Cardiac and Skeletal Muscle Mitochondria

1L-01.    MitoPathways and mitochondrial respiration in top gear – an introduction to the concept of the ‘Q-junction’ and respiratory control with multiple substrates.

               Erich Gnaiger (Innsbruck, AT)

Medical University of Innsbruck, Dept. General and Transplant Surgery, D. Swarovski Research Laboratory, A-6020 Innsbruck, Austria. – erich.gnaiger@i-med.ac.at

    Oxidative phosphorylation (OXPHOS) is a key element of bioenergetics, extensively studied to resolve the mechanisms of energy transduction in the mitochondrial electron transport system and analyze various modes of mitochondrial respiratory control. OXPHOS flux control is exerted by (i) coupling of electron transport to proton translocation and ATP synthesis mediated by the proton motive force, (ii) catalytic capacities of respiratory complexes, carriers and transporters, (iii) kinetic regulation by ADP, oxygen and various substrates feeding electrons into the respiratory chain, and (iv) specific inhibitors such as NO. Electrons flow to oxygen along linear thermodynamic cascades (electron transport chains) from either Complex I with three coupling sites, or from Complex II with two coupling sites. These pathways of electron transport are conventionally separated by using either NADH-linked substrates, such as pyruvate+malate, or the classical succinate+rotenone combination, to analyze site-specific H⁺/e and P/O ratios or defects of specific respiratory complexes in functional diagnosis. The experimental separation of various electron transport pathways is common to the extent of establishing a bioenergetic paradigm in studies of OXPHOS. This bioenergetic paradigm is extended by a perspective of mitochondrial physiology emerging from a series of studies based on high-resolution respirometry (OROBOROS Oxygraph-2k).

    Proper understanding and evaluation of the functional design of the OXPHOS system requires a transition from analyses of the electron transport chain (ETC) to a perspective of the convergent structure of electron flow to the Q-junction in the mitochondrial electron transport system (ETS). Electron transport capacity of cells in vivo is generally underestimated on the basis of the ‘State 3 paradigm’ and conventional respiratory protocols applied with isolated mitochondria, permeabilized cells or tissues, with profound implications on studies of biochemical thresholds, excess capacities and flux control coefficients of various mitochondrial enzymes.

    The present overview on ‘Mitochondrial Pathways and Respiratory Control’ combines concepts of bioenergetics and cell metabolism leading to a new understanding of mitochondrial respiratory physiology and application of substrate combinations in novel Oxygraph assays. (i) Convergent electron flow through respiratory Complexes I and II (CI+II e-input) and through glycerophosphate dehydrogenase and electron-transferring flavoprotein exert additive effects on respiratory flux, increasing coupled respiration 1.3- to 2-fold relative to State 3 with Complex I substrates. (ii) Uncoupled respiration in living cells represents OXPHOS capacity only in cases when the phosphorylation system (adenylate nucleotide translocase, phosphate carrier, ATP synthase) does not exert control over coupled respiration. Mitochondrial oxidation in vivo is coupled to phosphorylation. A shift towards control by the phosphorylation system is observed when electron transport capacity is increased by convergent electron input into the Q-junction. (iii) Respiratory control ratios and uncoupling control ratios need to be combined for proper evaluation of coupling in OXPHOS.

    Convergent CI+II e-input corresponds to the operation of the citric acid cycle and mitochondrial substrate supply in vivo. By establishing the reference state of maximum coupled respiration, convergent CI+II e-input provides the proper basis for (i) quantifying excess capacities of Complexes III and IV, (ii) interpreting flux control by various components such as the phosphorylation system or COX, and (iii) for evaluation of specific enzymatic defects in the context of mitochondrial respiratory physiology and pathology. Applicaton of substrate combinations in multiple substrate/inhibitor titration protocols extends conventional bioenergetic studies to the level of mitochondrial physiology applied for the diagnosis of respiratory control in health and disease.

Gnaiger E (2007) Mitochondrial pathways through Complexes I+II:  Convergent electron transport at the Q-junction and additive effect of substrate combinations. In: Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications, Innsbruck: pp. 21-37.

