Bioenerg Commun 2020.1 https://doi:10.26124/bec:2020-0001.vTest06

Consortium communication

Mitochondrial physiology

Gnaiger Erich et al (MitoEAGLE Task Group)*

Living Communication: extended resource of Mitochondrial respiratory states and rates. (Gnaiger et al, Nat Metab, in review); MtoFit preprint doi:10.26124/mitofit:190001.v6.

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 or internal

cellular O2 consumption, JrO2, takes 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 (mt) mitochondrial catabolism; mt-Rox (); non-mt O2 consumption by catabolic reactions, particularly peroxisomal oxidases and microsomal cytochrome P450 systems (); and 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 (). O2 is transported from the environment across the respiratory cascade, i.e., circulation between tissues and diffusion across cell membranes, to the intracellular compartment. The respiratory quotient, RQ, is the molar CO2/O2 exchange ratio; when combined with the respiratory 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 combination of O2 and CO2 exchange, as do lungs, gills, the skin and other surfaces on the morphological level. Respiratory states are defined in Table 1. Rates are illustrated in Figure 5. Consult Table 8 for a list of terms and symbols.

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Table of contents

Abstract – Executive summary – Box 1: In brief: Mitochondria and Bioblasts 1. Introduction 2. Coupling states and rates in mitochondrial preparations 2.1. Cellular and mitochondrial respiration

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2.1.1. Aerobic and anaerobic catabolism and ATP turnover 2.1.2. Specification of biochemical dose 2.2. Mitochondrial preparations 2.3. Electron transfer pathways 2.4. Respiratory coupling control 2.4.1. Coupling 2.4.2. Phosphorylation, P», and P»/O2 ratio 2.4.3. Uncoupling 2.5. Coupling states and respiratory rates 2.5.1. LEAK-state 2.5.2. OXPHOS-state 2.5.3. Electron transfer-state 2.5.4. ROX state and Rox 2.5.5. Quantitative relations 2.5.6. The steady-state 2.6. Classical terminology for isolated mitochondria 2.6.1. – 2.6.5. State 1 – State 5 2.7. Control and regulation 3. What is a rate? – Box 2: Metabolic flows and fluxes: vectoral, vectorial, and scalar 4. Normalization of rate per sample 4.1. Flow: per object 4.1.1. Number concentration 4.1.2. Flow per object 4.2. Size-specific flux: per sample size 4.2.1. Sample concentration 4.2.2. Size-specific flux 4.3. Marker-specific flux: per mitochondrial content 4.3.1. Mitochondrial concentration and mitochondrial markers 4.3.2. mt-Marker-specific flux 5. Normalization of rate per system 5.1. Flow: per chamber 5.2. Flux: per chamber volume 5.2.1. System-specific flux 5.2.2. Advancement per volume 6. Conversion of units 7. Conclusions – Box 3: Recommendations for studies with mitochondrial preparations References Authors (MitoEAGLE Task Group) – Author contributions Acknowledgements – Competing financial interests – Correspondence

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

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BEC2020.1 doi:10.26124/bec:2020-0001.vTest05 standards for evaluation of respiratory maintained between the mitochondrial states and rates will ultimately contribute matrix and intermembrane compartment or to reproducibility between laboratories outer mitochondrial space. Compartmental and thus support the development of coupling depends on ion translocation databases of mitochondrial respiratory across a semipermeable membrane, which is function in species, tissues, and cells. defined as vectorial metabolism and Clarity of concept and consistency of distinguishes OXPHOS from cytosolic nomenclature facilitate effective fermentation as counterparts of cellular core transdisciplinary communication, energy metabolism (Overview). Cell education, and ultimately further respiration is thus distinguished from discovery. fermentation: (1) Electron acceptors are supplied by external respiration for the Keywords: Mitochondrial respiratory maintenance of redox balance, whereas control, coupling control, mitochondrial fermentation is characterized by an internal preparations, protonmotive force, electron acceptor produced in intermediary uncoupling, oxidative phosphorylation: metabolism. In aerobic cell respiration, OXPHOS, efficiency, electron transfer: ET, redox balance is maintained by O2 as the electron transfer system: ETS, proton leak, electron acceptor. (2) Compartmental ion leak and slip compensatory state: LEAK, coupling in vectorial OXPHOS contrasts to residual oxygen consumption: ROX, State 2, exclusively scalar substrate-level State 3, State 4, normalization, flow, flux, phosphorylation in fermentation. oxygen: O2 2. When measuring mitochondrial metabolism, the contribution of Executive summary fermentation and other cytosolic

In view of the broad implications for health interactions must be excluded from analysis care, mitochondrial researchers face an by disrupting the barrier function of the increasing responsibility to disseminate plasma membrane. Selective removal or their fundamental knowledge and novel permeabilization of the plasma membrane discoveries to a wide range of stakeholders yields mitochondrial preparations— and scientists beyond the group of including isolated mitochondria, tissue and specialists. This requires implementation of cellular preparations—with structural and a commonly accepted terminology within functional integrity. Subsequently, the discipline and standardization in the extramitochondrial concentrations of fuel translational context. Authors, reviewers, substrates, 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 coupling control 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 respiratory states. following concepts in mitochondrial physiology: 3. Mitochondrial coupling states are defined

according to the control of respiratory 1. Aerobic respiration is the O2 flux in catabolic reactions coupled to oxygen flux by the protonmotive force, pmF, in an interaction of the electron transfer phosphorylation of ADP to ATP, and O2 flux system generating the pmF and the in a variety of O2 consuming reactions apart 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 the absence of Complex IV inhibitors such as

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

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 is caused by a variety of Archaea (Margulis 1970; Lane 2005; Roger physiological, pathological, toxicological, et al 2017). Richard Altmann (1894) pharmacological and environmental described the ‘bioblasts’, which include not conditions that exert an influence not only on only mitochondria as presently defined, but the proton leak and cation cycling, but also also symbiotic and free-living bacteria. The on proton slip within the proton pumps and word ‘mitochondria’ (Greek mitos: thread; the structural integrity of the mitochondria. chondros: granule) was introduced by Carl A more loosely coupled state is induced by Benda (1898). Mitochondrion is singular and stimulation of mitochondrial superoxide mitochondria is plural. Abbreviation: mt, as formation and the bypass of proton pumps. generally used in mtDNA. In addition, the use of protonophores Contrary to current textbook dogma, represents an experimental uncoupling which 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 microtubules. Mitochondrial size and 5. Respiratory oxygen consumption rates number can change in response to energy have to be carefully normalized to enable requirements of the cell via processes known meta-analytic studies beyond the question of as fusion and fission; these interactions a particular experiment. Therefore, all raw allow mitochondria to communicate within a data on rates and variables for normalization network (Chan 2006). Mitochondria can should be published in an open access data even traverse cell boundaries in a process repository. Normalization of rates for: (1) known as horizontal mitochondrial transfer the number of objects (cells, organisms); (2) (Torralba et al 2016). Another defining the volume or mass of the experimental morphological characteristic of sample; and (3) the concentration of mitochondria is the double membrane. The mitochondrial markers in the instrumental mitochondrial inner membrane (mtIM) chamber are sample-specific normalizations, forms dynamic tubular to disk-shaped which are distinguished from system- cristae that separate the mitochondrial specific normalization for the volume of the matrix, i.e., the negatively charged internal instrumental chamber (the measuring mitochondrial compartment, from the system). intermembrane space; the latter being 6. The consistent use of terms and symbols enclosed by the mitochondrial outer facilitates transdisciplinary communication membrane (mtOM) and positively charged and will support the further development of with respect to the matrix. a collaborative database on bioenergetics Intracellular stress factors may cause and mitochondrial physiology. shrinking or swelling of the mitochondrial matrix that can ultimately result in

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BEC2020.1 doi:10.26124/bec:2020-0001.vTest05 permeability transition (mtPT; Lemasters et mammalian mitochondrial proteome can be al 1998). The mtIM contains the non-bilayer used to discover and characterize the genetic phospholipid cardiolipin, which is also basis of mitochondrial diseases (Williams et involved in the mtOM (Gebert et al 2009) but al 2016; Palmfeldt and Bross 2017). is not present in any other eukaryotic Numerous cellular processes are cellular membrane. Cardiolipin has many orchestrated by a constant crosstalk regulatory functions (Oemer et al 2018); it between mitochondria and other cellular promotes and stabilizes the formation of components. For example, the crosstalk supercomplexes (SCInIIInIVn) based on between mitochondria and the endoplasmic dynamic interactions between specific reticulum is involved in the regulation of respiratory complexes (McKenzie et al 2006; calcium homeostasis, cell division, Greggio et al 2017; Lenaz et al 2017), and it autophagy, differentiation, and anti-viral supports proton transfer on the mtIM from signaling (Murley and Nunnari 2016). the electron transfer system to F1FO-ATPase Mitochondria contribute to the formation of (ATP synthase; Yoshinaga et al 2016). The peroxisomes, which are hybrids of mtIM is plastic and exerts an influence on the mitochondrial and ER-derived precursors functional properties of incorporated (Sugiura et al 2017). Cellular mitochondrial proteins (Waczulikova et al 2007). homeostasis (mitostasis) is maintained Mitochondria constitute the structural through regulation at transcriptional, post- and functional elementary components of translational and epigenetic levels (Ling and cell respiration. Aerobic respiration is the Rönn 2018; Lisowski et al 2018), resulting in reduction of molecular oxygen by electron dynamic regulation of mitochondrial transfer coupled to electrochemical proton turnover by biogenesis of new mitochondria translocation across the mtIM. In the process and removal of damaged mitochondria by of OXPHOS, the catabolic reaction of oxygen fusion, fission and mitophagy (Singh et al consumption is electrochemically coupled to 2018). Cell signalling modules contribute to the transformation of energy in the homeostatic regulation throughout the cell phosphorylation of ADP to adenosine cycle or even cell death by activating triphosphate (ATP; Mitchell 1961, 2011). proteostatic modules, e.g., the ubiquitin- Mitochondria are the powerhouses of the cell proteasome and autophagy- that contain the machinery of the OXPHOS- lysosome/vacuole pathways; specific pathways, including transmembrane proteases like LON, and genome stability respiratory complexes (proton pumps with modules in response to varying energy FMN, Fe-S and cytochrome b, c, aa3 redox demands and stress cues (Quiros et al 2016). systems); alternative dehydrogenases and In addition, several post-translational oxidases; the coenzyme ubiquinone (Q); modifications, including acetylation and F1FO-ATPase or ATP synthase; the enzymes nitrosylation, are capable of influencing the of the tricarboxylic acid cycle (TCA), fatty bioenergetic response, with clinically acid and amino acid oxidation; transporters significant implications for health and of ions, metabolites and co-factors; disease (Carrico et al 2018). iron/sulphur cluster synthesis; and Mitochondria of higher eukaryotes mitochondrial kinases related to catabolic typically maintain several copies of their pathways. TCA cycle intermediates are vital own circular genome known as precursors for macromolecule biosynthesis mitochondrial DNA (mtDNA; hundred to (Diebold et al 2019). The mitochondrial thousands per cell; Cummins 1998), which is proteome comprises over 1,200 types of maternally inherited in many species. protein (Calvo et al 2015; 2017), mostly However, biparental mitochondrial encoded by nuclear DNA (nDNA), with a inheritance is documented in some variety of functions, many of which are exceptional cases in humans (Luo et al relatively well known, e.g., proteins 2018), is widespread in birds, fish, reptiles regulating mitochondrial biogenesis or and invertebrate groups, and is even the apoptosis, while others are still under norm in some bivalve taxonomic groups investigation, or need to be identified, e.g., (Breton et al 2007; White et al 2008). The mtPT pore and alanine transporter. The mitochondrial genome of the angiosperm

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Figure 1.

