochondrial elementary marker, JO2/mtE the sample of X (Figure 5). The experimental [54; 105]. mt-concentration, CmtE = mtE∙V-1 in the (2) If mitochondrial quality does not chamber volume V, is the product of mt- change and only the amount of mitochondria -1 density per mass, DmtE/mX = mtE∙mX , times varies as a determinant of mass-specific flux, -1 sample mass concentration, CmX = mX∙V ; or any marker is equally qualified in principle; -1 CmtE is mt-content, mtEUce = mtE∙Nce per cell, then in practice selection of the optimum multiplied by cell-count concentration, Cce = marker depends only on the accuracy and Nce∙V-1 in the chamber (Table 4). precision of measurement of the mitochondrial marker.

4.3.2. mt-Marker-specific flux JO2/mtE: (3) If mitochondrial flux control ratios Volume-specific metabolic O2 flux depends change, then there may not be any best on: (1) the sample concentration in the mitochondrial marker. In general, volume of the experimental chamber, CmX or measurement of multiple mitochondrial CX; (2) the mitochondrial density DmtE/VX = markers enables a comparison and -1 -1 evaluation of normalization for these mtE∙VX or content mtEUX = mtE∙NX ; and (3) the specific mitochondrial activity or mitochondrial markers. Particularly during performance per mitochondrial elementary postnatal development, the activity of -1 •1 -1 marker enzymes—such as cytochrome c marker, JO2/mtE = JV,O2∙CmtE [mol∙s ∙mtEU ]

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Consortium Communication oxidase and citrate synthase—follows various mitochondrial markers and citrate different time courses [34]. Evaluation of synthase activity [8; 94; 112] are expected in mitochondrial markers in healthy controls is a specific tissue of healthy persons and in insufficient for providing guidelines for disease states not specifically targeting application in the diagnosis of pathological citrate synthase. Citrate synthase activity is states and specific treatments [125]. acutely modifiable by exercise [76; 132]. In line with the concept of the respiratory Evaluation of mitochondrial markers related acceptor control ratio RCR [19], the most to a selected age and sex cohort cannot be readily applied normalization is that of flux extrapolated to provide recommendations control ratios and flux control factors [53; for normalization in respirometric diagnosis 54]. Then, instead of a specific mt-enzyme of disease, in different states of development activity, the respiratory rate in a reference and aging, different cell types, tissues, and state serves as the mtE, yielding a species. mtDNA normalized to nDNA via nondimensional ratio of two fluxes qPCR is correlated to functional measured consecutively in the same mitochondrial markers including OXPHOS- respirometric titration protocol. Selection of and ET capacity in some cases [8; 36; 88; the state of maximum flux in a protocol as the 108; 137], but lack of such correlations have reference state ― e.g., ET state in L/E and P/E been reported [90; 105; 126]. Several studies flux control ratios [53] ― has the advantages indicate a strong correlation between of: (1) elimination of experimental cardiolipin content and increase in variability in additional measurements, such mitochondrial function with exercise [40; as determination of enzyme activity or tissue 73; 89; 90], but it has not been evaluated as a mass; (2) statistically validated linearization general mitochondrial biomarker in disease. of the response in the range of 0 to 1; and (3) With no single best mitochondrial marker, a consideration of maximum flux for good strategy is to quantify several different integrating a large number of metabolic biomarkers to minimize the decorrelating steps in the OXPHOS- or ET pathways. This effects caused by diseases, treatments, or reduces the risk of selecting a functional other factors. Determination of multiple marker that is specifically altered by the markers, particularly a matrix marker and a treatment or pathology, yet increases the marker from the mtIM, allows tracking chance that the highly integrative pathway is changes in mitochondrial quality defined by disproportionately affected, e.g., the their ratio. OXPHOS- rather than ET pathway in case of an enzymatic defect in the phosphorylation 5. Normalization of rate per system pathway. In this case, additional information can be obtained by reporting flux control 5.1. Flow: per chamber ratios based on a reference state that indicates stable tissue mass-specific flux The experimental system (chamber) is part [125]. of the measurement instrument, separated Stereological measurement of from the environment as a closed, open, mitochondrial content via two-dimensional isothermal or non-isothermal system transmission electron microscopy is (Table 4). Reporting O2 flows per -1 considered as the gold standard in respiratory chamber, IO2 [nmol∙s ], restricts determination of mitochondrial volume the analysis to intra-experimental fractions in cells and tissues [139]. Accurate comparison of relative differences. determination of three-dimensional volume by two-dimensional microscopy, however, is 5.2. Flux: per chamber volume both time consuming and statistically challenging [73]. The validity of using 5.2.1. System-specific flux JV,O2: We mitochondrial marker enzymes (citrate distinguish between (1) the system with synthase activity, CI to CIV amount or volume V and mass m defined by the system activity) for normalization of flux is limited boundaries and its total contents, and (2) the in part by the same factors that apply to flux sample of X with volume VX and mass mX control ratios. Strong correlations between enclosed in the experimental chamber

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Table 6. Conversion of various formats and units used in respirometry and ergometry to SI units (International System of Units [11]). zB is the charge number, which is the number of electrons e- or reducing equivalents Ne per elementary entity UB.

Format 1 Unit ∙ Multiplication factor SI-unit Notes

n ng.atom O∙s-1 (zO = 2) 0.5 nmol O2∙s•1

n ng.atom O∙min-1 (zO = 2) 8.333 pmol O2∙s•1

n natom O∙min-1 (zO = 2) 8.333 pmol O2∙s•1 -1 •1 n nmol O2∙min (zO2 = 4) 16.67 pmol O2∙s -1 •1 n nmol O2∙h (zO2 = 4) 0.2778 pmol O2∙s

V to n mL O2∙min-1 at STPD 0.7443 µmol O2∙s•1 1

e to n W = J∙s-1 at -470 kJ∙mol-1 O2 -2.128 µmol O2∙s•1

e to n mA = mC∙s-1 (zH+ = 1) 10.36 nmol H+∙s•1 2 -1 •1 e to n mA = mC∙s (zO2 = 4) 2.591 nmol O2∙s 2

n to e nmol H+∙s•1 (zH+ = 1) 0.09649 mA 3 •1 n to e nmol O2∙s (zO2 = 4) 0.3859 mA 3 1 At standard temperature and pressure dry (STPD: 0 °C = 273.15 K and 1 atm = 101.325

kPa = 760 mmHg), the molar volume of an ideal gas Vm is 22.414 and Vm,O2 is 22.392 L∙mol-1. Rounded to three decimal places, both values yield the conversion factor of

0.744. For comparison at normal temperature and pressure dry (NTPD: 20 °C), Vm,O2 is 24.038 L∙mol-1. Note that the SI standard pressure is 100 kPa. 2 The multiplication factor is 106/(zB∙F). 3 The multiplication factor is zB∙F/106.

