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Capacity of oxidative phosphorylation in human skeletal muscle: New perspectives of mitochondrial physiology

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Abstract

Maximal ADP-stimulated mitochondrial respiration depends on convergent electron flow through Complexes I + II to the Q-junction of the electron transport system (ETS). In most studies of respiratory control in mitochondrial preparations, however, respiration is limited artificially by supplying substrates for electron input through either Complex I or II. High-resolution respirometry with minimal amounts of tissue biopsy (1–3 mg wet weight of permeabilized muscle fibres per assay) provides a routine approach for multiple substrate-uncoupler-inhibitor titrations. Under physiological conditions, maximal respiratory capacity is obtained with glutamate + malate + succinate, reconstituting the operation of the tricarboxylic acid cycle and preventing depletion of key metabolites from the mitochondrial matrix. In human skeletal muscle, conventional assays with pyruvate + malate or glutamate + malate yield submaximal oxygen fluxes at 0.50–0.75 of capacity of oxidative phosphorylation (OXPHOS). Best estimates of muscular OXPHOS capacity at 37 °C (pmol O2 s−1 mg−1 wet weight) with isolated mitochondria or permeabilized fibres, suggest a range of 100–150 and up to 180 in healthy humans with normal body mass index and top endurance athletes, but reduction to 60–120 in overweight healthy adults with predominantly sedentary life style. The apparent ETS excess capacity (uncoupled respiration) over ADP-stimulated OXPHOS capacity is high in skeletal muscle of active and sedentary humans, but absent in mouse skeletal muscle. Such differences of mitochondrial quality in skeletal muscle are unexpected and cannot be explained at present. A comparative database of mitochondrial physiology may provide the key for understanding the functional implications of mitochondrial diversity from mouse to man, and evaluation of altered mitochondrial respiratory control patterns in health and disease.

Introduction

Oxidative phosphorylation (OXPHOS) is a key element of bioenergetics, extensively studied to resolve the mechanisms of energy transduction in the mitochondrial electron transport system (ETS) and analyze various modes of mitochondrial respiratory control. Electrons flow to oxygen along linear thermodynamic cascades (electron transport chains (ETC)) from Complex I (CI) with three coupling sites, or from Complex II (CII) with two coupling sites (Chance and Williams, 1956). These pathways of electron transport are conventionally separated in studies of mitochondrial preparations, by using either NADH-linked substrates such as pyruvate + malate, or the classical succinate + rotenone combination, for analysis of site-specific H+/e and ADP:O ratios (Chance and Williams, 1955, Lemasters, 1984, Mitchell and Moyle, 1967) and functional diagnosis of enzymatic defects in mitochondrial diseases. In mitochondrial physiology and pathology, maximal mitochondrial respiration in the coupled state is measured for quantitative determination of OXPHOS capacity, JO2,P (see Table 1 for definition of symbols). Increasing evidence is available that mitochondrial density and thus JO2,P per muscle mass is related to training and endurance exercise capacity (Andersen and Saltin, 1985, Blomstrand et al., 1997, di Prampero, 2003, Hoppeler et al., 1985, Starritt et al., 1999, Turner et al., 1997, Weibel and Hoppeler, 2005, Wibom et al., 1992, Zoll et al., 2002).

The methods and concepts established in bioenergetics are directly applied in numerous studies with a physiological perspective, considering that fluxes measured with ‘succinate in the presence of ADP and phosphate should reflect the maximal physiological respiratory activity of mitochondria, since the rate-controlling steps of adenine nucleotide translocation and cytochrome c oxidation are involved’ (Schwerzmann et al., 1989). Although it is generally appreciated that ‘it is essential to define both the substrate and ADP levels in order to identify the steady-state condition of the mitochondria during the experiment’ (Chance and Williams, 1956), surprisingly little attention is being payed in the majority of investigations to the question as to the most appropriate substrate for estimation of physiological oxidative capacity. Irrespective of the type of mitochondrial preparation, i.e. permeabilized fibres (Pfi) or isolated mitochondria (Imt), used to determine ‘maximal ADP-stimulated respiration’ (Saks et al., 1998, N’Guessan et al., 2004), some research groups prefer – appart from comparison with fatty acid oxidation – strictly pyruvate + malate as substrates (Tonkonogi et al., 1999, Mogensen et al., 2006), whereas others use only glutamate + malate ‘to establish maximal oxidative capacities of mitochondria in muscles’ (Daussin et al., 2008b). These and many other studies suggest (Table 2) that an important finding remained largely unnoticed, namely that ADP-stimulated respiration with pyruvate + malate and glutamate + malate yields only 66% and 75%, respectively, of JO2,P observed with the substrate combination glutamate + succinate in pig skeletal muscle (Rasmussen et al., 1996). Identical results in human skeletal muscle (Rasmussen and Rasmussen, 2000) challenge the paradigm that pyruvate + malate or glutamte + malate are suitable and sufficient substrate combinations for evaluation of maximal physiological OXPHOS capacity, or that ‘these measurements require the integrated function of the citric acid cycle’ (Tonkonogi et al., 1999). On the contrary, the TCA cycle is functionally not ‘closed’ when using the substrate combination pyruvate + malate or glutamate + malate; depletion of citrate, isocitrate, 2-oxoglutarate and succinate into the incubation medium prevents any significant contribution of succinate oxidation to respiratory flux (Gnaiger, 2007). Reconstitution of TCA cycle function in mitochondrial preparations requires addition of succinate together with the conventional substrates for Complex I, to support the simultaneous, convergent electron flow through CI + II into the Q-junction (Fig. 1).