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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. The H+pos/O2 ratio is 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. Modified from Lemieux et al (2017) and Rich (2013). (c) OXPHOS-coupling: The H+ circuit couples O2 flux through the catabolic ET-

pathway, JkO2, to flux through the phosphorylation-pathway of ADP to ATP, JP». (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 Gnaiger (2020). Amborella contains a record of six has no mitochondrion whatsoever and lacks mitochondrial genome equivalents acquired all typical nuclear-encoded mitochondrial by horizontal transfer of entire genomes, two proteins, showing that while in 99 % of from angiosperms, three from algae and one organisms mitochondria play a vital role, this from mosses (Rice et al 2016). In unicellular organelle is not indispensable (Karnkowska organisms, i.e., protists, the structural et al 2016). organization of mitochondrial genomes is In vertebrates, but not all invertebrates, highly variable and includes circular and mtDNA is compact (16.5 kB in humans) and linear DNA (Zikova et al 2016). While some encodes 13 protein subunits of the of the free-living flagellates exhibit the transmembrane respiratory Complexes CI, largest known gene coding capacity, e.g., CIII, CIV and ATP synthase (F1FO-ATPase), 22 jakobid Andalucia godoyi mtDNA codes for tRNAs, and two ribosomal RNAs. Additional 106 genes (Burger et al 2013), some protist gene content has been suggested to include groups, e.g., alveolates, possess microRNAs, piRNA, smithRNAs, repeat mitochondrial genomes with only three associated RNA, long noncoding RNAs, and protein-coding genes and two rRNAs (Feagin even additional proteins or peptides et al 2012). The complete loss of (Rackham et al 2011; Duarte et al 2014; Lee mitochondrial genome is observed in the et al 2015; Cobb et al 2016). The highly reduced mitochondria of mitochondrial genome requires nuclear- Cryptosporidium species (Liu et al 2016). encoded mitochondrially targeted proteins, Reaching the final extreme, the microbial e.g., TFAM, for its maintenance and eukaryote, oxymonad Monocercomonoides, expression (Rackham et al 2012). The

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Consortium Communication nuclear and the mitochondrial genomes With an emphasis on quality of research, encode peptides of the membrane spanning published data can be useful far beyond the redox pumps (CI, CIII and CIV) and F1FO- specific question of a particular experiment. ATPase, leading to strong constraints in the For example, collaborative data sets support coevolution of both genomes (Blier et al the development of open-access databases 2001). such as those for National Institutes of Given the multiple roles of mitochondria, Health sponsored research in genetics, it is perhaps not surprising that proteomics, and metabolomics. Indeed, mitochondrial dysfunction is associated with enabling meta-analysis is the most economic a wide variety of genetic and degenerative way of providing robust answers to diseases. Robust mitochondrial function is biological questions (Cooper et al 2009). supported by physical exercise and caloric However, the reproducibility of quantitative balance, and is central for sustained results depend on accurate measurements metabolic health throughout life. Therefore, under strictly-defined conditions. Likewise, a more consistent set of definitions for meaningful interpretation and comparability mitochondrial physiology will increase our of experimental outcomes requires understanding of the etiology of disease and harmonization of protocols between improve the diagnostic repertoire of research groups at different institutes. In mitochondrial medicine with a focus on addition to quality control, a conceptual protective medicine, evolution, lifestyle, framework is also required to standardise environment, and healthy aging. and harmonise terminology and methodology. Vague or ambiguous jargon can lead to confusion and may convert valuable signals to wasteful noise. For this 1. Introduction reason, measured values must be expressed in standard units for each parameter used to Mitochondria are the powerhouses of the cell define mitochondrial respiratory function. A with numerous morphological, consensus on fundamental nomenclature physiological, molecular, and genetic and conceptual coherence, however, are functions (Box 1). Every study of missing in the expanding field of mitochondrial health and disease faces mitochondrial physiology. To fill this gap, the Evolution, Age, Gender and sex, Lifestyle, present communication provides an in- and Environment (MitoEAGLE) as essential depth review on harmonization of background conditions intrinsic to the nomenclature and definition of technical individual person or cohort, species, tissue terms, which are essential to improve the and to some extent even cell line. As a large awareness of the intricate meaning of and coordinated group of laboratories and current and past scientific vocabulary. This is researchers, the mission of the global important for documentation and MitoEAGLE Network is to generate the integration into data repositories in general, necessary scale, type, and quality of and quantitative modelling in particular consistent data sets and conditions to (Beard 2005). address this intrinsic complexity. In this review, we focus on coupling states Harmonization of experimental protocols and fluxes through metabolic pathways of and implementation of a quality control and aerobic energy transformation in data management system are required to mitochondrial preparations in the attempt to interrelate results gathered across a establish a conceptually-oriented spectrum of studies and to generate a nomenclature in bioenergetics and rigorously monitored database focused on mitochondrial physiology in a series of mitochondrial respiratory function. In this communications, prepared in the frame of way, researchers from a variety of the EU COST Action MitoEAGLE open to disciplines can compare their findings using global bottom-up input. clearly defined and accepted international standards.

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2. Coupling states and rates in selective permeability of ions, organic mitochondrial preparations molecules, and particles across the cell boundary. The intact plasma membrane ‘Every professional group develops its own prevents the passage of many water-soluble technical jargon for talking about matters mitochondrial substrates and inorganic of critical concern ... People who know a ions—such as succinate, adenosine word can share that idea with other diphosphate (ADP) and inorganic phosphate members of their group, and a shared (Pi) that must be precisely controlled at vocabulary is part of the glue that holds kinetically-saturating concentrations for the people together and allows them to create analysis of mitochondrial respiratory a shared culture’ (Miller 1991). capacities (Figure 2). Respiratory capacities delineate, comparable to channel capacity in 2.1. Cellular and mitochondrial respiration information theory (Schneider 2006), the upper boundary of the rate of O2 2.1.1. Aerobic and anaerobic catabolism consumption measured in defined and ATP turnover: In respiration, electron respiratory states. The intact plasma transfer is coupled to the phosphorylation of membrane limits the scope of investigations ADP to ATP, with energy transformation into mitochondrial respiratory function in mediated by the protonmotive force, pmF living cells, despite the activity of solute (Figure 2). Anabolic reactions are coupled to carriers, e.g., the sodium-dependent catabolism, both by ATP as the intermediary dicarboxylate transporter SLC13A3 and the energy currency and by small organic sodium-dependent phosphate transporter precursor molecules as building blocks for SLC20A2, which transport specific biosynthesis (Diebold et al 2019). Glycolysis metabolites across the plasma membrane of involves substrate-level phosphorylation of various cell types, and the availability of ADP to ATP in fermentation without plasma membrane-permeable succinate utilization of O2, studied mainly in living cells (Ehinger et al 2016). These limitations are and organisms. Many cellular fuel substrates overcome by the use of mitochondrial are catabolized to acetyl-CoA or to preparations. 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 protonmotive force and phosphorylation of ADP (Figure 1b and 1c). In contrast,extramitochondrial oxidation of Figure 2. Four-compartment model of fatty acids and amino acids proceeds oxidative phosphorylation partially in peroxisomes without coupling to Respiratory states (ET, OXPHOS, LEAK; ATP production: acyl-CoA oxidase catalyzes Table 1) and corresponding rates (E, P, L) are the oxidation of FADH with electron 2 connected by the protonmotive force, pmF. (1) transfer to O ; amino acid oxidases oxidize 2 ET-capacity, E, is partitioned into (2) flavin mononucleotide FMNH2 or FADH2 dissipative LEAK-respiration, L, when the (Figure 1a). Gibbs energy change of catabolic O2 flux is The plasma membrane separates the irreversibly lost, (3) net OXPHOS-capacity, P- intracellular compartment including the L, with partial conservation of the capacity to cytosol, nucleus, and organelles from the perform work, and (4) the ET-excess capacity, extracellular environment. Cell membranes E-P. Modified from Gnaiger (2020). include the plasma membrane and organellar membranes. The plasma membrane consists of a lipid bilayer with 2.1.2. Specification of biochemical dose: embedded proteins and attached organic Substrates, uncouplers, inhibitors, and other molecules that collectively control the chemical reagents are titrated to analyse cellular and mitochondrial function. Nominal

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Consortium Communication concentrations of these substances are 2.2. Mitochondrial preparations usually reported as initial amount of substance concentration [mol∙L-1] in the Mitochondrial preparations are defined as incubation medium. either isolated mitochondria or tissue and Kinetically-saturating conditions are cellular preparations in which the barrier evaluated by substrate kinetics to obtain the function of the plasma membrane is maximum reaction velocity or maximum disrupted. Since this entails the loss of cell pathway flux, in contrast to solubility- viability, mitochondrial preparations are not saturated conditions. When aiming at the studied in vivo. In contrast to isolated measurement of kinetically-saturated mitochondria and tissue homogenate processes—such as OXPHOS-capacities— preparations, mitochondria in the concentrations for substrates can be permeabilized tissues and cells are in situ chosen according to half-saturating relative to the plasma membrane. When substrate concentrations, c50, for metabolic studying mitochondrial preparations, pathways, or the Michaelis constant, Km, for substrate-uncoupler-inhibitor-titration enzyme kinetics. In the case of hyperbolic (SUIT) protocols are used to establish kinetics, only 80 % of maximum respiratory respiratory coupling control states (CCS) and capacity is obtained at a substrate pathway control states (PCS) that provide concentration of four times the c50, whereas reference values for various output variables substrate concentrations of 5, 9, 19 and 49 (Table 1). Physiological conditions in vivo times the c50 are theoretically required for deviate from these experimentally obtained reaching 83 %, 90 %, 95 % or 98 % of the states; this is because kinetically-saturating maximal rate (Gnaiger 2001). concentrations, e.g., of ADP, oxygen (O2; Other reagents are chosen to inhibit or dioxygen) or fuel substrates, may not apply alter a particular process. The amount of to physiological intracellular conditions. these chemicals in an experimental Further information is obtained in studies of incubation is selected to maximize effect, kinetic responses to variations in fuel avoiding unacceptable off-target substrate concentrations, [ADP], or [O2] in consequences that would adversely affect the range between kinetically-saturating the data being sought. Specifying the amount concentrations and anoxia (Gnaiger 2001). of substance in an incubation as nominal The cholesterol content of the plasma concentration in the aqueous incubation membrane is high compared to medium can be ambiguous (Doskey et al mitochondrial membranes (Korn 1969). 2015), particularly for cations (TPP+; Therefore, mild detergents—such as fluorescent dyes such as safranin, TMRM; digitonin and saponin—can be applied to Chowdhury et al 2015) and lipophilic selectively permeabilize the plasma substances (oligomycin, uncouplers, membrane via interaction with cholesterol; permeabilization agents; Doerrier et al this allows free exchange of organic 2018), which accumulate in the molecules and inorganic ions between the mitochondrial matrix or on biological cytosol and the immediate cell environment, membranes, respectively. Generally, dose while maintaining the integrity and can be specified per unit of biological sample, localization of organelles, cytoskeleton, and i.e., (nominal moles of xenobiotic)/(number the nucleus. Application of permeabilization of cells) [mol∙cell•1] or, as appropriate, per agents (mild detergents or toxins) leads to mass of biological sample [mol∙kg-1]. This washout of cytosolic marker enzymes—such approach to specification of dose provides a as lactate dehydrogenase—and results in the scalable parameter that can be used to complete loss of cell viability (tested by design experiments, help interpret a wide nuclear staining using plasma membrane- variety of experimental results, and provide impermeable dyes), while mitochondrial absolute information that allows researchers function remains intact (tested by worldwide to make the most use of cytochrome c stimulation of respiration). published data (Doskey et al 2015). The Digitonin concentrations have to be duration of exposure adds the dimension of optimized according to cell type, particularly time in contact with a particular dose. since mitochondria from cancer cells contain

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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 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)

OXPHOS P high, ADP- max. high kinetically- JP» by phosphorylation-

stimulated saturating [ADP] pathway capacity; or JkO2 by respiration, and [Pi] ET-capacity OXPHOS- 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 significantly higher contents of cholesterol in differential centrifugation, entailing the loss both membranes (Baggetto and Testa- of mitochondria at typical recoveries ranging Perussini, 1990). For example, a dose of from 30 % to 80 % of total mitochondrial digitonin of 8 fmol∙cell•1 (10 pg∙cell•1; 10 content (Lai et al 2018). Using Percoll or µg∙10•6 cells) is optimal for permeabilization sucrose density gradients to maximize the of endothelial cells, and the concentration in purity of isolated mitochondria may the incubation medium has to be adjusted compromise the mitochondrial yield or according to the cell concentration (Doerrier structural and functional integrity. et al 2018). Respiration of isolated Therefore, mitochondrial isolation protocols mitochondria remains unaltered after the need to be optimized according to each addition of low concentrations of digitonin study. The term mitochondrial preparation or saponin. In addition to mechanical cell neither includes living cells, nor disruption during homogenization of tissue, submitochondrial particles and further permeabilization agents may be applied to fractionated mitochondrial components. ensure permeabilization of all cells in tissue homogenates. 2.3. Electron transfer pathways Suspensions of cells permeabilized in the respiration chamber and crude tissue Mitochondrial electron transfer (ET) homogenates contain all components of the pathways are fuelled by diffusion and cell at highly dilute concentrations. All transport of substrates across the mtOM and mitochondria are retained in chemically- mtIM. In addition, the mitochondrial permeabilized mitochondrial preparations electron transfer system (ETS) consists of and crude tissue homogenates. In the the matrix-ETS and membrane-ETS (Figure preparation of isolated mitochondria, 1b). Upstream sections of ET-pathways however, the mitochondria are separated converge at the NADH-junction (N-junction). from other cell fractions and purified by NADH is mainly generated in the