(Figure 5). O2 flow per object, IO2/X, is the 5.2.2. Advancement per volume: When the total O2 flow in the system divided by the reactor volume does not change during the number NX of objects in the system. IO2/X reaction, which is typical for liquid phase increases as the mass MUX per object X is reactions, the volume-specific flux of a increased. Sample mass-specific O2 flux, chemical reaction r is the time derivative of

JO2/ms, should be independent of the mass- the advancement of the reaction per unit concentration of the subsample obtained volume, JV,rB = drξB/dt∙V-1 [(mol∙s•1)∙L•1]. The from the same tissue or cell culture, but rate of concentration change is dcB/dt [(mol∙L•1)∙s•1], where concentration is cB = system volume-specific O2 flux JV,O2 (per -1 liquid volume of the experimental chamber) nB∙V . There is a difference between (1) JV,rO2 increases in proportion to the mass of the [mol∙s•1∙L•1] and (2) rate of concentration change [mol∙L•1∙s•1]. These merge into a sample in the chamber. Although JV,O2 depends on mass-concentration of the single expression only in closed systems. In sample in the chamber, it should be open systems, internal transformations independent of the chamber (system) (catabolic flux, O2 consumption) are volume at constant sample mass- distinguished from external flux (such as O2 concentration. There are practical supply). External fluxes of all substances are limitations to increasing the mass- zero in closed systems. In a closed chamber -1 concentration of the sample in the chamber, O2 consumption (internal flow IkO2 [pmol∙s ] when one is concerned about crowding of catabolic reactions k) causes a decline in effects and instrumental time resolution. The the amount nO2 [nmol] of O2 in the system. wall of the instrumental chamber and the Normalization of these quantities for the enclosed solid stirrer are not counted as part of volume V [L] of the system yields volume- -1 •1 -1 the experimental chamber volume. specific O2 flux, JV,kO2 = IkO2∙V [nmol∙s ∙L ], -1 and O2 concentration, [O2] or cO2 = nO2∙V [µmol∙L•1 = µM = nmol∙mL•1]. Instrumental

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Consortium Communication background O2 flux is due to external flux For studies of cells, we recommend that into a non-ideal closed respirometer, so total respiration be expressed, as far as possible, volume-specific flux has to be corrected for as: (1) O2 flux normalized for a mitochondrial instrumental background O2 flux—O2 marker, for separation of the effects of diffusion into or out of the experimental mitochondrial quality and content on cell chamber. JV,kO2 is relevant mainly for respiration (this includes FCRs as a methodological reasons and should be normalization for a functional mitochondrial compared with the accuracy of instrumental marker); (2) O2 flux in units of cell volume or resolution of background-corrected flux, e.g., mass, for comparison of respiration of cells ±1 nmol∙s•1∙L-1 [51]. ‘Catabolic’ indicates O2 with different cell size [113] and with studies flux JkO2 corrected for: (1) instrumental on tissue preparations, and (3) O2 flow in -18 background O2 flux; (2) chemical units of attomole (10 mol) of O2 consumed background O2 flux due to autoxidation of per second by each individual cell chemical components added to the [amol∙s•1∙x•1], numerically equivalent to •1 6 •1 incubation medium; and (3) Rox of O2- [pmol∙s ∙(10 x) ]. This convention allows consuming side reactions unrelated to the information to be easily used when catabolic pathway k. designing experiments in which O2 flow must be considered. For example, to estimate the 6. Conversion of units volume-specific O2 flux in an experimental chamber that would be expected at a Many different units have been used to particular cell-count concentration, one report the O2 consumption rate OCR (Table simply needs to multiply the flow per cell by 6). SI base units provide the common the number of cells per volume of interest. reference to introduce the theoretical This provides the amount of O2 [mol] principles (Figure 5), and are used with consumed per time [s-1] per unit volume appropriately chosen SI prefixes to express [L•1]. At an O2 flow of 100 amol∙s•1∙x-1 and a numerical data in the most practical format, cell-count concentration of 109 x∙L•1 (= 106 with an effort towards unification within x∙mL•1), the chamber volume-specific O2 flux specific areas of application (Table 7). is 100 nmol∙s-1∙L-1 (= 100 pmol∙s•1∙mL•1). Reporting data in SI units—including the ET capacity in human cell types including mole [mol], coulomb [C], joule [J], and second HEK 293, primary HUVEC, and fibroblasts [s]—should be encouraged, particularly by ranges from 50 to 180 amol∙s-1∙x-1, measured journals that propose the use of SI units. in living cells in the noncoupled state [54]. At Although volume is expressed as m3 using 100 amol∙s-1∙x-1 corrected for Rox, the the SI base unit, the liter [L = dm3] is a current across the mt- membranes, Iel, conventional unit of volume for approximates 193 pA∙x-1 or 0.2 nA per cell. concentration and is used for most solution See Rich [115] for an extension of chemical kinetics. If one multiplies IO2/X by CX, quantitative bioenergetics from the then the result will not only be the amount of molecular to the human scale, with a O2 [mol] consumed per time [s-1] in one liter transmembrane proton flux equivalent to [L•1], but also the change in O2 concentration 520 A in an adult at a catabolic power of •110 per second (for any volume of an ideally W∙x-1. Modelling approaches illustrate the closed system). This is ideal for kinetic link between protonmotive force and modeling as it blends with chemical rate currents [143]. equations where concentrations are We consider isolated mitochondria as typically expressed in mol∙L-1 [136]. In powerhouses and proton pumps as studies of multinuclear cells—such as molecular machines to relate experimental differentiated skeletal muscle cells—it is results to energy metabolism of living cells. easy to determine the number of nuclei but The cellular P»/O2 based on oxidation of not the total number of cells. A generalized glycogen is increased by the glycolytic concept, therefore, is obtained by (fermentative) substrate-level substituting cells by nuclei as the elementary phosphorylation of 3 P»/Glyc or 0.5 mol P» entity. This does not hold, however, for non- for each mol O2 consumed in the complete nucleated platelets. oxidation of a mol glycosyl-unit (Glyc).