Extending the concept on mitochondrial respiratory control by multiple substrate supply (Rasmussen et al., 1996, Rasmussen et al., 2001a, Rasmussen et al., 2001b, Rasmussen and Rasmussen, 2000), a novel perspective of mitochondrial respiratory physiology emerges from a series of studies based on high-resolution respirometry (HRR; Gnaiger et al., 1995, Gnaiger, 2001, Gnaiger, 2008). Application of permeabilized muscle fibres (Veksler et al., 1987, Letellier et al., 1992) has become successful to minimize the size of needle biopsies and reduce the amount of tissue required for HRR. Long-term stability of the instrumental signal and of mitochondrial preparations in mitochondrial respiration medium MiR05 (Gnaiger et al., 2000a), and high dilution of the sample in the oxygraph chamber provide the experimental basis for application of sequential, multiple substrate-uncoupler-inhibitor titration (SUIT) protocols (Gnaiger et al., 2005). The design of SUIT protocols places a balanced emphasis on coupling control and substrate control of OXPHOS capacity, for exploring the functional consequences of convergent mitochondrial pathways at the Q-junction (Gnaiger, 2007). Following this integrated systems approach, combining HRR with application of permeabilized fibres, incubation in optimized medium MiR05, measurement at physiological temperature and strategic design of CI + II SUIT protocols, a rapidly growing number of studies confirms the importance of the additive effect of substrate combinations on JO2,P (Aragonés et al., 2008, Boushel et al., 2007, Hütter et al., 2007, Jüllig et al., 2008, Lemieux et al., 2006, Phielix et al., 2008, Rabøl et al., 2009, Wijers et al., 2008).

Section snippets

High-resolution respirometry compared to specialized microchamber system

Respiration of permeabilized muscle fibres was measured by high-resolution respirometry with the Oxygraph-2k (OROBOROS INSTRUMENTS, Innsbruck, Austria) at 37 °C, using 1–3 mg tissue wet weight in each 2 ml glass chamber (Boushel et al., 2007). Respiration of isolated mitochondria was measured in a 36.5 μl glass microchamber at 25 °C, with 3–15 μg mitochondrial protein (Pmt) (Rasmussen and Rasmussen, 2000). At a yield in mitochondrial isolation of 45%, and a mitochondrial protein concentration of 10 μg

Reconstitution of TCA cycle function

In addition to coupling and substrate supply, metabolite depletion of TCA cycle intermediates plays a fundamental role in OXPHOS flux control in isolated mitochondria or permeabilized cells. Using classical NADH-related substrates such as pyruvate, malate and glutamate, the TCA cycle metabolites citrate and 2-oxoglutarate are exchanged rapidly for malate by the tricarboxylate and 2-oxoglutarate carrier, and succinate is lost from the matrix space in exchange for inorganic phosphate catalyzed by

Tissue-OXPHOS capacity in human permeabilized muscle fibres and isolated mitochondria

Few studies on human muscle mitochondria apply physiological conditions for estimating mitochondrial OXPHOS capacity. In Table 2, a physiological reference state is defined with CI + II substrate supply (Fig. 2) at experimental temperature close to body temperature, for quantitative evaluation of JO2,P in mammalian tissues. The further the experimental conditions differ from the physiological reference state, the larger the error becomes which may result from adjustment to the CI + II substrate

Conclusions: tissue-OXPHOS capacity and functional diversity

Substrate control is complementary to coupling control of oxygen flux in mitochondrial preparations, applied for assessment of physiological OXPHOS capacities and diagnosis of qualitative properties of mitochondrial respiratory control. The capacity of the electron transport system is generally underestimated on the basis of the ‘State 3 paradigm’ and conventional respiratory protocols applied with isolated mitochondria, permeabilized cells or tissues. The integrated function of the TCA cycle

Acknowledgements

I thank Robert Boushel, Hélène Lemieux, Steven Hand, Kathrin Renner, Dominique Votion, Bengt Kayser, Richard Haas, Charles Hoppel, Rodrigue Rossignol, Guy Brown and many unnamed colleagues for sharing their views on mitochondrial physiology during joint projects, numerous courses on high-resolution respirometry and Mitochondrial Physiology Conferences.

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