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Consortium Communication tricarboxylic acid (TCA) cycle and is oxidized be produced by mitochondria, nor is there by Complex I (CI), with further electron entry any internal process without energy into the coenzyme Q-junction (Q-junction). conservation. Exergy or Gibbs energy (‘free Similarly, succinate is formed in the TCA energy’) is the part of energy that can cycle and oxidized by CII to fumarate. CII is potentially be transformed into work under part of both the TCA cycle and the ETS, and conditions of constant temperature and reduces FAD to FADH2 with further pressure. Coupling is the interaction of an reduction of ubiquinone to ubiquinol exergonic process (spontaneous, negative downstream of the TCA cycle in the Q- exergy change) with an endergonic process junction. Thus FADH2 is not a substrate but is (positive exergy change) in energy the product of CII, in contrast to erroneous transformations which conserve part of the metabolic maps shown in many publications. exergy change. Exergy is not completely β-oxidation of fatty acids (FA) supplies conserved, however, except at the limit of reducing equivalents via (1) FADH2 as the 100 % efficiency of energy transformation in substrate of electron transferring a coupled process. The exergy or Gibbs flavoprotein complex (CETF); (2) acetyl-CoA energy change that is not conserved by generated by chain shortening; and (3) copling is irreversibly lost or dissipated, and NADH generated via 3-hydroxyacyl-CoA is accounted for as the entropy change of the dehydrogenases. The ATP yield depends on surroundings and the system, multiplied by whether acetyl-CoA enters the TCA cycle, or the temperature of the irreversible process. is for example used in ketogenesis. Pathway control states (PCS) and Selected mitochondrial catabolic coupling control states (CCS) are pathways of electron transfer from the complementary, since mitochondrial oxidation of fuel substrates to the reduction preparations depend on (1) an exogenous of O2 are stimulated by addition of fuel supply of pathway-specific fuel substrates substrates to the mitochondrial respiration and oxygen, and (2) exogenous control of medium after depletion of endogenous phosphorylation (Figure 1). substrates (Figure 1b). Substrate combinations and specific inhibitors of ET- 2.4.2. Phosphorylation, P», and P»/O2 pathway enzymes are used to obtain defined ratio: Phosphorylation in the context of pathway control states in mitochondrial OXPHOS is defined as phosphorylation of preparations (Gnaiger 2020). ADP by Pi to form ATP. On the other hand, the term phosphorylation is used generally in 2.4. Respiratory coupling control many contexts, e.g., protein phosphorylation. This provides the argument for introducing a 2.4.1. Coupling: Coupling of electron transfer symbol more discriminating and specific (ET) to phosphorylation of ADP to ATP is than P as used in the P/O ratio (phosphate to mediated by vectorial translocation of protons atomic oxygen ratio), where P indicates across the mtIM. Proton pumps generate or phosphorylation of ADP to ATP or GDP to utilize the electrochemical pmF (Figure 1). GTP (Figure 1): The symbol P» indicates the The pmF is the sum of two partial forces, the endergonic (uphill) direction of electric force (electric potential difference) and phosphorylation ADP→ATP, and likewise P« chemical force (proton chemical potential the corresponding exergonic (downhill) difference, related to ΔpH; Mitchell 1961, hydrolysis ATP→ADP. P» refers mainly to 2011). The catabolic flux of scalar reactions is electrontransfer phosphorylation but may collectively measured as O2 flux, JkO2. also involve substrate-level phosphorylation Thus mitochondria are elementary as part of the TCA cycle (succinyl-CoA ligase, components of energy transformation. phosphoglycerate kinase) and Energy is a conserved quantity and cannot be phosphorylation of ADP catalyzed by lost or produced in any internal process pyruvate kinase, and of GDP phosphorylated (First Law of Thermodynamics). Open and by phosphoenolpyruvate carboxykinase. closed systems can gain or lose energy only Transphosphorylation is performed by by external fluxes—by exchange with the adenylate kinase, creatine kinase (mtCK), environment. Therefore, energy can neither hexokinase and nucleoside diphosphate

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BEC2020.1 doi:10.26124/bec:2020-0001.vTest05 kinase. In isolated mammalian mitochondria, (Watt et al 2010), in agreement with the ATP production catalyzed by adenylate measured P»/O ratio for succinate of 1.58 ± kinase (2 ADP ↔ ATP + AMP) proceeds 0.02 (Gnaiger et al 2000). without fuel substrates in the presence of ADP (Komlódi and Tretter 2017). Kinase 2.4.3. Uncoupling: The effective P»/O2 flux cycles are involved in intracellular energy ratio (YP»/O2 = JP»/JkO2) is diminished relative transfer and signal transduction for to the mechanistic P»/O2 ratio by intrinsic regulation of energy flux.The P»/O2 ratio and extrinsic uncoupling or dyscoupling (P»/4 e-) is two times the ‘P/O’ ratio (P»/2 (Figure 3). This is distinct from switching e•). P»/O2 is a generalized symbol, not between mitochondrial pathways that specific for reporting Pi consumption (Pi/O2 involve fewer than three proton pumps flux ratio), ADP depletion (ADP/O2 flux (‘coupling sites’: Complexes CI, CIII and CIV), ratio), or ATP production (ATP/O2 flux bypassing CI through multiple electron ratio). The mechanistic P»/O2 ratio—or entries into the Q-junction, or bypassing CIII P»/O2 stoichiometry—is calculated from the and CIV through AOX (Figure 1b). proton–to–O2 and proton–to– Reprogramming of mitochondrial pathways phosphorylation coupling stoichiometries leading to different types of substrates being (Figure 1c): oxidized may be considered as a switch of + Hpos/O2 gears (changing the stoichiometry by P»/O2 = + (1) Hneg/P» altering the substrate that is oxidized) rather

The H+pos/O2 coupling stoichiometry than uncoupling (loosening the tightness of (referring to the full four electron reduction coupling relative to a fixed stoichiometry). In of O2) depends on the relative involvement of addition, YP»/O2 depends on several the three coupling sites (respiratory experimental conditions of flux control, Complexes CI, CIII and CIV) in the catabolic increasing as a hyperbolic function of [ADP] ET-pathway from reduced fuel substrates to a maximum value (Gnaiger 2001). (electron donors) to the reduction of O2 Uncoupling of mitochondrial (electron acceptor). This varies with: (1) a respiration is a general term comprising bypass of CI by single or multiple electron diverse mechanisms (Figure 3): input into the Q-junction; and (2) a bypass of CIV by involvement of alternative oxidases, 1. Proton leak across the mtIM from the AOX. AOX are expressed in all plants, some positive to the negative compartment (H+ fungi, many protists, and several animal leak-uncoupled); phyla, but are not expressed in vertebrate 2. Cycling of other cations, strongly mitochondria (McDonald et al 2009). stimulated by mtPT; comparable to the The H+pos/O2 coupling stoichiometry use of protonophores, cation cycling is equals 12 in the ET-pathways involving CIII experimentally induced by valinomycin in and CIV as proton pumps, increasing to 20 the presence of K+ for the NADH-pathway through CI (Figure 3. Decoupling by proton slip in the redox 1b). A general consensus on H+pos/O2 proton pumps (CI, CIII and CIV) when stoichiometries, however, remains to be protons are effectively not pumped in the reached (Hinkle 2005; Wikström and ETS, or are not driving phosphorylation + Hummer 2012; Sazanov 2015). The H neg/P» (F1FO-ATPase) coupling stoichiometry (3.7; Figure 1b) is 4. Loss of vesicular (compartmental) the sum of 2.7 H+neg required by the F1FO- integrity when electron transfer is ATPase of vertebrate and most invertebrate acoupled species (Watt et al 2010) and the proton 5. Electron leak in the loosely coupled balance in the translocation of ADP, ATP and univalent reduction of O2 to superoxide Pi (Figure 1c). Taken together, the (O2•­; superoxide anion radical) mechanistic P»/O2 ratio is calculated at 5.4 and 3.3 for the N- and S-pathway, Differences of terms—uncoupled vs. respectively (Eq. 1). The corresponding noncoupled—are easily overlooked, classical P»/O ratios (referring to the 2 although they relate to different meanings of electron reduction of 0.5 O2) are 2.7 and 1.6 uncoupling (Table 2 and Figure 3).

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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; Canton et al 1995) (Fig. 2b-d) loosely 0 component of L, lower coupling due to superoxide coupled formation and bypass of proton pumps by electron

intrinsic, no protonophoreadded 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. Coupling states and respiratory rates substrates and inhibitors of specific branches of the ET-pathway. Concept-driven To extend the classical nomenclature on nomenclature aims at mapping the meaning mitochondrial coupling states (Section 2.6) and concept behind the words and acronyms by a concept-driven terminology that onto the forms of words and acronyms explicitly incorporates information on the (Miller 1991). The focus of concept-driven meaning of respiratory states, the nomenclature is primarily the conceptual terminology must be general and not why, along with clarification of the restricted to any particular experimental experimental how (Table 1). protocol or mitochondrial preparation (Gnaiger 2009). Diagnostically meaningful LEAK: The contribution of intrinsically and reproducible conditions are defined for uncoupled O2 consumption is studied by measuring mitochondrial function and preventing the stimulation of respiratory capacities of core energy phosphorylation either in the absence of metabolism. Standard respiratory coupling ADP or by inhibition of the states are obtained while maintaining a phosphorylation-pathway. The defined ET-pathway state with constant fuel corresponding states are collectively

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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.

classified as LEAK-states when O2 metabolic rates; for example: ET- consumption compensates mainly for ion pathways, ET-states, and ET-capacities, E, leaks, including the proton leak. respectively (Table 1). The protonmotive OXPHOS: The ET- and phosphorylation- force, pmF, is maximum in the LEAK-state pathways comprise coupled segments of of coupled mitochondria, driven by LEAK- the OXPHOS-system and provide respiration at a minimum back-flux of reference values of respiratory capacities. cations to the matrix side, high in the The OXPHOS-capacity is measured at OXPHOS-state when it drives kinetically-saturating concentrations of phosphorylation, and very low in the ET- ADP and Pi. state when uncouplers short-circuit the ET: Compared to OXPHOS-capacity, the proton cycle (Table 1). oxidative ET-capacity reveals the limitation of OXPHOS-capacity mediated 2.5.1. LEAK-state (Figure 4a): The LEAK- by the phosphorylation-pathway. By state is defined as a state of mitochondrial application of external uncouplers, ET- respiration when O2 flux mainly capacity is measured as noncoupled compensates for ion leaks in the absence of respirationThe three coupling states, ATP synthesis, at kinetically-saturating LEAK, OXPHOS, and ET are shown concentrations of O2 and respiratory fuel schematically with the corresponding substrates. LEAK-respiration is measured to respiratory rates, abbreviated as L, P, and obtain an estimate of intrinsic uncoupling E, respectively (Figure 2). We distinguish without addition of an experimental between metabolic pathways and uncoupler: (1) in the absence of adenylates, metabolic states with the corresponding i.e., AMP, ADP and ATP; (2) after depletion of

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ADP at a maximum ATP/ADP ratio; or (3) translocated by a redox proton pump of after inhibition of the phosphorylation- the ET-pathways and slip back to the pathway by inhibitors of F1FO-ATPase original vesicular compartment. The (oligomycin), or adenine nucleotide proton leak is the dominant contributor translocase (carboxyatractyloside). to the overall leak current in mammalian Adjustment of the nominal concentration of mitochondria incubated under these inhibitors to the concentration of physiological conditions at 37 °C, whereas biological sample applied can minimize or proton slip increases at lower avoid inhibitory side-effects exerted on ET- experimental temperature (Canton et al capacity or even some dyscoupling. The 1995). Proton slip can also happen in chelator EGTA is added to mt-respiration association with the F1FO-ATPase, in media to bind free Ca2+, thus limiting cation which the proton slips downhill across cycling. The LEAK-rate is a function of the pump to the matrix without respiratory state, hence it depends on (1) the contributing to ATP synthesis. In each barrier function of the mtIM (‘leakiness’), (2) case, proton slip is a property of the the electrochemical potential differences proton pump and increases with the and concentration differences across the pump turnover rate. mtIM, and (3) the H+/O2 ratio of the ET- • Electron leak and loosely coupled pathway (Figure 1b). respiration: Superoxide production by • Proton leak and uncoupled the ETS leads to a bypass of redox proton respiration: The intrinsic proton leak is pumps and correspondingly lower P»/O2 the uncoupled leak current of protons in ratio. This depends on the actual site of which protons diffuse across the mtIM in electron leak and the scavenging of the dissipative direction of the downhill superoxide by cytochrome c, whereby protonmotive force without coupling to electrons may re-enter the ETS with phosphorylation (Figure 4a). The proton proton translocation by CIV. leak flux depends non-linearly on the • Dyscoupled respiration: Mitochondrial protonmotive force (Garlid et al 1989; injuries may lead to dyscoupling as a Divakaruni and Brand 2011), which is a pathological or toxicological cause of temperature-dependent property of the uncoupled respiration. Dyscoupling may mtIM and may be enhanced due to involve any type of uncoupling possible contamination by free fatty acids. mechanism, e.g., opening the mtPT pore. Inducible uncoupling mediated by Dyscoupled respiration is distinguished uncoupling protein 1 (UCP1) is from experimentally induced noncoupled physiologically controlled, e.g., in brown respiration in the ET-state (Table 2). adipose tissue. UCP1 is a member of the • Protonophore titration and mitochondrial carrier family that is noncoupled respiration: involved in the translocation of protons Protonophores are uncouplers which are across the mtIM (Jezek et al 2018). titrated to obtain maximum noncoupled Consequently, this short-circuit lowers respiration as a measure of ET-capacity. the pmF and stimulates electron transfer, • Loss of compartmental integrity and respiration, and heat dissipation in the acoupled respiration: Electron transfer absence of phosphorylation of ADP. and catabolic O2 flux proceed without • Cation cycling: There can be other cation compartmental proton translocation in contributors to leak current including disrupted mitochondrial fragments. Such Ca2+ and probably magnesium. Ca2+ influx fragments are an artefact of is balanced by mitochondrial Na+/Ca2+ or mitochondrial isolation, and may not fully H+/Ca2+ exchange, which is balanced by fuse to re-establish structurally intact Na+/H+ or K+/H+ exchanges. This is mitochondria. Loss of mtIM integrity, another effective uncoupling mechanism therefore, is the cause of acoupled different from proton leak (Table 2). respiration, which is a nonvectorial • Proton slip and decoupled respiration: dissipative process without control by the Proton slip is the decoupled process in protonmotive force. which protons are only partially