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Table 7. Conversion of units with preservation of numerical values.

Name Frequently used unit Equivalent unit Notes

-1 -1 -1 -1 volume-specific flux JV,O2 pmol∙s ∙mL nmol∙s ∙L 1 mmol∙s-1∙L-1 mol∙s-1∙m-3 2 -1 -1 -1 -1 flow per cell IO2/ce pmol∙s ∙Mx amol∙s ∙x 3 pmol∙s-1∙Gx-1 zmol∙s-1∙x-1 4

cell-count concentration Cce 106 x∙mL-1 109 x∙L-1

mitochondrial protein concentration CmtE 0.1 mg∙mL-1 0.1 g∙L-1 -1 -1 -1 -1 sample mass-specific flux JO2/mX pmol∙s ∙mg nmol∙s ∙g 5 catabolic power Pk µW∙Mx-1 pW∙x-1 1, 3 volume V 1000 L m3 (1000 kg) L dm3 (kg) mL cm3 (g) µL mm3 (mg) fL µm3 (pg) 6

amount of substance concentration, nB M = mol∙L-1 mol∙dm-3 1 pmol: picomole = 10-12 mol 4 zmol: zeptomole = 10-21 mol; 1 Gx = 109 x 2 mmol: millimole = 10-3 mol 5 nmol: nanomole = 10-9 mol 3 amol: attomole = 10-18 mol; 1 Mx = 106 x 6 fL: femtoliter = 10-15 L; µL: microliter = 10-6 L

Adding 0.5 to the mitochondrial P»/O2 ratio conserved in the phosphorylation of ADP to of 5.4 yields a bioenergetic cell physiological ATP or in ion pumping, or dissipated in an P»/O2 ratio close to 6. Two NADH electrochemical short-circuit. Respiration is equivalents are formed during glycolysis and thus clearly distinguished from fermentation transported from the cytosol into the as the counterparts of cellular core energy mitochondrial matrix, either by the malate- metabolism. O2 flux balance schemes aspartate shuttle or by the glycerophosphate illustrate the relationships and general shuttle (Figure 1a) resulting in different definitions (Overview, Figure 1). theoretical yields of ATP generated by Experimentally, respiration is separated mitochondria, the energetic cost of which in mitochondrial preparations from the potentially must be taken into account. interactions with the fermentative pathways Considering also substrate-level of the living cell. OXPHOS analysis is based on phosphorylation in the TCA cycle, this high the study of mitochondrial preparations P»/O2 ratio not only reflects proton complementary to bioenergetic translocation and OXPHOS studied in investigations of (1) submitochondrial isolation, but integrates mitochondrial particles and molecular structures, (2) living physiology with energy transformation in cells, and (3) organisms—from model the living cell [49]. organisms to the human species including healthy and diseased persons (patients). 7. Conclusions

Catabolic cell respiration is the process of Box 3: Recommendations for studies exergonic and exothermic energy with mitochondrial preparations transformation in which scalar redox reactions are coupled to vectorial ion ● Normalization of respiratory rates should translocation across a semipermeable be provided as far as possible: membrane, which separates the small A. Sample normalization volume of a bacterial cell or mitochondrion from the larger volume of its surroundings. 1. Object count-specific biophysical The electrochemical exergy can be partially normalization: O2 flow on a per single cell

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Consortium Communication

or per single organism basis; this may not substrate competition that may occur be possible when dealing with coenocytic when providing physiological substrate organisms, e.g., filamentous fungi, or cocktails. tissues without cross-walls separating ● In studies of isolated mitochondria, the individual cells, e.g., muscle fibers. mitochondrial recovery and yield should 2. Size-specific cellular normalization: per g be reported. Experimental criteria such as protein; per organism-, cell- or tissue- transmission electron microscopy for mass as mass-specific O2 flux; per cell evaluation of purity versus integrity volume as cell volume-specific flux. should be considered. Mitochondrial 3. Mitochondrial normalization: per markers—such as citrate synthase mitochondrial marker as mt-specific flux. activity as an enzymatic matrix marker—

B. Chamber normalization provide a link to the tissue of origin on the basis of calculating the mitochondrial 1. Chamber volume-specific flux JV recovery, i.e., the fraction of [pmol∙s•1∙mL-1] is reported for quality mitochondrial marker obtained from a control in relation to instrumental unit mass of tissue. Total mitochondrial sensitivity and limit of detection of protein is frequently applied as a volume-specific flux. mitochondrial marker, which is restricted 2. Sample concentration in the experimental to isolated mitochondria. chamber is reported as count concentration, mass concentration, or ● In studies of permeabilized cells, the mitochondrial concentration; this is a viability of the cell culture or cell component of the measuring conditions. suspension of origin should be reported. With information on cell size and the use Normalization should be evaluated for total cell count or viable cell count. of multiple normalizations, maximum potential information is available [54; ● Terms and symbols are summarized in 113; 136]. Reporting exclusively flow in a Table 8. Their use will facilitate respiratory chamber [nmol∙s-1] is transdisciplinary communication and discouraged, since it restricts the analysis support further development of a to intra-experimental comparison of consistent theory of bioenergetics and relative (qualitative) differences. mitochondrial physiology. Technical

● Catabolic mitochondrial respiration is terms related to and defined with practical words can be used as index distinguished from residual O2 consumption. Fluxes in mitochondrial terms in data repositories, support the coupling states should be, as far as creation of ontologies towards semantic information processing (MitoPedia), and possible, corrected for residual O2 consumption. help in communicating analytical findings as impactful data-driven stories. ‘Making ● Different mechanisms of uncoupling data available without making it should be distinguished by defined terms. understandable may be worse than not The tightness of coupling relates to these making it available at all’ [99]. Success uncoupling mechanisms, whereas the will depend on taking further steps: (1) coupling stoichiometry varies as a exhaustive text-mining considering function the substrate type involved in ET Omics data and functional data; (2) pathways with either three or two redox network analysis of Omics data with proton pumps operating in series. bioinformatics tools; (3) cross-validation Separation of tightness of coupling from with distinct bioinformatics approaches; the pathway-dependent coupling (4) correlation with physiological data; stoichiometry is possible only when the (5) guidelines for biological validation of substrate type undergoing oxidation network data. This is a call to carefully remains the same for respiration in contribute to FAIR principles (Findable, LEAK•, OXPHOS-, and ET states. In studies Accessible, Interoperable, Reusable) for of the tightness of coupling, therefore, the sharing of scientific data. simple substrate•inhibitor combinations should be applied to exlcude a shift in

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Table 8. Terms, symbols, and units. SI base units are used, except for the liter [L = dm3]. SI refers to ref. [11], IUPAC refers to ref. [24].

Term Symbol Unit Links and comments

adenosine diphosphate ADP Tab. 1; Fig. 1, 2, 5 adenosine monophosphate AMP 2 ADP ↔ ATP+AMP adenosine triphosphate ATP Fig. 2, 5 adenylates AMP, ADP, ATP Section 2.5.1 alternative quinol oxidase AOX Fig. 1B amount of substance B nB or n(B) [mol] IUPAC

ATP yield per O2 YP»/O2 1 YP»/O2 = JP»/JkO2; P»/O2 measured in any respiratory state catabolic rate of respiration JkO2; IkO2 varies Fig. 1, 3; see flux J and flow I catabolic reaction k Fig. 1, 3 cell count Nce [x] Tab. 4; Fig. 5; see number of cells cell-count concentration Cce [x∙L•1] Tab. 4; Cce = Nce∙V-1 cell mass mce [kg] Tab. 5; Fig. 5 •1 cell mass, mass per cell MUce [kg∙x ] Tab. 5; Fig. 5 •1 -1 cell-mass concentration Cmce [kg∙L ] Cmce = mce∙V ; Tab. 4; see CmX cell viability index VIce VIce = Nvce∙Nce•1 = 1- Ndce∙Nce•1 -1 charge number per UB zB 1 Tab. 6; zO2 = 4; (zB = QB∙e [24]) Complexes I to IV CI to CIV respiratory Complexes CI to CVI are redox proton pumps of the ETS, but not F1FO-ATPase; hence the term CV is not recommended; Fig. 1B concentration of B, amount cB = nB∙V•1 [mol∙L•1] SI: amount of substance concentration (IUPAC) •1 •1 concentration of O2, amount cO2 = nO2∙V [mol∙L ] [O2]; Box 2 concentration of X, count CX = NX∙V•1 [x∙L-1] Tab. 4 (IUPAC: number concentration) count format N [x] Tab. 4, 5; Fig. 5 count of entity-type X NX [x] SI; see number of entities X coupling-control state CCS Section 2.4.1 dead cells dce Tab. 5 electrical format e [C] Tab. 6 electron transfer, state ET Tab. 1; Fig. 2B, 4 (State 3u) electron transfer system ETS Fig. 2B, 4 (electron transport chain) elementary entity of entity-type X UX [x] single countable object; Tab. 4, 5 ET capacity E varies rate; Tab. 1; Fig. 2 ET-excess capacity E-P varies Fig. 2 •1 flow, for O2 IO2 [mol∙s ] system-related or count-specific extensive quantity; Fig. 5 flux, for O2 JO2 varies size-specific quantity; Fig. 5 flux control ratio FCR 1 background/reference flux; Fig.5 inorganic phosphate Pi Fig. 1C inorganic phosphate carrier PiC Fig. 1C isolated mitochondria imt Tab. 5 LEAK respiration L varies rate; Tab. 1; Fig. 2 LEAK state LEAK Tab. 1; Fig. 2 (compare State 4) living cells, entity-type ce Tab. 5 (intact cells) mass, dry mass md [kg] Fig. 5 (dry weight) [56]

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Consortium Communication mass, wet mass mw [kg] Fig. 5 (wet weight) -1 mass concentration in a mixture CmX [kg∙L ] Tab. 4 mass format m [kg] Tab. 4 mass of sample of X in a mixture mX [kg] SI: mass mS of pure sample S •1 mass per elementary entity UX, MUX [kg∙x ] body mass; Fig. 5; Tab. 4; SI: mass per individual object m(X) (compare molar mass MB [kg∙mol•1]) MITOCARTA https://www.broadinstitute.org/scientific- community/science/programs/ metabolic-disease- program/publications/mitocarta /mitocarta-in-0 [13] mitochondria or mitochondrial mt Box 1 mitochondrial concentration CmtE = mtE∙V-1 [mtEU∙L•1] per chamber volume; Tab. 4 •1 -1 mitochondrial content per UX mtEUX [mtEU∙x ] mtEUX = mtE∙NX ; Tab. 4 •1 -1 mitochondrial density per mX DmtE/mX [mtEU∙kg ] DmtE/mX = mtE∙mX ; Tab. 4 •1 -1 mitochondrial density per VX DmtE/VX [mtEU∙L ] DmtE/VX = mtE∙VX ; Tab. 4 mitochondrial DNA mtDNA Box 1 mitochondrial elementary marker mtE [mtEU] quantity of mt-marker; Tab. 4 mitochondrial elementary unit mtEU varies specific units for mt-marker; Tab. 4 mitochondrial inner membrane mtIM Fig. 1; Box 1 (MIM) mitochondrial outer membrane mtOM Fig. 1; Box 1 (MOM) mitochondrial recovery YmtE 1 fraction of mtE recovered from the tissue sample in imt-stock -1 mitochondrial yield YmtE/mX [mtEU∙kg ] mt-yield in imt-stock per mass of tissue sample; YmtE/mX=YmtE∙DmtE MitoPedia http://www.bioblast.at/index.php/MitoPedia molar format n [mol] Tab. 6 -1 -1 molar mass MB [kg∙mol ] compare MUB [kg∙x ]; SI M(X) negative neg Fig. 4 number of cells Nce [x] total cell count of living cells, Nce = Nvce + Ndce; Tab. 4, 5 number of dead cells Ndce [x] non-viable cell count, loss of plasma membrane barrier function; Tab. 5 number of entities B, count NB = N∙UB [x] Tab. 4 (IUPAC) number of elementary entities, NX = N∙UX [x] ‘count’ is an SI quantity [11], but count of X the elementary unit [x] is not in the SI [95]; Tab. 4; Fig. 5 number of viable cells, count Nvce [x] viable cell count, intact plasma membrane barrier function; Tab. 5 organisms, entity-type org Tab. 5 oxidative phosphorylation OXPHOS Tab. 1 OXPHOS capacity P varies rate; Tab. 1; Fig. 2 OXPHOS state OXPHOS Tab. 1; Fig. 2; OXPHOS state distinguished from the process OXPHOS (State 3 at kinetically- saturating [ADP] and [Pi]) •1 •1 oxygen concentration cO2 = nO2∙V [mol∙L ] [O2]; Section 3.2 oxygen flux, in reaction r JrO2 varies Overview pathway-control state PCS Section 2.2