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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 coupled to phosphorylation, JP», which is stimulated by kinetically- saturating [ADP] and [Pi], supported by a high protonmotive force maintained by pumping of protons to the positive + compartment, JmH pos. O2 flux, JkO2,P, is well-coupled at a P»/O2 flux ratio of -1 JP»,P∙JO2,P . Extramitochondrial ATPases may recycle ATP, 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. 2.5.2. OXPHOS-state (Figure 4b): The (Rostovtseva et al 2008) or other OXPHOS-state is defined as the respiratory intracellular structures (Birkedal et al 2014). state with kinetically-saturating In addition, kinetically-saturating ADP concentrations of O2, respiratory and concentrations need to be evaluated under phosphorylation substrates, and absence of different experimental conditions such as exogenous uncoupler, which provides an temperature (Lemieux et al 2017) and with estimate of the maximal respiratory capacity different animal models (Blier and Guderley in the OXPHOS-state for any given ET- 1993). In permeabilized muscle fiber pathway state. Respiratory capacities at bundles of high respiratory capacity, the kinetically-saturating substrate apparent Km for ADP increases up to 0.5 mM concentrations provide reference values or (Saks et al 1998), consistent with upper limits of performance, aiming at the experimental evidence that >90 % kinetic generation of data sets for comparative saturation is reached only at >5 mM ADP purposes. Physiological activities and effects (Pesta and Gnaiger 2012). Similar ADP of substrate kinetics can be evaluated concentrations are also required for accurate relative to the OXPHOS-capacity. determination of OXPHOS-capacity in human As discussed previously, 0.2 mM ADP clinical cancer samples and permeabilized does not kinetically-saturate flux in isolated cells (Klepinin et al 2016; Koit et al 2017). 2.5 mitochondria (Gnaiger 2001; Puchowicz et to 5 mM ADP is sufficient to obtain the actual al 2004); greater [ADP] is required, OXPHOS-capacity in many types of particularly in permeabilized muscle fibers permeabilized tissue and cell preparations, and cardiomyocytes, to overcome limitations but experimental validation is required in by intracellular diffusion and by the reduced each specific case. conductance of the mtOM (Jepihhina et al 2011; Illaste et al 2012; Simson et al 2016), 2.5.3. Electron transfer-state (Figure 4c): either through interaction with tubulin O2 flux determined in the ET-state yields an

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Consortium Communication estimate of ET-capacity. The ET-state is (Vamecq et al 1987). Rox represents a defined as the noncoupled state with baseline used to correct respiration kinetically-saturating concentrations of O2, measured in defined coupling control states. respiratory substrate and optimum Rox-corrected L, P and E are not only lower exogenous uncoupler concentration for than total fluxes, but also change the flux maximum O2 flux. Uncouplers are weak lipid- control ratios L/P and L/E. Rox is not soluble acids which function as necessarily equivalent to non-mitochondrial protonophores. These disrupt the barrier reduction of O2, considering O2-consuming function of the mtIM and thus short-circuit reactions in mitochondria that are not the protonmotive system, functioning like a related to ET—such as O2 consumption in clutch in a mechanical system. As a reactions catalyzed by monoamine oxidases consequence of the nearly collapsed (type A and B), monooxygenases protonmotive force, the driving force is (cytochrome P450 monooxygenases), insufficient for phosphorylation, and JP» = 0. dioxygenases (trimethyllysine dioxygenase), The most frequently used uncouplers are and several hydoxylases. Isolated carbonyl cyanide m-chloro phenyl mitochondrial fractions, especially those hydrazone (CCCP), carbonyl cyanide p- obtained from liver, may be contaminated by trifluoromethoxyphenylhydrazone (FCCP), peroxisomes, as shown by transmission or dinitrophenol (DNP). Stepwise titration of electron microscopy. This fact makes the uncouplers stimulates respiration up to or exact determination of mitochondrial O2 above the level of O2 consumption rates in consumption and mitochondria-associated the OXPHOS-state; respiration is inhibited, generation of reactive oxygen species however, above optimum uncoupler complicated (Schönfeld et al 2009; Speijer concentrations (Mitchell 2011). Data 2016; Figure 1). The variability of ROX- obtained with a single dose of uncoupler linked O2 consumption needs to be studied in must be evaluated with caution, particularly relation to non-ET enzyme activities, when a fixed uncoupler concentration is availability of specific substrates, O2 used in studies exploring a treatment or concentration, and electron leakage leading disease that may alter the mitochondrial to the formation of reactive oxygen species. content or mitochondrial sensitivity to inhibition by uncouplers. There is a need for 2.5.5. Quantitative relations: E may exceed new protonophoric uncouplers that drive or be equal to P. E > P is observed in many maximal respiration across a broad dosing types of mitochondria, varying between range and do not inhibit respiration at high species, tissues and cell types (Gnaiger concentrations (Kenwood et al 2013). The 2009). E-P is the ET-excess capacity pushing effect on ET-capacity of the reversed the phosphorylation-flux to the limit of its function of F1FO-ATPase (JP«; Figure 4c) can capacity for utilizing the protonmotive force be evaluated in the presence and absence of (Figure 2). In addition, the magnitude of E-P extramitochondrial ATP. depends on the tightness of respiratory coupling or degree of uncoupling, since an 2.5.4. ROX state: The state of residual O2 increase of L causes P to increase towards consumption, ROX, is not a coupling state, the limit of E (Lemieux et al 2011). The ET- but is relevant to assess respiratory function excess capacity, E-P, therefore, provides a (Overview). The rate of residual oxygen sensitive diagnostic indicator of specific consumption, Rox, is defined as O2 injuries of the phosphorylation-pathway, consumption due to oxidative reactions under conditions when E remains constant measured after inhibition of ET with but P declines relative to controls. Substrate rotenone, malonic acid and antimycin A. cocktails supporting simultaneous Cyanide and azide inhibit not only CIV but convergent electron transfer to the Q- catalase and several peroxidases involved in junction for reconstitution of TCA cycle Rox, whereas AOX is not inhibited (Figure function establish pathway control states 1b). High concentrations of antimycin A, but with high ET-capacity, and consequently not rotenone or cyanide, inhibit peroxisomal increase the sensitivity of the E-P assay. acyl-CoA oxidase and D-amino acid oxidase

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E cannot theoretically be lower than P. E experimental conditions, we cannot separate < P must be discounted as an artefact, which the tightness of coupling versus coupling may be caused experimentally by: (1) loss of stoichiometry as the mechanisms of oxidative capacity during the time course of respiratory control in a shift of L/P ratios. the respirometric assay, since E is measured The tightness of coupling and fully coupled subsequently to P; (2) using insufficient O2 flux, P-L (Table 2), therefore, are obtained uncoupler concentrations; (3) using high from measurements of coupling control of uncoupler concentrations which inhibit ET LEAK-respiration, OXPHOS- and ET- (Gnaiger 2008); (4) high oligomycin capacities in well-defined pathway states, concentrations applied for measurement of L using either pyruvate and malate as before titrations of uncoupler, when substrates or the classical succinate and oligomycin exerts an inhibitory effect on E. rotenone substrate-inhibitor combination On the other hand, the ET-excess capacity is (Figure 1b). overestimated if kinetically non-saturating [ADP] or [Pi] are used. See State 3 in the next 2.5.6. The steady-state: Mitochondria section. represent a thermodynamically open system The net OXPHOS-capacity is calculated by in non-equilibrium states of biochemical subtracting L from P (Figure 2). The net energy transformation. State variables P»/O2 equals P»/(P-L), wherein the (protonmotive force; redox states) and dissipative LEAK component in the OXPHOS- metabolic rates (fluxes) are measured in state may be overestimated. This can be defined mitochondrial respiratory states. avoided by measuring LEAK-respiration in a Steady-states can be obtained only in open state when the protonmotive force is systems, in which changes by internal adjusted to its slightly lower value in the transformations, e.g., O2 consumption, are OXPHOS-state by titration of an ET-inhibitor instantaneously compensated for by (Divakaruni and Brand 2011). Any turnover- external fluxes across the system boundary, dependent components of proton leak and e.g., O2 supply, preventing a change of O2 slip, however, are underestimated under concentration in the system (Gnaiger these conditions (Garlid et al 1993). In 1993b). Mitochondrial respiratory states general, it is inappropriate to use the term monitored in closed systems satisfy the ATP production or ATP turnover for the criteria of pseudo-steady states for limited difference of O2 flux measured in the periods of time, when changes in the system OXPHOS- and LEAK-states. P-L is the upper (concentrations of O2, fuel substrates, ADP, limit of OXPHOS-capacity that is freely Pi, H+) do not exert significant effects on available for ATP production (corrected for metabolic fluxes (respiration, LEAK-respiration) and is fully coupled to phosphorylation). Such pseudo-steady states phosphorylation with a maximum require respiratory media with sufficient mechanistic stoichiometry (Figure 2). buffering capacity and substrates LEAK-respiration and OXPHOS-capacity maintained at kinetically-saturating depend on (1) the tightness of coupling concentrations, and thus depend on the under the influence of the respiratory kinetics of the processes under investigation. uncoupling mechanisms (Figure 3), and (2) the coupling stoichiometry, which varies as a 2.6. Classical terminology for isolated function of the substrate type undergoing mitochondria oxidation in ET-pathways with either two or ‘When a code is familiar enough, it ceases three coupling sites (Figure 1b). When appearing like a code; one forgets that substrate cocktails are used supporting the there is a decoding mechanism. The convergent NADH- and succinate-pathways message is identical with its meaning’ simultaneously, the relative contribution of (Hofstadter 1979). ET-pathways with three or two coupling sites cannot be controlled experimentally, is Chance and Williams (1955; 1956) difficult to determine, and may shift in introduced five classical states of transitions between LEAK-, OXPHOS- and mitochondrial respiration and cytochrome ET-states (Gnaiger 2020). Under these redox states. Table 3 shows a protocol with

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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 isolated mitochondria in a closed transitions of isolated mitochondria in a respirometric chamber, defining a sequence closed respirometric chamber. Repeated of respiratory states. States and rates are not ADP titration re-establishes State 3 at ‘high distinguished in this nomenclature. ADP’. Starting at O2 concentrations near air- saturation (193 or 238 µM O2 at 37 °C or 25 2.6.1. State 1 is obtained after addition of °C and sea level at 1 atm or 101.32 kPa, and isolated mitochondria to air-saturated an oxygen solubility of respiration medium isoosmotic/isotonic respiration medium at 0.92 times that of pure water; Forstner containing Pi, but no fuel substrates and no and Gnaiger 1983), the total ADP adenylates. concentration added must be low enough (typically 100 to 300 µM) to allow 2.6.2. State 2 is induced by addition of a phosphorylation to ATP at a coupled O2 flux ‘high’ concentration of ADP (typically 100 to that does not lead to O2 depletion during the 300 µM), which stimulates respiration transition to State 4. In contrast, kinetically- transiently on the basis of endogenous fuel saturating ADP concentrations usually are substrates and phosphorylates only a small 10-fold higher than 'high ADP', e.g., 2.5 mM in portion of the added ADP. State 2 is then isolated mitochondria. The abbreviation obtained at a low respiratory activity limited State 3u is occasionally used in by exhausted endogenous fuel substrate bioenergetics, to indicate the state of availability (Table 3). If addition of specific respiration after titration of an uncoupler, inhibitors of respiratory complexes such as without sufficient emphasis on the rotenone does not cause a further decline of fundamental difference between OXPHOS- O2 flux, State 2 is equivalent to the ROX state capacity (well-coupled with an endogenous (Table 1). Undefined endogenous fuel uncoupled component) and ET-capacity substrates are a confounding factor of (noncoupled). pathway control, contributing to the effect of subsequently externally added substrates 2.6.4. State 4 is a LEAK-state that is obtained and inhibitors. In an alternative sequence of only if the mitochondrial preparation is titration steps, the second state is induced by intact and well-coupled. Depletion of ADP by addition of fuel substrate without ADP or phosphorylation to ATP causes a decline of ATP (Estabrook 1967). In contrast to the O2 flux in the transition from State 3 to State original State 2 defined in Table 1 as a ROX 4. Under the conditions of State 4, a state, the alternative ‘State 2’ is a LEAK-state maximum protonmotive force and high with L(n). Some researchers have called this ATP/ADP ratio are maintained. The gradual condition as ‘pseudostate 4’. decline of YP»/O2 towards diminishing [ADP] at State 4 must be taken into account for 2.6.3. State 3 is the state stimulated by calculation of P»/O2 ratios (Gnaiger 2001). addition of fuel substrates while the ADP State 4 respiration, L(T) (Table 1), reflects concentration in the original State 2 is still intrinsic proton leak and ATP hydrolysis high (Table 3) and supports coupled energy activity. O2 flux in State 4 is an transformation. 'High ADP' is a overestimation of LEAK-respiration if the concentration of ADP specifically selected to contaminating ATP hydrolysis activity allow the measurement of State 3 to State 4 recycles some ATP to ADP, JP«, which