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BEC 2020.1 doi:10.26124/bec:2020-0001.v1 permeability transition mtPT Fig. 3; Section 2.4.3 (MPT) permeabilized cells, entity-type pce experimental permeabilization of plasma membrane; Tab. 5 permeabilized muscle fibers pfi Tab. 5 permeabilized tissue pti Tab. 5 phosphorylation of ADP to ATP P» Tab. 1, 2; Fig. 1, 4

P»/O2 ratio P»/O2 1 mechanistic YP»/O2, calculated from pump stoichiometries; Fig. 1c positive pos Fig. 4 proton in the neg compartment H+neg [x] Fig. 4 proton in the pos compartment H+pos [x] Fig. 4 protonmotive force pmF [V] Overview; Tab. 1; Fig 1a, 2, 4 rate in ET state E varies ET capacity; Tab. 1; Fig. 2, 4 rate in LEAK state L varies Tab. 1: L(n), L(T), L(Omy); Fig. 2 rate in OXPHOS state P varies OXPHOS capacity; Tab.1; Fig. 2, 4 rate in ROX state Rox varies Overview; Tab. 1 residual oxygen consumption ROX; Rox state ROX; rate Rox; Tab. 1 respiration JrO2 varies rate of reaction r; Overview respiratory supercomplex SCInIIInIVn supramolecular assemblies with variable copy numbers (n) of CI, CIII and CIV; Box 1 sample of X in a mixture s VX < V; Tab. 4, 5 substrate concentration at half-maximal rate c50 [mol∙L•1] Section 2.1.2 substrate-uncoupler-inhibitor- titration SUIT Section 2.2 tissue homogenate thom Tab. 5 viable cells vce Tab. 5 volume format V [L] Tab. 6 volume of experimental chamber V [L] V with s; Tab. 4, 7; Fig. 5 volume of sample of X in a mixture VX [L] Fig. 5; Tab. 5

Different mechanisms of respiratory normalized for defined quantities of sample. uncoupling have to be distinguished (Figure For some samples it is informative, if 3). Metabolic fluxes measured in defined quantification is possible in terms of a count coupling- and pathway-control states (number of countable objects). Using cells as (Figures 1, 2 and 4) provide insights into an example, a distinction is made between the meaning of cellular and organismic sample type (cells) and the quantity of cells respiration. (count, mass, volume). Using the unit The optimal choice for expressing [mol∙s•1∙cell•1] has been common but is mitochondrial and cell respiration as O2 flow ambiguous. This is resolved by (1) not only per biological sample, and normalization for indicating the entity-type (cell), but (2) specific tissue-markers (volume, mass, additionally defining the quantity (count [x], protein) and mitochondrial markers mass [kg], volume [L]) in which the entity is (volume, protein, content, mtDNA, activity of expressed with corresponding units. marker enzymes, respiratory reference Similarly, substance concentrations can be state) is guided by the scientific question expressed in various formats with under study. Interpretation of the data corresponding units, including molecular -1 -1 depends critically on appropriate count concentration, CO2 = NO2∙V [x∙L ], and -1 normalization (Figure 5). molar amount concentration, cO2 = nO2∙V Results are comparable between studies [mol∙L-1], whereas it does not make sense to only, if respirometric measurements are write [O2∙L-1]. In conclusion, expressions