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BEC2020.1 doi:10.26124/bec:2020-0001.vTest05 stimulates respiration coupled to protonmotive force, redox states, flux–force phosphorylation, JP» > 0. Some degree of relationships, coupling and efficiency; (5) mechanical disruption and loss of Ca2+ and other ions including H+; (6) mitochondrial integrity allows the exposed inhibitors, e.g., nitric oxide or intermediary mitochondrial F1FO-ATPases to hydrolyze metabolites such as oxaloacetate; (7) the ATP synthesized by the fraction of signalling pathways and regulatory proteins, coupled mitochondria. This can be tested by e.g., insulin resistance, transcription factor inhibition of the phosphorylation-pathway hypoxia inducible factor 1. using oligomycin, ensuring that JP» = 0 (State Mechanisms of respiratory control and 4o). On the other hand, the State 4 regulation include adjustments of: (1) respiration reached after exhaustion of enzyme activities by allosteric mechanisms added ADP is a more physiological condition, and phosphorylation; (2) enzyme content, i.e., presence of ATP, ADP and even AMP. concentrations of cofactors and conserved Sequential ADP titrations re-establish State moieties such as adenylates, nicotinamide 3, followed by State 3 to State 4 transitions adenine dinucleotide [NAD+/NADH], while sufficient O2 is available. Anoxia may coenzyme Q, cytochrome c; (3) metabolic be reached, however, before exhaustion of channeling by supercomplexes; and (4) ADP (State 5). mitochondrial density (enzyme concentrations) and morphology 2.6.5. State 5 ‘may be obtained by antimycin (membrane area, cristae folding, fission and A treatment or by anaerobiosis’ (Chance and fusion). Mitochondria are targeted directly Williams, 1955). These definitions give State by hormones, e.g., progesterone and 5 two different meanings: ROX or anoxia. glucacorticoids, which affect their energy Anoxia is obtained after exhaustion of O2 in a metabolism (Lee et al 2013; Gerö and Szabo closed respirometric chamber. Diffusion of 2016; Price and Dai 2016; Moreno et al 2017; O2 from the surroundings into the aqueous Singh et al 2018). Evolutionary or acquired solution may be a confounding factor differences in the genetic and epigenetic preventing complete anoxia (Gnaiger 2001). basis of mitochondrial function (or In Table 3, only States 3 and 4 are dysfunction) between individuals; age; coupling control states, with the restriction biological sex, and hormone concentrations; that rates in State 3 may be limited life style including exercise and nutrition; kinetically by non-saturating ADP and environmental issues including thermal, concentrations. atmospheric, toxic and pharmacological factors, exert an influence on all control 2.7. Control and regulation mechanisms listed above. For reviews, see Brown 1992; Gnaiger 1993a, 2009; 2020; The terms metabolic control and regulation Paradies et al 2014; Morrow et al 2017. are frequently used synonymously, but are Lack of control by a metabolic pathway, distinguished in metabolic control analysis: e.g., phosphorylation-pathway, means that “We could understand the regulation as the there will be no response to a variable mechanism that occurs when a system activating it, e.g., [ADP]. The reverse, maintains some variable constant over time, however, is not true as the absence of a in spite of fluctuations in external conditions response to [ADP] does not exclude the (homeostasis of the internal state). On the phosphorylation-pathway from having some other hand, metabolic control is the power to degree of control. The degree of control of a change the state of the metabolism in component of the OXPHOS-pathway on an response to an external signal” (Fell 1997). output variable, such as O2 flux, will in Respiratory control may be induced by general be different from the degree of experimental control signals that exert an control on other outputs, such as influence on: (1) ATP demand and ADP phosphorylation-flux or proton leak flux. phosphorylation-rate; (2) fuel substrate Therefore, it is necessary to be specific as to composition, pathway competition; (3) which input and output are under available amounts of substrates and O2, e.g., consideration (Fell 1997). starvation and hypoxia; (4) the

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Respiratory control refers to the ability of quantity (J; per system size). Flows, Itr, are mitochondria to adjust O2 flux in response to defined for all transformations as extensive external control signals by engaging various quantities. This is a generalization derived mechanisms of control and regulation. from electrical terms: Electric charge per Respiratory control is monitored in a unit time is electric flow or current, Iel = mitochondrial preparation under conditions dQel∙dt-1 [A≡ C∙s•1]. When dividing Iel by size defined as respiratory states, preferentially of the system (cross-sectional area of a under near-physiological conditions of ‘wire’), we obtain flux as a size-specific temperature, pH, and medium ionic quantity; this is the current density (surface- composition, to generate data of higher density of flow) perpendicular to the biological relevance. When phosphorylation direction of flux, Jel = Iel∙A-1 [A∙m•2] (Cohen et of ADP to ATP is stimulated or depressed, an al 2008). Fluxes with spatial geometric increase or decrease is observed in electron direction and magnitude are vectors. Vector transfer measured as O2 flux in respiratory and scalar fluxes are related to flows as Jtr = coupling states of intact mitochondria Itr∙A-1 [mol∙s-1∙m-2] and Jtr = Itr∙V-1 (‘controlled states’ in the classical [mol∙s•1∙m•3], expressing flux as an area- terminology of bioenergetics). Alternatively, specific vector or volume-specific vectorial coupling of electron transfer with or scalar quantity, respectively (Gnaiger phosphorylation is diminished by 1993b). We use the metre–kilogram– uncouplers. The corresponding coupling second–ampere (MKSA) international control state is characterized by a high system of units (SI) for general cases ([m], respiratory rate without control by P» [kg], [s] and [A]), with decimal SI prefixes for (noncoupled or ‘uncontrolled state’). specific applications (Table 4). We suggest defining: (1) vectoral fluxes, 3. What is a rate? which are translocations as functions of gradients with direction in geometric space The term rate is not adequately defined to be in continuous systems; (2) vectorial fluxes, useful for reporting data. Normalization of which describe translocations in rates leads to a diversity of formats. discontinuous systems and are restricted to Application of common and defined units is information on compartmental differences required for direct transfer of reported (transmembrane proton flux); and (3) scalar results into a data repository. The second [s] fluxes, which are localized transformations is the SI unit for the base quantity time. It is without translocation, such as chemical also the standard time-unit used in solution reactions in a homogenous system (catabolic chemical kinetics. O2 flux, JkO2). The inconsistency of the meanings of rate becomes apparent when considering Galileo • Extensive quantities: An extensive Galilei’s famous principle, that ‘bodies of quantity increases proportionally with different weight all fall at the same rate (have system size. For example, mass and a constant acceleration)’ (Coopersmith volume are extensive quantities. Flow is 2010). A rate may be an extensive quantity, an extensive quantity. The magnitude of which is a flow, I, when expressed per object an extensive quantity is completely (per number of cells or organisms) or per additive for non-interacting subsystems. chamber (per instrumental system). System The magnitude of these quantities is defined as the open or closed chamber of depends on the extent or size of the the measuring device. A rate is a flux, J, when system (Cohen et al 2008). expressed as a size-specific quantity (Figure • Size-specific quantities: ‘The adjective 5A; Box 2). specific before the name of an extensive

quantity is often used to mean divided by Box 2: Metabolic flows and fluxes: mass’ (Cohen et al 2008). The term vectoral, vectorial, and scalar specific has different meanings in three particular contexts: (1) In the system- Flow is an extensive quantity (I; per system), paradigm, (a) mass-specific flux is flow distinguished from flux as a size-specific divided by mass of the system (the mass

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Figure 5. Flow and flux, and normalization in structure- function analysis (A) A fundamental distinction is made between metabolic rate related to the experimental sample (left) or to the instrumental chamber (right). The different meanings of rate need to be specified by the type of normalization. Left: Results are expressed as mass-specific flux, JmX, per mg protein, dry or wet mass. Cell volume, Vce, may be used for normalization (volume-specific flux, JVce). Normalization per mitochondrial elementary marker, mtE, relies on determination of mt- markers expressed in various mitochondrial elementary units [mtEU]. Right: Flow per instrumental chamber, I, or flux per chamber volume, JV, are merely reported for methodological reasons.

(B) O2 flow per cell, IO2/Nce, is the product of mitochondria- specific flux, mt-density and mass per cell. Unstructured analysis: performance is the product of mass- •1 •1 specific flux, JO2/mce [mol∙s ∙kg ], and size (mass per cell). Structured analysis: performance is the product of mitochondrial function (mt-specific flux) and structure (mt-content). Modified from Gnaiger (2020). For further details see Table 4. of everything contained in the specific compared to brain-specific instrumental chamber or reactor). (b) properties). Rates are frequently expressed as • Intensive quantities: In contrast to size- volume-specific flux (volume of the specific properties, forces are intensive instrumental chamber). A mass-specific quantities defined as the change of an or volume-specific quantity is extensive quantity per advancement of an independent of the extent of non- energy transformation (Gnaiger 1993b). interacting homogenous subsystems. (2) • Formats: The quantity of a sample X can In the context of sample size, tissue- be expressed in different formats. nX, NX, specific quantities are related to the mass and mX are the molar amount, number, or volume of the sample in contrast to the and mass of X, respectively. When mass or volume of the system (e.g., muscle different formats are indicated in symbols mass-specific or cell volume-specific of derived quantities, the format (n, N, m) normalization; Figure 5). (3) An entirely is shown as a subscript (underlined italic), different meaning is implied in the such as in IO2/NX and JO2/mX. As of 2019 May context of sample type (e.g., muscle- 20, the definition of the SI unit mole [mol] is based on a natural constant, namely

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Table 4. Sample concentratio ns and normalization of flux.

Expression Symbol Definition Unit Notes

Sample identity of sample X object: cell, tissue, animal, patient number of sample entities X NX number of objects x 1

mass of sample X mX kg 2

mass of object X mNX mNX = mX∙NX-1 kg∙x•1 2 Mitochondria mitochondria mt X = mt amount of mt-elementary marker mtE quantity of mt-marker mtEU Concentrations

object number concentration CNX CNX = NX∙V-1 x∙m•3 3

sample mass concentration CmX CmX = mX∙V-1 kg∙m•3

mitochondrial concentration CmtE CmtE = mtE∙V-1 mtEU∙m•3 4

specific mitochondrial density DmtE DmtE = mtE∙mX-1 mtEU∙kg•1 5 mitochondrial content, mtE per number of sample mtENX mtENX = mtE∙NX-1 mtEU∙x•1 6 entities (objects) X O2 flow and flux 7 -1 flow, system IO2 internal flow mol∙s 8 -1 -1 -3 volume-specific flux JV,O2 JV,O2 = IO2∙V mol∙s ∙m 9 -1 -1 -1 flow per object X IO2/NX IO2/NX = JV,O2∙CNX mol∙s ∙x 10 -1 -1 -1 mass-specific flux JO2/mX JO2/mX = JV,O2∙CmX mol∙s ∙kg -1 -1 -1 mt-marker-specific flux JO2/mtE JO2/mtE = JV,O2∙CmtE mol∙s ∙mtEU 11 1 The unit x for a number is not used by IUPAC. To avoid confusion, the units [kg∙x-1] and [kg] distinguish the mass per object from the mass of a sample that may contain any number of objects. Similarly, the units for flow per system versus flow per object are [mol∙s-1] (Note 8) and [mol∙s-1∙x-1] (Note 10). 2 Units are given in the MKSA system (Box 2). The SI prefix k is used for the SI base unit of mass (kg = 1,000 g). In praxis, various SI prefixes are used for convenience, to make numbers easily readable, e.g., 1 mg tissue, cell or mitochondrial mass instead of 0.000001 kg. 3 In case of cells (sample X = cells), the object number concentration is CNce = Nce∙V-1, and volume may be expressed in [dm3 ≡ L] or [cm3 = mL]. See Table 5 for different object types. 4 mt-concentration is an experimental variable, dependent on sample concentration: (1) CmtE = mtE∙V•1; (2) CmtE = mtEX∙CNX; (3) CmtE = CmX∙DmtE. 5 If the amount of mitochondria, mtE, is expressed as mitochondrial mass, then DmtE is the mass fraction of mitochondria in the sample. If mtE is expressed as mitochondrial volume, Vmt, and the mass of sample, mX, is replaced by volume of sample, VX, then DmtE is the volume fraction of mitochondria in the sample. 6 mtENX = mtE∙NX-1 = CmtE∙CNX-1. 7 O2 can be replaced by other chemicals to study different reactions, e.g., ATP, H2O2, or vesicular compartmental translocations, e.g., Ca2+. 8 IO and V are defined per instrumental chamber as a system of constant volume (and constant temperature), which 2 may be closed or open. IO is abbreviated for IrO , i.e., the metabolic or internal O2 flow of the chemical reaction r in 2 2 which O2 is consumed, hence the negative stoichiometric number, νO = -1. IrO = drnO /dt∙νO -1. If r includes all chemical 2 2 2 2 reactions in which O2 participates, then drnO = dnO – denO , where dnO is the change in the amount of O2 in the 2 2 2 2 instrumental chamber and denO is the amount of O2 added externally to the system. At steady state, by definition dnO 2 2 = 0, hence drnO = –denO . Note that in this context ‘external’, e, refers to the instrumental system, whereas in respiratory 2 2 physiology ‘external’, ext, refers to the organism (Overview). 9 JV,O is an experimental variable, expressed per volume of the instrumental chamber. 2 10 IO /NX is a physiological variable, depending on the size of entity X. 2 11 There are many ways to normalize for a mitochondrial marker, that are used in different experimental approaches: (1) JO /mtE = JV,O ∙CmtE-1; (2) JO /mtE = JV,O ∙CmX-1∙DmtE-1 = JO /mX∙DmtE•1; (3) JO /mtE = JV,O ∙CNX•1∙mtENX•1 = IO /NX∙mtENX-1; (4) JO /mtE 2 2 2 2 2 2 2 2 2 = IO ∙mtE-1. The mt-elementary unit [mtEU] varies depending on the mt-marker. 2 24 of 44 Gnaiger E et al (2020) Bioenerg Commun 2020.1

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Avogadro’s constant: one mole contains respiratory dysfunction in dead cells should exactly 6.02214076∙1023 elementary be eliminated as a confounding factor. entities, in contrast to the former definition in terms of the number of 4.2. Size-specific flux: per sample size molaratoms in the mass of 0.012 kilogram of carbon 12 (Gibney 2018). Metabolic 4.2.1. Sample concentration, CmX: oxygen flow and flux are expressed in the Considering permeabilized tissue,

format, nO2 [mol], but in the volume homogenate or cells as the sample, X, the 3 sample mass is mX [mg], which is frequently format, VO2 [m ], in ergometry. These measured as wet or dry mass, mw or md [mg], formats are distinguished as JnO2/mX and respectively, or as mass of protein, mProtein. The J , respectively, for mass-specific flux. VO2/mX sample concentration is the mass of the Further examples are given in Table 4 subsample per volume of the instrumental -1 -1 •1 and Figure 5. chamber, CmX = mX∙V [g∙L = mg∙mL ]. X is the type of sample—isolated mitochondria, 4. Normalization of rate per sample tissue homogenate, permeabilized muscle fibers or cells (Table 4). mce [mg] is the total mass of The challenges of measuring mitochondrial all cells in an instrumental chamber, whereas -1 -1 respiratory flux are matched by those of mNce = mce∙Nce [mg∙x ] is the (average) mass of normalization. Normalization (Table 4) is an individual cell (Table 5). guided by physicochemical principles, methodological considerations, and 4.2.2. Size-specific flux: Cellular O2 flow conceptual strategies (Figure 5). can be compared between cells of identical size. To take into account changes and 4.1. Flow: per object differences in cell size (Renner et al 2003), normalization is required to obtain cell size- 4.1.1. Number concentration, CNX: specific or mitochondrial marker-specific O2 Normalization per sample concentration is flux (Figure 5). routinely required to report respiratory -1 -1 data. CNX is the experimental number • Mass-specific flux, JO2/mX [mol∙s ∙kg ]: concentration of sample X. In the case of Mass-specific flux is the expression of animals NX is the number of organisms in the respiration per mass of sample, mX [mg]. chamber, e.g., nematodes, CNX = NX∙V-1 [x∙L-1]. Chamber volume-specific flux, JV,O2, is Similarly, the number of cells per chamber divided by mass concentration of X in the volume is the number concentration of cells, chamber, JO /mX = JV,O ∙CmX-1. Cell mass- C = N ∙V•1 [x∙L•1], where N is the number 2 2 Nce ce ce specific flux is obtained by dividing flow of cells in the chamber (Table 4). per cell by mass per cell, J = O2/mce -1 IO2/Nce∙mNce . 4.1.2. Flow per object, IO2/NX: O2 flow per cell is calculated from volume-specific O2 • Cell volume-specific flux, JO2/VX •1 •3 flux, JV,O [nmol∙s•1∙L•1] (per V of the [mol∙s ∙m ]: Sample volume-specific flux 2 is obtained by expressing respiration per instrumental chamber [L]), divided by the volume of sample. number concentration of cells. The total cell count is the sum of viable and dead cells, N ce If size-specific O flux is constant and = N +N (Table 5). The cell viability index, 2 vce dce independent of sample size, then there is no VI = N ∙N •1, is the ratio of the number of vce ce interaction between the subsystems. For viable cells, N , per total number of living vce example, 1.5 mg and 3.0 mg sub-samples of cells in the population. After experimental muscle tissue respire at identical mass- permeabilization, all cells are permeabilized, specific flux. If mass-specific O flux, N = N . The cell viability index can be used 2 pce ce however, changes as a function of the mass of to normalize respiration for the number of a tissue sample, cells or isolated cells that have been viable before mitochondria in the instrumental chamber, experimental permeabilization, I = O2/Nvce then the nature of the interaction becomes I ∙VI•1, considering that mitochondrial O2/Nce an issue. Therefore, cell concentration must

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Table 5. Sample types, X, abbreviations, and quantification.

Identity of sample X NX Massa Volume mt-Marker mitochondrial preparation [x] [kg] [kg∙x-1] [m3] [m3∙x-1] [mtEU]

isolated mitochondria imt mmt Vmt mtE tissue homogenate thom mthom mtEthom permeabilized tissue pti mpti mtEpti permeabilized muscle fibers pfi mpfi mtEpfi permeabilized cells pce Npce mpce mNpce Vpce VNpce mtEpce living cellsb ce Nce mce mNce Vce VNce mtEce viable cells vce Nvce mvce mNvce Vvce VNvce dead cells dce Ndce mdce mNdce Vdce VNdce organisms org Norg morg mNorg Vorg VNorg

a Instead of mass, the wet weight or dry weight is frequently stated, Ww or Wd. mX is mass of -1 the sample [kg], mNX is mass of the object [kg∙x ] (Table 4). b 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. be optimized, particularly in experiments bias should be taken into account when carried out in wells, considering the planning experiments using isolated confluency of the cell monolayer or clumps of mitochondria. Different sizes of cells (Salabei et al 2014). mitochondria are enriched at specific The complexity changes when centrifugation speeds, which can be used considering the scaling law of respiratory strategically for isolation of mitochondrial physiology. Strong interactions are revealed subpopulations. between O2 flow and body mass of an Part of the mitochondrial content of a individual organism: basal metabolic rate tissue is lost during preparation of isolated (flow) does not increase linearly with body mitochondria. The fraction of isolated mass, whereas maximum mass-specific O2 mitochondria obtained from a tissue sample flux, 푉̇O2max or 푉̇O2peak, is approximately is expressed as mitochondrial recovery. At a constant across a large range of individual high mitochondrial recovery, the fraction of body mass (Weibel and Hoppeler 2005). isolated mitochondria is more Individuals, breeds and species, however, representative of the total mitochondrial deviate substantially from this relationship. population than in preparations characterized by low recovery. 푉̇O2peak of human endurance athletes is 60 to Determination of the mitochondrial recovery 80 mL O2·min•1·kg•1 body mass, converted to and yield is based on measurement of the JO peak/mNorg of 45 to 60 nmol·s-1·g-1 (Gnaiger 2 concentration of a mitochondrial marker in 2020; Table 6). the stock suspension of isolated

mitochondria, C , and crude tissue 4.3. Marker-specific flux: per mitochondrial mtE,stock homogenate, C , which together content mtE,thom provide information on the specific

mitochondrial density in the sample, D Tissues can contain multiple cell populations mtE (Table 4). that may have distinct mitochondrial When discussing concepts of subtypes. Mitochondria undergo dynamic normalization, it is essential to consider the fission and fusion cycles, and can exist in question posed by the study. If the study multiple stages and sizes that may be altered aims at comparing tissue performance— by a range of factors. The isolation of such as the effects of a treatment on a specific mitochondria (often achieved through tissue, then normalization for tissue mass or differential centrifugation) can therefore protein content is appropriate. However, if yield a subsample of the mitochondrial types the aim is to find differences in present in a tissue, depending on the mitochondrial function independent of isolation protocols utilized. This possible

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BEC2020.1 doi:10.26124/bec:2020-0001.vTest05 mitochondrial density (Table 4), then measurement of mitochondrial marker normalization to a mitochondrial marker is enzyme activity to mitochondrial pathway imperative (Figure 5). One cannot assume capacity, ET- or OXPHOS-capacity can be that quantitative changes in various considered as an integrative functional markers—such as mitochondrial proteins— mitochondrial marker. necessarily occur in parallel with one Depending on the type of mitochondrial another. It should be established that the marker, the mitochondrial elementary marker chosen is not selectively altered by entity, mtE, is expressed in marker-specific the performed treatment. In conclusion, the units. Mitochondrial concentration in the normalization must reflect the question instrumental chamber and mitochondrial under investigation to reach a satisfying density in the tissue of origin are quantified answer. On the other hand, the goal of as (1) a quantity for normalization in comparing results across projects and functional analyses, CmtE, and (2) a institutions requires standardization on physiological output that is the result of normalization for entry into a databank. mitochondrial biogenesis and degradation, DmtE, respectively (Table 4). It is 4.3.1. Mitochondrial concentration, CmtE, recommended, therefore, to distinguish and mitochondrial markers: experimental mitochondrial concentration, Mitochondrial organelles compose a CmtE = mtE∙V-1 and physiological dynamic cellular reticulum in various states mitochondrial density, DmtE = mtE∙mX-1. Then of fusion and fission. Hence, the definition of mitochondrial density is the amount of an ‘amount’ of mitochondria is often mitochondrial elementary components per misconceived: mitochondria cannot be mass of tissue, which is a biological variable counted reliably as a number of occurring (Figure 5). The experimental variable is elementary components. Therefore, mitochondrial density multiplied by sample quantification of the amount of mitochondria mass concentration in the measuring depends on the measurement of chosen chamber, CmtE = DmtE∙CmX, or mitochondrial mitochondrial markers. ‘Mitochondria are content multiplied by sample number the structural and functional elementary units concentration, CmtE = mtEX∙CNX (Table 4). of cell respiration’ (Gnaiger 2020). The quantity of a mitochondrial marker can 4.3.2. mt-Marker-specific flux, JO2/mtE: reflect the amount of mitochondrial Volume-specific metabolic O2 flux depends elementary components, mtE, expressed in on: (1) the sample concentration in the various mitochondrial elementary units volume of the instrumental chamber, CmX, or [mtEU] specific for each measured mt- CNX; (2) the mitochondrial density in the marker (Table 4). However, since sample, DmtE = mtE∙mX-1 or mtEX = mtE∙NX-1; mitochondrial quality may change in and (3) the specific mitochondrial activity or response to stimuli—particularly in performance per mitochondrial elementary mitochondrial dysfunction (Campos et al -1 -1 -1 marker, JO2/mtE = JV,O2∙CmtE [mol∙s ∙mtEU ] 2017) and after exercise training (Pesta et al (Table 4). Obviously, the numerical results 2011) and during aging (Daum et al 2013)— for JO /mtE vary with the type of mitochondrial some markers can vary while others are 2 unchanged: (1) Mitochondrial volume and marker chosen for measurement of mtE and -1 -3 membrane area are structural markers, CmtE = mtE∙V [mtEU∙m ]. whereas mitochondrial protein mass is Different methods for the quantification commonly used as a marker for isolated of mitochondrial markers have different mitochondria. (2) Molecular and enzymatic strengths and weaknesses. Some problems mitochondrial markers (amounts or are common for all mitochondrial markers, activities) can be selected as matrix markers, mtE: (1) Accuracy of measurement is crucial, e.g., citrate synthase activity, mtDNA; mtIM- since even a highly accurate and markers, e.g., cytochrome c oxidase activity, reproducible measurement of chamber volume-specific O2 flux results in an aa3 content, cardiolipin, or mtOM-markers, e.g., the voltage-dependent anion channel inaccurate and noisy expression if (VDAC), TOM20. (3) Extending the normalized by a biased and noisy measurement of a mitochondrial marker.

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This problem is acute in mitochondrial titration protocol. Selection of the state of respiration because the denominators used maximum flux in a protocol as the reference (the mitochondrial markers) are often small state ― e.g., ET-state in L/E and P/E flux moieties of which accurate and precise control ratios (Gnaiger 2009) ― has the determination is difficult. In contrast, an advantages of: (1) elimination of internal marker is used when O2 fluxes experimental variability in additional measured in substrate-uncoupler-inhibitor measurements, such as determination of titration protocols are normalized for flux in enzyme activity or tissue mass; (2) a defined respiratory reference state within statistically validated linearization of the the assay, which yields flux control ratios, response in the range of 0 to 1; and (3) FCRs. FCRs are independent of externally consideration of maximum flux for measured markers and, therefore, are integrating a large number of metabolic statistically robust, considering the steps in the OXPHOS- or ET-pathways. This limitations of ratios in general (Jasienski and reduces the risk of selecting a functional Bazzaz 1999). FCRs indicate qualitative marker that is specifically altered by the changes of mitochondrial respiratory treatment or pathology, yet increases the control, with highest quantitative resolution, chance that the highly integrative pathway is separating the effect of mitochondrial disproportionately affected, e.g., the density on JO2/mX and IO2/NX from that of OXPHOS- rather than ET-pathway in case of function per mitochondrial elementary an enzymatic defect in the phosphorylation- pathway. In this case, additional information marker, JO2/mtE (Pesta et al 2011; Gnaiger 2020). (2) If mitochondrial quality does not can be obtained by reporting flux control change and only the amount of mitochondria ratios based on a reference state that varies as a determinant of mass-specific flux, indicates stable tissue-mass specific flux. any marker is equally qualified in principle; Stereological measurement of then in practice selection of the optimum mitochondrial content via two-dimensional marker depends only on the accuracy and transmission electron microscopy is precision of measurement of the considered as the gold standard in mitochondrial marker. (3) If mitochondrial determination of mitochondrial volume flux control ratios change, then there may fractions in cells and tissues (Weibel, not be any best mitochondrial marker. In Hoppeler, 2005). Accurate determination of general, measurement of multiple three-dimensional volume by two- mitochondrial markers enables a dimensional microscopy, however, is both comparison and evaluation of normalization time consuming and statistically challenging for these mitochondrial markers. (Larsen et al 2012). The validity of using Particularly during postnatal development, mitochondrial marker enzymes (citrate the activity of marker enzymes—such as synthase activity, CI to CIV amount or cytochrome c oxidase and citrate synthase— activity) for normalization of flux is limited follows different time courses (Drahota et al in part by the same factors that apply to flux 2004). Evaluation of mitochondrial markers control ratios. Strong correlations between in healthy controls is insufficient for various mitochondrial markers and citrate providing guidelines for application in the synthase activity (Reichmann et al 1985; diagnosis of pathological states and specific Boushel et al 2007; Mogensen et al 2007) are treatments. expected in a specific tissue of healthy In line with the concept of the respiratory persons and in disease states not specifically acceptor control ratio (Chance and Williams targeting citrate synthase. Citrate synthase 1955a), the most readily applied activity is acutely modifiable by exercise normalization is that of flux control ratios (Tonkonogi et al 1997; Leek et al 2001). and flux control factors (Gnaiger 2009; Evaluation of mitochondrial markers related 2020). Then, instead of a specific mt-enzyme to a selected age and sex cohort cannot be activity, the respiratory activity in a extrapolated to provide recommendations reference state serves as the mtE, yielding a for normalization in respirometric diagnosis dimensionless ratio of two fluxes measured of disease, in different states of development consecutively in the same respirometric and aging, different cell types, tissues, and