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Consortium Communication such as [cells∙L•1] or [mol∙s-1∙cell-1] should be effect of temperature on the ADP- replaced by [x∙L•1] or [mol∙s•1∙x•1]. Symbols dependence of ATP synthesis. J Exp Biol 176:145-58. for quantities, such as CX for count concentration, gain meaning only in context 8. Boushel RC, Gnaiger E, Schjerling P, et al with specification of the entity-type, e.g., cell (2007) Patients with Type 2 diabetes have normal mitochondrial function in skeletal types, growth conditions. Simple symbols muscle. Diabetologia 50:790-6. -1 can be used, e.g., M for body mass [kg∙x ], if 9. Breton S, Beaupré HD, Stewart DT, et al clarity of definition is provided in the text. (2007) The unusual system of doubly MitoEAGLE can serve as a gateway to uniparental inheritance of mtDNA: isn't one better diagnose mitochondrial respiratory enough? Trends Genet 23:465-74. adaptations and defects linked to genetic 10. Brown GC (1992) Control of respiration and variation, age-related health risks, sex- ATP synthesis in mammalian mitochondria specific mitochondrial performance, lifestyle and cells. Biochem J 284:1-13. with its effects on degenerative diseases, and 11. Bureau International des Poids et Mesures thermal and chemical environment. The (2019) The International System of Units (SI). 9th edition:117-216 ISBN 978-92-822- present recommendations on coupling- 2272-0. control states and rates are focused on 12. Burger G, Gray MW, Forget L, Lang BF studies using mitochondrial preparations (2013) Strikingly bacteria-like and gene- (Box 3). These will be extended in a series of rich mitochondrial genomes throughout reports on pathway control of mitochondrial jakobid protists. Genome Biol Evol 5:418-38. respiration, respiratory states and rates in 13. Calvo SE, Klauser CR, Mootha VK (2016) living cells, respiratory flux control ratios, MitoCarta2.0: an updated inventory of and harmonization of experimental mammalian mitochondrial proteins. Nucleic procedures. Acids Research 44:D1251-7. 14. Calvo SE, Julien O, Clauser KR, et al (2017) Comparative analysis of mitochondrial N- References termini from mouse, human, and yeast. Mol Cell Proteomics 16:512-23. 1. Altmann R (1894) Die 15. Campos JC, Queliconi BB, Bozi LHM, et al Elementarorganismen und ihre (2017) Exercise reestablishes autophagic Beziehungen zu den Zellen. Zweite flux and mitochondrial quality control in vermehrte Auflage. Verlag Von Veit & Comp, heart failure. Autophagy 13:1304-317. Leipzig:160 pp. 16. Canton M, Luvisetto S, Schmehl I, Azzone GF 2. Baggeto LG, Testa-Perussini R (1990) Role (1995) The nature of mitochondrial of acetoin on the regulation of intermediate respiration and discrimination between metabolism of Ehrlich ascites tumor membrane and pump properties. Biochem J mitochondria: its contribution to membrane 310:477-81. cholesterol enrichment modifying passive 17. Carrico C, Meyer JG, He W, et al (2018) The proton permeability. Arch Biochem Biophys mitochondrial acylome emerges: 283:341-8. proteomics, regulation by Sirtuins, and 3. Beard DA (2005) A biophysical model of the metabolic and disease implications. Cell mitochondrial respiratory system and Metab 27:497-512. oxidative phosphorylation. PLoS Comput 18. Chan DC (2006) Mitochondria: dynamic Biol 1(4):e36. organelles in disease, aging, and 4. Benda C (1898) Weitere Mitteilungen über development. Cell 125:1241-52. die Mitochondria. Verh Dtsch Physiol 19. Chance B, Williams GR (1955a) Respiratory Ges:376-83. enzymes in oxidative phosphorylation. I. 5. Birkedal R, Laasmaa M, Vendelin M (2014) Kinetics of oxygen utilization. J Biol Chem The location of energetic compartments 217:383-93. affects energetic communication in 20. Chance B, Williams GR (1955b) Respiratory cardiomyocytes. Front Physiol 5:376. enzymes in oxidative phosphorylation: III. 6. Blier PU, Dufresne F, Burton RS (2001) The steady state. J Biol Chem 217:409-27. Natural selection and the evolution of 21. Chance B, Williams GR (1956) The mtDNA-encoded peptides: evidence for respiratory chain and oxidative intergenomic co-adaptation. Trends Genet phosphorylation. Adv Enzymol Relat Subj 17:400-6. Biochem 17:65-134. 7. Blier PU, Guderley HE (1993) Mitochondrial 22. Chowdhury SK, Djordjevic J, Albensi B, activity in rainbow trout red muscle: the Fernyhough P (2015) Simultaneous

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Christos, Chowdhury Subir Kumar, Haas Clarissa, Haavik Jan, Hachmo Yafit, Cizmarova Beata, Clementi Emilio, Coen Paul Haendeler Judith, Haider Markus, M, Cohen Bruce H, Coker Robert H, Collin- Hajrulahovic Anesa, Hamann Andrea, Han Chenot Anne, Coughlan Melinda T, Coxito Jin, Han Woo Hyun, Hancock Chad R, Hand Pedro, Crisostomo Luis, Crispim Marcell, Steven C, Handl Jiri, Hansikova Hana, Hardee Crossland Hannah, Dahdah Norma Ramon, Justin P, Hargreaves Iain P, Harper Mary- Dalgaard Louise T, Dambrova Maija, Ellen, Harrison David K, Hassan Hazirah, Danhelovska Tereza, Darveau Charles-A, Hatokova Zuzana, Hausenloy Derek J, Heales Darwin Paula M, Das Anibh Martin, Dash Simon JR, Hecker Matthias, Heiestad Ranjan K, Davidova Eliska, Davis Michael S, Christina, Hellgren Kim T, Henrique Dayanidhi Sudarshan, De Bem Andreza Alexandrino, Hepple Russell T, Hernansanz- Fabro, De Goede Paul, De Palma Clara, De Agustin Pablo, Hewakapuge Sudinna, Hickey Pinto Vito, Dela Flemming, Dembinska-Kiec Anthony J, Ho Dieu Hien, Hoehn Kyle L, Hoel Aldona, Detraux Damian, Devaux Yvan, Di Fredrik, Holland Olivia J, Holloway Graham P, Marcello Marco, Di Paola Floriana Jessica, Holzner Lorenz, Hoppel Charles L, Hoppel Dias Candida, Dias Tania R, Diederich Marc, Florian, Hoppeler Hans, Houstek Josef, Distefano Giovanna, Djafarzadeh Siamak, Huete-Ortega Maria, Hyrossova Petra, Doermann Niklas, Doerrier Carolina, Dong Iglesias-Gonzalez Javier, Indiveri Cesare, Lan-Feng, Donnelly Chris, Drahota Zdenek, Irving Brian A, Isola Raffaella, Iyer Shilpa, Duarte Filipe Valente, Dubouchaud Herve, Jackson Christopher Benjamin, Jadiya Pooja, Duchen Michael R, Dumas Jean-Francois, Jana Prado Fabian, Jandeleit-Dahm Karin, Durham William J, Dymkowska Dorota, Jang David H, Jang Young Charles, Janowska Dyrstad Sissel E, Dyson Alex, Dzialowski Joanna, Jansen Kirsten M, Jansen-Duerr Edward M, Eaton Simon, Ehinger Johannes K, Pidder, Jansone Baiba, Jarmuszkiewicz Elmer Eskil, Endlicher Rene, Engin Ayse Wieslawa, Jaskiewicz Anna, Jaspers Richard Basak, Escames Germaine, Evinova Andrea, T, Jedlicka Jan, Jerome Estaquier, Jespersen Ezrova Zuzana, Falk Marni J, Fell David A, Nichlas Riise, Jha Rajan Kumar, Jones John G, Ferdinandy Peter, Ferko Miroslav, Joseph Vincent, Juhasz Laszlo, Jurczak Fernandez-Ortiz Marisol, Fernandez-Vizarra Michael J, Jurk Diana, Jusic Amela, Kaambre Erika, Ferreira Julio Cesar B, Ferreira Rita Tuuli, Kaczor Jan Jacek, Kainulainen Heikki, Maria P, Ferri Alessandra, Fessel Joshua Kampa Rafal Pawel, Kandel Sunil Mani, Kane Patrick, Festuccia William T, Filipovska Daniel A, Kapferer Werner, Kapnick Senta, Aleksandra, Fisar Zdenek, Fischer Christine, Kappler Lisa, Karabatsiakis Alexander, Fischer Michael J, Fisher Gordon, Fisher Karavaeva Iuliia, Karkucinska-Wieckowska Joshua J, Fontanesi Flavia, Forbes-Hernandez Agnieszka, Kaur Sarbjot, Keijer Jaap, Keller Tamara Y, Ford Ellen, Fornaro Mara, Fuertes Markus A, Keppner Gloria, Khamoui Andy V, Agudo Marina, Fulton Montana, Galina Kidere Dita, Kilbaugh Todd, Kim Hyoung Antonio, Galkin Alexander, Gallee Leon, Galli Kyu, Kim Julian KS, Kimoloi Sammy, Klepinin Gina L J, Gama Perez Pau, Gan Zhenji, Aleksandr, Klepinina Lyudmila, Klingenspor Ganetzky Rebecca, Gao Yun, Garcia Geovana Martin, Klocker Helmut, Kolassa Iris, S, Garcia-Rivas Gerardo, Garcia-Roves Pablo Komlodi Timea, Koopman Werner JH, Miguel, Garcia-Souza Luiz F, Garlid Keith D, Kopitar-Jerala Natasa, Kowaltowski Alicia J, Garrabou Gloria, Garten Antje, Gastaldelli Kozlov Andrey V, Krajcova Adela, Krako Amalia, Gayen Jiaur, Genders Amanda J, Jakovljevic Nina, Kristal Bruce S, Krycer Genova Maria Luisa, Giampieri Francesca, James R, Kuang Jujiao, Kucera Otto, Kuka Giovarelli Matteo, Glatz Jan FC, Goikoetxea Janis, Kwak Hyo Bum, Kwast Kurt E, Kwon Usandizaga Naroa, Goncalo Teixeira da Silva Oh Sung, Laasmaa Martin, Labieniec-Watala Rui, Goncalves Debora Farina, Gonzalez- Magdalena, Lagarrigue Sylviane, Lai Nicola, Armenta Jenny L, Gonzalez-Franquesa Alba, Lalic Nebojsa M, Land John M, Lane Nick, Gonzalez-Freire Marta, Gonzalo Hugo, Laner Verena, Lanza Ian R, Laouafa Sofien, Goodpaster Bret H, Gorr Thomas A, Gourlay Laranjinha Joao, Larsen Steen, Larsen Terje Campbell W, Grams Bente, Granata Cesare, S, Lavery Gareth G, Lazou Antigone, Ledo Ana Grefte Sander, Grilo Luis, Guarch Meritxell Margarida, Lee Hong Kyu, Leeuwenburgh Espino, Gueguen Naig, Gumeni Sentiljana, Christiaan, Lehti Maarit, Lemieux Helene,

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Lenaz Giorgio, Lerfall Joergen, Li Pingan Puurand Marju, Radenkovic Filip, Ramzan Andy, Li Puma Lance, Liang Liping, Liepins Rabia, Rattan Suresh IS, Reano Simone, Edgars, Lin Chien-Te, Liu Jiankang, Lopez Reboredo-Rodriguez Patricia, Rees Bernard Garcia Luis Carlos, Lucchinetti Eliana, Ma B, Renner-Sattler Kathrin, Rial Eduardo, Tao, Macedo Maria Paula, Machado Ivo F, Robinson Matthew M, Roden Michael, Maciej Sarah, MacMillan-Crow Lee Ann, Rodrigues Ana Sofia, Rodriguez Enrique, Magalhaes Jose, Magri Andrea, Majtnerova Rodriguez-Enriquez Sara, Roesland Gro Pavlina, Makarova Elina, Makrecka-Kuka Vatne, Rohlena Jakub, Rolo Anabela Pinto, Marina, Malik Afshan N, Marcouiller Ropelle Eduardo R, Roshanravan Baback, Francois, Marechal Amandine, Markova Rossignol Rodrigue, Rossiter Harry B, Michaela, Markovic Ivanka, Martin Daniel S, Rousar Tomas, Rubelj Ivica, Rybacka- Martins Ana Dias, Martins Joao D, Maseko Mossakowska Joanna, Saada Reisch Ann, Tumisang Edward, Maull Felicia, Mazat Jean- Safaei Zahra, Salin Karine, Salvadego Desy, Pierre, McKenna Helen T, McKenzie Sandi Carmen, Saner Nicholas, Santos Diana, Matthew, McMillan Duncan GG, McStay Gavin Sanz Alberto, Sardao Vilma, Sarlak Saharnaz, P, Mendham Amy, Menze Michael A, Mercer Sazanov Leonid A, Scaife Paula, Scatena John R, Merz Tamara, Messina Angela, Roberto, Schartner Melanie, Scheibye- Meszaros Andras, Methner Axel, Michalak Knudsen Morten, Schilling Jan M, Schlattner Slawomir, Mila Guasch Maria, Minuzzi Uwe, Schmitt Sabine, Schneider Gasser Edith Luciele M, Misirkic Marjanovic Maja, Mariane, Schoenfeld Peter, Schots Pauke C, Moellering Douglas R, Moisoi Nicoleta, Schulz Rainer, Schwarzer Christoph, Scott Molina Anthony JA, Montaigne David, Moore Graham R, Selman Colin, Sendon Pamella Anthony L, Moore Christy, Moreau Kerrie, Marie, Shabalina Irina G, Sharma Pushpa, Moreira Bruno P, Moreno-Sanchez Rafael, Sharma Vipin, Shevchuk Igor, Shirazi Reza, Mracek Tomas, Muccini Anna Maria, Munro Shiroma Jonathan G, Siewiera Karolina, Daniel, Muntane Jordi, Muntean Danina M, Silber Ariel Mariano, Silva Ana Maria, Sims Murray Andrew James, Musiol Eva, Nabben Carrie A, Singer Dominique, Singh Brijesh Miranda, Nair K Sreekumaran, Nehlin Jan O, Kumar, Skolik Robert A, Smenes Benedikte Nemec Michal, Nesci Salvatore, Neufer P Therese, Smith James, Soares Felix Darrell, Neuzil Jiri, Neviere Remi, Newsom Alexandre Antunes, Sobotka Ondrej, Sean A, Norman Jennifer, Nozickova Sokolova Inna, Solesio Maria E, Soliz Jorge, Katerina, Nunes Sara, Nuoffer Jean-Marc, Sommer Natascha, Sonkar Vijay K, Sova O'Brien Kristin, O'Brien Katie A, O'Gorman Marina, Sowton Alice P, Sparagna Genevieve Donal, Olgar Yusuf, Oliveira Ben, Oliveira C, Sparks Lauren M, Spinazzi Marco, Jorge, Oliveira Marcus F, Oliveira Marcos Stankova Pavla, Starr Jonathan, Stary Creed, Tulio, Oliveira Pedro Fontes, Oliveira Paulo J, Stefan Eduard, Stelfa Gundega, Stepto Nigel Olsen Rolf Erik, Orynbayeva Zulfiya, K, Stevanovic Jelena, Stiban Johnny, Stier Osiewacz Heinz D, Paez Hector, Pak Youngmi Antoine, Stocker Roland, Storder Julie, Kim, Pallotta Maria Luigia, Palmeira Carlos, Sumbalova Zuzana, Suomalainen Anu, Parajuli Nirmala, Passos Joao F, Passrugger Suravajhala Prashanth, Svalbe Baiba, Manuela, Patel Hemal H, Pavlova Nadia, Swerdlow Russell H, Swiniuch Daria, Szabo Pavlovic Kasja, Pecina Petr, Pedersen Tina M, Ildiko, Szewczyk Adam, Szibor Marten, Perales Jose Carles, Pereira da Silva Grilo da Tanaka Masashi, Tandler Bernard, Silva Filomena, Pereira Rita, Pereira Susana Tarnopolsky Mark A, Tausan Daniel, P, Perez Valencia Juan Alberto, Perks Kara L, Tavernarakis Nektarios, Teodoro Joao Pesta Dominik, Petit Patrice X, Pettersen Soeiro, Tepp Kersti, Thakkar Himani, Thapa Nitschke Ina Katrine, Pichaud Nicolas, Maheshwor, Thyfault John P, Tomar Pichler Irene, Piel Sarah, Pietka Terri A, Dhanendra, Ton Riccardo, Torp May-Kristin, Pinho Sonia A, Pino Maria F, Pirkmajer Torres-Quesada Omar, Towheed Atif, Sergej, Place Nicolas, Plangger Mario, Porter Treberg Jason R, Tretter Laszlo, Trewin Craig, Porter Richard K, Preguica Ines, Adam J, Trifunovic Aleksandra, Trivigno Prigione Alessandro, Procaccio Vincent, Catherine, Tronstad Karl Johan, Trougakos Prochownik Edward V, Prola Alexandre, Ioannis P, Truu Laura, Tuncay Erkan, Turan Pulinilkunnil Thomas, Puskarich Michael A, Belma, Tyrrell Daniel J, Urban Tomas, Urner