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BEC2020.1 doi:10.26124/bec:2020-0001.vTest05 species. mtDNA normalized to nDNA via mass-concentration of the subsample qPCR is correlated to functional obtained from the same tissue or cell culture, mitochondrial markers including OXPHOS- but system volume-specific O2 flux, JV,O2 (per and ET-capacity in some cases (Puntschart et liquid volume of the instrumental chamber), al 1995; Wang et al 1999; Menshikova et al increases in proportion to the mass of the 2006; Boushel et al 2007; Ehinger et al sample in the chamber. Although JV,O2 2015), but lack of such correlations have depends on mass-concentration of the been reported (Menshikova et al 2005; sample in the chamber, it should be Schultz and Wiesner 2000; Pesta et al 2011). independent of the chamber (system) Several studies indicate a strong correlation volume at constant sample mass- between cardiolipin content and increase in concentration. There are practical mitochondrial function with exercise limitations to increasing the mass- (Menshikova et al 2005; Menshikova et al concentration of the sample in the chamber, 2007; Larsen et al 2012; Faber et al 2014), when one is concerned about crowding but it has not been evaluated as a general effects and instrumental time resolution. The mitochondrial biomarker in disease. With no wall of the chamber and the enclosed solid single best mitochondrial marker, a good stirrer are not counted as part of the chamber strategy is to quantify several different volume. biomarkers to minimize the decorrelating effects caused by diseases, treatments, or 5.2.2. Advancement per volume: When the other factors. Determination of multiple reactor volume does not change during the markers, particularly a matrix marker and a reaction, which is typical for liquid phase marker from the mtIM, allows tracking reactions, the volume-specific flux of a changes in mitochondrial quality defined by chemical reaction r is the time derivative of their ratio. the advancement of the reaction per unit

volume, JV,rB = drξB/dt∙V-1 [(mol∙s•1)∙L•1]. The 5. Normalization of rate per system rate of concentration change is dcB/dt [(mol∙L•1)∙s•1], where concentration is cB = 5.1. Flow: per chamber nB∙V-1. There is a difference between (1) JV,rO 2 [mol∙s•1∙L•1] and (2) rate of concentration The instrumental system (chamber) is part change [mol∙L•1∙s•1]. These merge into a of the measurement instrument, separated single expression only in closed systems. In from the environment as an isolated, closed, open systems, internal transformations open, isothermal or non-isothermal system (catabolic flux, O2 consumption) are (Table 4). Reporting O2 flows per distinguished from external flux (such as O2 respiratory chamber, I [nmol∙s-1], restricts O2 supply). External fluxes of all substances are the analysis to intra-experimental zero in closed systems. In a closed chamber comparison of relative differences. O2 consumption (internal flux of catabolic reactions k; IkO [pmol∙s-1]) causes a decline 5.2. Flux: per chamber volume 2 in the amount of O in the system, n [nmol]. 2 O2 Normalization of these quantities for the 5.2.1. System-specific flux, JV,O : We 2 volume of the system, V [L ≡ dm3], yields distinguish between (1) the system with volume-specific O flux, J = I /V volume V and mass m defined by the system 2 V,kO2 kO2 •1 -1 boundaries, and (2) the sample or objects [nmol∙s ∙L ], and O2 concentration, [O2] or cO = nO ∙V-1 [µmol∙L•1 = µM = nmol∙mL•1]. with volume VX and mass mX that are 2 2 enclosed in the instrumental chamber Instrumental background O2 flux is due to (Figure 5). Metabolic O2 flow per object, external flux into a non-ideal closed respirometer, so total volume-specific flux IO2/NX, is the total O2 flow in the system has to be corrected for instrumental divided by the number of objects, NX, in the background O2 flux—O2 diffusion into or out system. IO2/NX increases as the mass of the of the instrumental chamber. JV,kO2 is relevant object is increased. Sample mass-specific O2 mainly for methodological reasons and flux, JO /mX should be independent of the 2 should be compared with the accuracy of

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Table 6. Conversion of various formats and units used in respirometry and ergometry. e- is the number of electrons or reducing equivalents. zB is the charge number of entity B.

Format 1 Unit ∙ Multiplication factor SI-unit Notes

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

n ng.atom O∙min-1 (2 e-) 8.33 pmol O2∙s•1

n natom O∙min-1 (2 e-) 8.33 pmol O2∙s•1

n nmol O2∙min-1 (4 e-) 16.67 pmol O2∙s•1

n nmol O2∙h-1 (4 e-) 0.2778 pmol O2∙s•1

V to n mL O2∙min-1 at STPDa 0.744 µmol O2∙s•1 1

e to n W = J/s at -470 kJ/mol O2 -2.128 µmol O2∙s•1 -1 + •1 e to n mA = mC∙s (zH+ = 1) 10.36 nmol H ∙s 2 -1 •1 e to n mA = mC∙s (zO2 = 4) 2.59 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) 0.38594 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, and Vm,O2 is 22.414 and 22.392 L∙mol-1, respectively. 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), -1 Vm,O2 is 24.038 L∙mol . 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.

instrumental resolution of background- Although volume is expressed as m3 using corrected flux, e.g., ±1 nmol∙s•1∙L-1 (Gnaiger the SI base unit, the litre [dm3] is a

2001). ‘Catabolic’ indicates O2 flux, JkO2, conventional unit of volume for corrected for: (1) instrumental background concentration and is used for most solution

O2 flux; (2) chemical background O2 flux due chemical kinetics. If one multiplies IO2/Nce by to autoxidation of chemical components CNce, then the result will not only be the added to the incubation medium; and (3) Rox amount of O2 [mol] consumed per time [s-1] for O2-consuming side reactions unrelated to in one litre [L•1], but also the change in O2 the catabolic pathway k. concentration per second (for any volume of an ideally closed system). This is ideal for 6. Conversion of units kinetic modeling as it blends with chemical rate equations where concentrations are Many different units have been used to typically expressed in mol∙L-1 (Wagner et al report the O2 consumption rate, OCR (Table 2011). In studies of multinuclear cells—such 6). SI base units provide the common as differentiated skeletal muscle cells—it is reference to introduce the theoretical easy to determine the number of nuclei but principles (Figure 5), and are used with not the total number of cells. A generalized appropriately chosen SI prefixes to express concept, therefore, is obtained by numerical data in the most practical format, substituting cells by nuclei as the sample with an effort towards unification within entity. This does not hold, however, for non- specific areas of application (Table 7). nucleated platelets. Reporting data in SI units—including the For studies of cells, we recommend that mole [mol], coulomb [C], joule [J], and second respiration be expressed, as far as possible, [s]—should be encouraged, particularly by as: (1) O2 flux normalized for a mitochondrial journals that propose the use of SI units. marker, for separation of the effects of

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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 -1 -6 -1 -1 cell-specific flow, IO2/Nce pmol∙s ∙10 cells amol∙s ∙cell 2 pmol∙s-1∙10-9 cells zmol∙s-1∙cell-1 3

cell number concentration, CNce 106 cells∙mL-1 109 cells∙L-1

mitochondrial protein concentration, CmtE 0.1 mg∙mL-1 0.1 g∙L-1 -1 -1 -1 -1 mass-specific flux, JO2/m pmol∙s ∙mg nmol∙s ∙g 4 catabolic power, Pk µW∙10-6 cells pW∙cell-1 1 volume 1,000 L m3 (1,000 kg) L dm3 (kg) mL cm3 (g) µL mm3 (mg) fL µm3 (pg) 5 amount of substance concentration M = mol∙L-1 mol∙dm-3 1 pmol: picomole = 10-12 mol 4 nmol: nanomole = 10-9 mol 2 amol: attomole = 10-18 mol 5 fL: femtolitre = 10-15 L 3 zmol: zeptomole = 10-21 mol mitochondrial quality and content on cell corrected for Rox, the current across the mt- respiration (this includes FCRs as a membranes, IH+e, approximates 193 pA∙cell-1 normalization for a functional mitochondrial or 0.2 nA per cell. See Rich (2003) for an marker); (2) O2 flux in units of cell volume or extension of quantitative bioenergetics from mass, for comparison of respiration of cells the molecular to the human scale, with a with different cell size (Renner et al 2003) transmembrane proton flux equivalent to and with studies on tissue preparations, and 520 A in an adult at a catabolic power of •110 (3) O2 flow in units of attomole (10-18 mol) of W. Modelling approaches illustrate the link O2 consumed per second by each cell between protonmotive force and currents [amol∙s•1∙cell-1], numerically equivalent to (Willis et al 2016). [pmol∙s•1∙10•6 cells]. This convention allows We consider isolated mitochondria as information to be easily used when powerhouses and proton pumps as designing experiments in which O2 flow must molecular machines to relate experimental be considered. For example, to estimate the results to energy metabolism of living cells. volume-specific O2 flux in an instrumental The cellular P»/O2 based on oxidation of chamber that would be expected at a glycogen is increased by the glycolytic particular cell number concentration, one (fermentative) substrate-level simply needs to multiply the flow per cell by phosphorylation of 3 P»/Glyc or 0.5 mol P» the number of cells per volume of interest. for each mol O2 consumed in the complete This provides the amount of O2 [mol] oxidation of a mol glycosyl unit (Glyc). consumed per time [s-1] per unit volume [L- Adding 0.5 to the mitochondrial P»/O2 ratio 1]. At an O2 flow of 100 amol∙s•1∙cell-1 and a of 5.4 yields a bioenergetic cell physiological cell concentration of 109 cells∙L•1 (106 P»/O2 ratio close to 6. Two NADH cells∙mL•1), the volume-specific O2 flux is 100 equivalents are formed during glycolysis and nmol∙s-1∙L-1 (100 pmol∙s•1∙mL•1). transported from the cytosol into the ET-capacity in human cell types including mitochondrial matrix, either by the malate- HEK 293, primary HUVEC, and fibroblasts aspartate shuttle or by the glycerophosphate ranges from 50 to 180 amol∙s-1∙cell-1, shuttle (Figure 1a) resulting in different measured in living cells in the noncoupled theoretical yields of ATP generated by state (Gnaiger 2020). At 100 amol∙s-1∙cell-1 mitochondria, the energetic cost of which

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Consortium Communication potentially must be taken into account. sensitivity and limit of detection of Considering also substrate-level volume-specific flux. phosphorylation in the TCA cycle, this high 2. Sample concentration in the instrumental P»/O2 ratio not only reflects proton chamber is reported as number translocation and OXPHOS studied in concentration, mass concentration, or isolation, but integrates mitochondrial mitochondrial concentration; this is a physiology with energy transformation in component of the measuring conditions. the living cell (Gnaiger 1993a). With information on cell size and the use of multiple normalizations, maximum 7. Conclusions potential information is available (Renner et al 2003; Wagner et al 2011; Gnaiger Catabolic cell respiration is the process of 2020). Reporting flow in a respiratory exergonic and exothermic energy chamber [nmol∙s-1] is discouraged, since it transformation in which scalar redox restricts the analysis to intra- reactions are coupled to vectorial ion experimental comparison of relative translocation across a semipermeable (qualitative) differences. membrane, which separates the small ● Catabolic mitochondrial respiration is volume of a bacterial cell or mitochondrion distinguished from residual O2 from the larger volume of its surroundings. consumption. Fluxes in mitochondrial The electrochemical exergy can be partially coupling states should be, as far as conserved in the phosphorylation of ADP to possible, corrected for residual O2 ATP or in ion pumping, or dissipated in an consumption. electrochemical short-circuit. Respiration is ● Different mechanisms of uncoupling thus clearly distinguished from fermentation should be distinguished by defined terms. as the counterparts of cellular core energy The tightness of coupling relates to these metabolism. An O2 flux balance scheme uncoupling mechanisms, whereas the illustrates the relationships and general coupling stoichiometry varies as a definitions (Figures 1 and 2). function the substrate type involved in ET-pathways with either three or two Box 3: Recommendations for studies redox proton pumps operating in series. with mitochondrial preparations Separation of tightness of coupling from the pathway-dependent coupling

stoichiometry is possible only when the ● Normalization of respiratory rates should substrate type undergoing oxidation be provided as far as possible: remains the same for respiration in LEAK-

, OXPHOS-, and ET-states. In studies of the A. Sample normalization tightness of coupling, therefore, simple 1. Object-specific biophysical normalization: substrate•inhibitor combinations should on a per organism or per cell basis as O 2 be applied to exlcude a shift in substrate flow; this may not be possible when competition that may occur when dealing with coenocytic organisms, e.g., providing physiological substrate filamentous fungi, or tissues without cocktails. cross-walls separating individual cells, ● In studies of isolated mitochondria, the 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 O flux; per cell 2 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—

provide a link to the tissue of origin on the B. Chamber normalization basis of calculating the mitochondrial 1. Chamber volume-specific flux, J V 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

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unit mass of tissue. Total mitochondrial Omics data with bioinformatics tools; (3) protein is frequently applied as a cross-validation with distinct mitochondrial marker, which is restricted bioinformatics approaches; (4) to isolated mitochondria. correlation with physiological data; (5) ● In studies of permeabilized cells, the guidelines for biological validation of viability of the cell culture or cell network data. This is a call to carefully suspension of origin should be reported. contribute to FAIR principles (Findable, Normalization should be evaluated for Accessible, Interoperable, Reusable) for total cell count or viable cell count. the sharing of scientific data. ● Terms and symbols are summarized in Table 8. Their use will facilitate Experimentally, respiration is separated transdisciplinary communication and in mitochondrial preparations from the support further development of a interactions with the fermentative pathways consistent theory of bioenergetics and of the living cell. OXPHOS analysis is based on mitochondrial physiology. Technical the study of mitochondrial preparations terms related to and defined with normal complementary to bioenergetic words can be used as index terms in data investigations of (1) submitochondrial repositories, support the creation of particles and molecular structures, (2) living ontologies towards semantic information cells, and (3) organisms—from model processing (MitoPedia), and help in organisms to the human species including communicating analytical findings as healthy and diseased persons (patients). impactful data-driven stories. ‘Making Different mechanisms of respiratory data available without making it uncoupling have to be distinguished (Figure understandable may be worse than not 3). Metabolic fluxes measured in defined making it available at all’ (National coupling and pathway control states Academies of Sciences, Engineering, and (Figures 5 and 6) provide insights into the Medicine 2018). Success will depend on meaning of cellular and organismic taking further steps: (1) exhaustive text- respiration. mining considering Omics data and functional data; (2) network analysis of