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Sofia, Valentine Joseph Marco, Van Bergen (European Cooperation in Science and Nicole J, Van der Ende Miranda, Varricchio Technology), in cooperation with COST Frederick, Vaupel Peter, Vella Joanna, Actions CA16225 EU-CARDIOPROTECTION Vendelin Marko, Vercesi Anibal E, Verdaguer and CA17129 CardioRNA; K-Regio project Ignasi Bofill, Vernerova Andrea, Victor Victor MitoFit funded by the Tyrolian Government, Manuel, Vieira Ligo Teixeira Camila, Vidimce and project NextGen-O2k which has received Josif, Viel Christian, Vieyra Adalberto, Vilks funding from the European Union’s Horizon Karlis, Villena Josep A, Vincent Vinnyfred, 2020 research and innovation programme Vinogradov Andrey D, Viscomi Carlo, under grant agreement No. 859770. Vitorino Rui Miguel Pinheiro, Vlachaki Walker Julia, Vogt Sebastian, Volani Chiara, Volska Kristine, Votion Dominique-Marie, Vujacic-Mirski Ksenija, Wagner Brett A, Ward Marie Louise, Warnsmann Verena, Wasserman David H, Watala Cezary, Wei Yau-Huei, Weinberger Klaus M, Weissig Volkmar, White Sarah Haverty, Whitfield Jamie, Wickert Anika, Wieckowski Mariusz R, Competing financial interests: Erich Wiesner Rudolf J, Williams Caroline M, Gnaiger is founder and CEO of Oroboros Winwood-Smith Hugh, Wohlgemuth Instruments, Innsbruck, Austria.

Stephanie E, Wohlwend Martin, Wolff Jonci Corresponding author: Erich Gnaiger Nikolai, Wrutniak-Cabello Chantal, Wuest Chair COST Action CA15203 MitoEAGLE Rob CI, Yokota Takashi, Zablocki Krzysztof, http://www.mitoeagle.org Zanon Alessandra, Zanou Nadege, Zaugg Department of Visceral, Transplant and Kathrin, Zaugg Michael, Zdrazilova Lucie, Thoracic Surgery, D. Swarovski Research Zhang Yong, Zhang Yizhu, Zikova Alena, Laboratory, Medical University of Innsbruck, Zischka Hans, Zorzano Antonio, Zujovic Innrain 66/4, A-6020 Innsbruck, Austria Tijana, Zurmanova Jitka, Zvejniece Liga Email: [email protected]

Affiliations: Tel +43 512 566796, Fax +43 512 566796 20 https://www.bioenergetics- Copyright: © 2020 The authors. This is an communications.org/index.php/BEC_2020.1_ Open Access communication distributed doi10.26124bec2020-0001.v1 under the terms of the Creative Commons

Attribution License, which permits Author contributions: This manuscript unrestricted use, distribution, and developed as an open invitation to scientists reproduction in any medium, provided the and students to join as coauthors in the original authors and source are credited. © bottom-up spirit of COST, based on a first remains with the authors, who have granted draft written by the corresponding author, Bioenergetics Communications an Open who integrated coauthor contributions in a Access publication licence in perpetuity. sequence of Open Access versions. Coauthors contributed to the scope and Published online: 2020-05-20 quality of the manuscript, may have focused on a particular section, and are listed in alphabetical order. Coauthors confirm that they have read the final manuscript and agree to implement or discuss the recommendations in future manuscripts, presentations and teaching materials.

Acknowledgements: We thank Marija Beno for management assistance, and Peter R Rich for valuable discussions. This publication is based upon work from COST Action CA15203 MitoEAGLE, supported by COST

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