Table 8. Terms, symbols, and units. SI base units are used, except for the liter [L = dm3]

Term Symbol Unit Links and comments

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

ATP yield per O2 YP»/O2 P»/O2 ratio measured in any respiratory state catabolic reaction k Fig. 1, 3 catabolic respiration JkO2 varies Fig. 1, 3 •1 cell concentration (mass) Cmce [kg∙L ] Tab. 4 •1 cell concentration (number) CNce [x∙L ] Tab. 4 cell respiration JrO2 varies Overview •1 •1 cell viability index VI VI = Nvce∙Nce = 1- Ndce∙Nce charge number of entity B zB Tab. 6; zO2 = 4 Complexes I to IV CI to CIV respiratory ET Complexes are redox proton pumps; Fig. 1B; F1FO-ATPase is not a redox

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proton pump of the ETS, hence the term CV is not recommended •1 •1 concentration of substance B cB = nB∙V ; [B] [mol∙L ] Box 2 coupling control state CCS Section 2.4.1 dead cells dce Tab. 5 electric 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) ET-capacity E varies rate; Tab. 1; Fig. 2 ET-excess capacity E-P varies Fig. 2 •1 flow, for substance B IB [mol∙s ] system-related extensive quantity; Fig. 5 flux, for substance B JB varies size-specific quantitiy; Fig. 5 inorganic phosphate Pi Fig. 1C inorganic phosphate carrier PiC Fig. 1C isolated mitochondria imt Tab. 5 living cells ce Tab. 5 (intact cells) LEAK-state LEAK Tab. 1; Fig. 2 (compare State 4) LEAK-respiration L varies rate; Tab. 1; Fig. 2 mass format m [kg] Tab. 4; Fig. 5 •1 mass of sample or object X mX or mNX [kg] or [kg∙x ] Tab. 4 •1 mass, dry mass md [kg] or [kg∙x ] Fig. 5 (dry weight) •1 mass, wet mass mw [kg] or [kg∙x ] Fig. 5 (wet weight) MITOCARTA https://www.broadinstitute.org/scientific- community/science/programs/meta bolic-disease- program/publications/mitocarta/mi tocarta-in-0 MitoPedia http://www.bioblast.at/index.php/MitoPedia mitochondria or mitochondrial mt Box 1 mitochondrial DNA mtDNA Box 1 -1 •3 mitochondrial concentration CmtE = mtE∙V [mtEU∙m ] Tab. 4 •1 -1 mitochondrial content mtEX [mtEU∙x ] mtEX = mtE∙NX ; Tab. 4 -1 •1 mitochondrial density DmtE = mtE∙mX [mtEU∙kg ] specific mt-density in tissue; Tab. 4 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 fraction of mtE recovered from the tissue sample in imt-stock -1 mitochondrial yield YmtE/m [mtEU∙kg ] mt-yield in imt-stock per m of tissue sample; YmtE/m = YmtE ∙ DmtE molar format n [mol] Tab. 6 negative neg Fig. 2 -3 number concentration of X CNX [x∙m ] Tab. 4 number format N [x] Tab. 4; Fig. 5 number of cells Nce [x] total cell count of living cells, Nce = Nvce + Ndce; Tab. 5 number of dead cells Ndce [x] non-viable cells, loss of plasma membrane barrier function; Tab. 5 number of entities X NX [x] Tab. 4; Fig. 5

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BEC2020.1 doi:10.26124/bec:2020-0001.vTest05 number of entity B NB [x] Tab. 4 number of viable cells Nvce [x] viable cells, intact plasma mem- brane barrier function; Tab. 5 organisms org Tab. 5 oxidative phosphorylation OXPHOS Tab. 1; Fig. 2 OXPHOS-state OXPHOS Tab. 1 (State 3 at kinetically- saturating [ADP] and [Pi]) OXPHOS-capacity P varies rate; Tab. 1; Fig. 2 •1 •3 oxygen concentration cO2 = nO2∙V [mol∙m ] [O2]; Section 3.2 oxygen flux, in reaction r JrO2 varies Overview pathway control state PCS Section 2.2 permeability transition mtPT Fig. 3; Section 2.4.3 (MPT) permeabilized cells 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» Section 2.2

P»/O2 ratio P»/O2 mechanistic YP»/O2, calculated from pump stoichiometries; Fig. 2B positive pos Fig. 2 + proton in the negative compartment H neg Fig. 2 + proton in the positive compartment H pos Fig. 2 protonmotive force pmF [V] Fig 1, 2A, 4; Tab. 1 rate of electron transfer in ET-state E varies ET-capacity; Tab. 1 rate of LEAK-respiration L varies Tab. 1: L(n), L(T), L(Omy) rate of oxidative phosphorylation P varies OXPHOS-capacity; Tab. 1 rate of residual oxygen consumption Rox Tab. 1; Overview residual oxygen consumption ROX; Rox state ROX; rate Rox; Tab. 1 respiratory supercomplex SCInIIInIVn supramolecular assemblies with variable copy numbers (n) of CI, CIII and CIV; Box 1 substrate concentration at •3 half-maximal rate c50 [mol∙m ] Section 2.1.2 substrate-uncoupler-inhibitor- titration SUIT Section 2.2 tissue homogenate thom Tab. 5 viable cells vce Tab. 5 volume V [L] Tab. 7 volume format V [L] Tab. 6 •1 volume of sample or object X VX or VNX [L] or [L∙x ] Tab. 6

The optimal choice for expressing adaptations and defects linked to genetic mitochondrial and cell respiration as O2 flow variation, age-related health risks, sex- per biological sample, and normalization for specific mitochondrial performance, lifestyle specific tissue-markers (volume, mass, with its effects on degenerative diseases, and protein) and mitochondrial markers thermal and chemical environment. The (volume, protein, content, mtDNA, activity of present recommendations on coupling marker enzymes, respiratory reference control states and rates are focused on state) is guided by the scientific question studies using mitochondrial preparations under study. Interpretation of the data (Box 3). These will be extended in a series of depends critically on appropriate reports on pathway control of mitochondrial normalization (Figure 5). respiration, respiratory states and rates in MitoEAGLE can serve as a gateway to living cells, respiratory flux control ratios, better diagnose mitochondrial respiratory

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

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Laranjinha Joao, Larsen Steen, Larsen Terje Prochownik Edward V, Prola Alexandre, S, Lavery Gareth G, Lazou Antigone, Ledo Ana Pulinilkunnil Thomas, Puskarich Michael A, Margarida, Lee Hong Kyu, Leeuwenburgh Puurand Marju, Radenkovic Filip, Ramzan Christiaan, Lehti Maarit, Lemieux Helene, Rabia, Rattan Suresh IS, Reano Simone, Lenaz Giorgio, Lerfall Joergen, Li Pingan Reboredo-Rodriguez Patricia, Rees Bernard Andy, Li Puma Lance, Liang Liping, Liepins B, Renner-Sattler Kathrin, Rial Eduardo, Edgars, Lin Chien-Te, Liu Jiankang, Lopez Robinson Matthew M, Roden Michael, Garcia Luis Carlos, Lucchinetti Eliana, Ma Rodrigues Ana Sofia, Rodriguez Enrique, Tao, Macedo Maria Paula, Machado Ivo F, Rodriguez-Enriquez Sara, Roesland Gro Maciej Sarah, MacMillan-Crow Lee Ann, Vatne, Rohlena Jakub, Rolo Anabela Pinto, Magalhaes Jose, Magri Andrea, Majtnerova Ropelle Eduardo R, Roshanravan Baback, Pavlina, Makarova Elina, Makrecka-Kuka Rossignol Rodrigue, Rossiter Harry B, Marina, Malik Afshan N, Marcouiller Rousar Tomas, Rubelj Ivica, Rybacka- Francois, Markova Michaela, Markovic Mossakowska Joanna, Saada Reisch Ann, Ivanka, Martin Daniel S, Martins Ana Dias, Safaei Zahra, Salin Karine, Salvadego Desy, Martins Joao D, Maseko Tumisang Edward, Sandi Carmen, Saner Nicholas, Santos Diana, Maull Felicia, Mazat Jean-Pierre, McKenna Sanz Alberto, Sardao Vilma, Sarlak Saharnaz, Helen T, McKenzie Matthew, McMillan Sazanov Leonid A, Scaife Paula, Scatena Duncan GG, Mendham Amy, Menze Michael Roberto, Schartner Melanie, Scheibye- A, Mercer John R, Merz Tamara, Messina Knudsen Morten, Schilling Jan M, Schlattner Angelina, Meszaros Andras, Methner Axel, Uwe, Schmitt Sabine, Schneider Gasser Edith Michalak Slawomir, Mila Guasch Maria, Mariane, Schoenfeld Peter, Schots Pauke C, Minuzzi Luciele M, Moellering Douglas R, Schulz Rainer, Schwarzer Christoph, Scott Moisoi Nicoleta, Molina Anthony JA, Graham R, Selman Colin, Sendon Pamella Montaigne David, Moore Anthony L, Moore Marie, Shabalina Irina G, Sharma Pushpa, Christy, Moreau Kerrie, Moreira Bruno P, Sharma Vipin, Shevchuk Igor, Shirazi Reza, Moreno-Sanchez Rafael, Mracek Tomas, Shiroma Jonathan G, Siewiera Karolina, Muccini Anna Maria, Munro Daniel, Muntane Silber Ariel M, Silva Ana Maria, Sims Carrie A, Jordi, Muntean Danina M, Murray Andrew Singer Dominique, Singh Brijesh Kumar, James, Musiol Eva, Nabben Miranda, Nair K Skolik Robert A, Smenes Benedikte Therese, Sreekumaran, Nehlin Jan O, Nemec Michal, Smith James, Soares Felix Alexandre Nesci Salvatore, Neufer P Darrell, Neuzil Jiri, Antunes, Sobotka Ondrej, Sokolova Inna, Neviere Remi, Newsom Sean A, Norman Solesio Maria E, Soliz Jorge, Sommer Jennifer, Nozickova Katerina, Nunes Sara, Natascha, Sonkar Vijay K, Sova Marina, O'Brien Kristin, O'Brien Katie A, O'Gorman Sowton Alice P, Sparagna Genevieve C, Donal, Olgar Yusuf, Oliveira Ben, Oliveira Sparks Lauren M, Spinazzi Marco, Stankova Jorge, Oliveira Marcus F, Oliveira Marcos Pavla, Starr Jonathan, Stary Creed, Stefan Tulio, Oliveira Pedro Fontes, Oliveira Paulo J, Eduard, Stelfa Gundega, Stepto Nigel K, Olsen Rolf Erik, Orynbayeva Zulfiya, 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

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Ioannis P, Truu Laura, Tuncay Erkan, Turan Acknowledgements: We thank Marija Beno for Belma, Tyrrell Daniel J, Urban Tomas, Urner management assistance, and Peter R Rich for Sofia, Valentine Joseph Marco, Van Bergen valuable discussions. This publication is based Nicole J, Van der Ende Miranda, Varricchio upon work from COST Action MitoEAGLE, Frederick, Vaupel Peter, Vella Joanna, supported by COST (European Cooperation in Science and Technology), in cooperation with Vendelin Marko, Vercesi Anibal E, Verdaguer COST Actions CA16225 EU-CARDIOPROTECTION Ignasi Bofill, Vernerova Andrea, Victor Victor and CA17129 CardioRNA; K-Regio project Manuel, Vieira Ligo Teixeira Camila, Vidimce MitoFit funded by the Tyrolian Government, and Josif, Viel Christian, Vieyra Adalberto, Vilks project NextGen-O2k which has received funding Karlis, Villena Josep A, Vincent Vinnyfred, from the European Union’s Horizon 2020 Vinogradov Andrey D, Viscomi Carlo, research and innovation programme under grant Vitorino Rui Miguel Pinheiro, Vlachaki agreement No. 859770. Walker Julia, Vogt Sebastian, Volani Chiara, Volska Kristine, Votion Dominique-Marie, Competing financial interests: Erich Gnaiger is Vujacic-Mirski Ksenija, Wagner Brett A, founder and CEO of Oroboros Instruments, Innsbruck, Austria. Ward Marie Louise, Warnsmann Verena, Wasserman David H, Watala Cezary, Wei Corresponding author: Erich Gnaiger Yau-Huei, Weinberger Klaus M, Weissig Chair COST Action CA15203 MitoEAGLE – Volkmar, White Sarah Haverty, Whitfield http://www.mitoeagle.org Jamie, Wickert Anika, Wieckowski Mariusz R, Department of Visceral, Transplant and Thoracic Wiesner Rudolf J, Williams Caroline M, Surgery, D. Swarovski Research Laboratory, Winwood-Smith Hugh, Wohlgemuth Medical University of Innsbruck, Innrain 66/4, Stephanie E, Wohlwend Martin, Wolff Jonci A-6020 Innsbruck, Austria Nikolai, Wrutniak-Cabello Chantal, Wuest Email: [email protected]; Tel: +43 512 Rob CI, Yokota Takashi, Zablocki Krzysztof, 566796, Fax: +43 512 566796 20

Zanon Alessandra, Zanou Nadege, Zaugg Kathrin, Zaugg Michael, Zdrazilova Lucie, Zhang Yong, Zhang Yizhu, Zikova Alena, Zischka Hans, Zorzano Antonio, Zujovic Tijana, Zvejniece Liga

Affiliations: https://www.bioenergetics- communications.org/index.php/BEC2020.1_doi 10.26124bec2020-0001.v1

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