1L-04.    High excess capacity of cytochrome c oxidase in permeabilized fibers of the mouse heart.

               Hélène Lemieux, Erich Gnaiger

D. Swarovski Research Laboratory, Department of Transplant Surgery, Medical University of Innsbruck, Innrain 66/6, 6020 Innsbruck, Austria. - helene.lemieux@oroboros.at

    Metabolic flux control analysis and the concept of excess capacity of enzymes over pathway flux are related by the functional threshold, at which damage or inhibition of an enzyme reduces excess capacity to a minimum and start to affect overall flux of the pathway. Excess capacity of cytochrome c oxidase (COX) varies between tissues, but little is known about differences between species. In particular, information is lacking on mitochondrial respiratory function in the mouse heart, despite the fact that mutant mice provide increasingly important animal models. Permeabilized muscle fibers were prepared from the left ventricle of a single mouse heart, and measured in OROBOROS Oxygraph-2k instruments in parallel at 4, 25, 30, 37 and 40 °C (N³4)¹. Threshold plots were constructed from azide titrations of flux through the electron transport chain (parallel e-input into complexes I+II with malate+pyruvate+glutamate+succinate and uncoupling by FCCP), versus COX (0.5 mM TMPD+2 mM ascorbate after uncoupling and inhibition by rotenone+malonate+antimycin)² A). Azide was used, since inhibition of COX by cyanide is reversed by pyruvate particularly at low oxygen levels. The inhibition constant, K_i, of COX for azide was 0.1 mM at 37 °C, increasing from 4 to 40 °C over two orders of magnitude. COX velocity measured with TMPD+ascorbate was 1.3-fold of maximum electron transport capacity of the respiratory chain at 25 to 40 °C, and 3.3-fold at 4 °C. In contrast, linear extrapolations of the threshold plots revealed a COX excess capacity of 1.6-fold over pathway flux in the range of 30 to 40 °C, increasing to 1.8- and 7.6-fold at 25 °C and 4 °C, respectively. Application of complex I substrates only, would yield an apparent COX excess capacity of >3-fold over pathway flux (at 30 and 37 °C), since parallel e-input through complex I+II doubled flux compared to complex I substrates. Taken together, COX excess capacity in myocardial fibers of the mouse was significantly higher than in fibers of rat heart or human skeletal muscle. Results obtained under hypothermic incubation conditions of permeabilzed fibers may be extrapolated to physiological temperature of 37 °C with caution only. The very high COX excess capacity under hypothermia (4 °C) may compensate for hypothermic hypoxia by decreasing the p₅₀ of mitochondrial respiration in parallel to the decreased p₅₀ of hemoglobin and myoglobin. The present study yields an important baseline for further investigations of mitochondrial function in the mouse heart, including genetic models of acquired and inherited mitochondrial defects.

1.    Lemieux H, Garedew A, Blier PU, Tardif J-C, Gnaiger E (2006) Temperature effects on the control and capacity of mitochondrial respiration in permeabilized fibers of          the mouse heart. Biochim. Biophys. Acta 1757 (5-6, Suppl. 1): 201-202.

2.    Garedew A, Lemieux H, Schachner T, Blier PU, Tardif J-C, Gnaiger E (2006) High excess capacity of cytochrome c oxidase in permeabilized fibers of the mouse heart. Biochim. Biophys. Acta 1757 (5-6, Suppl. 1): 167-168

1P-01.   Preservation of cardiac mitochondrial function during ischemia and ischemia-reperfusion: Role of the cardioplegic protection by Celsior and Histidine buffer solution.

               Marco Alves², Paulo J. Oliveira², Rui A. Carvalho¹

¹ Department of Biochemistry, NMR Center, University of Coimbra, Portugal

²Center for Neurosciences and Cell Biology, University of Coimbra, Portugal - alvesmarc@gmail.com

    Cardiac ischemia occurs when a reduced coronary blood flow occurs, which results in a decrease of oxygen and nutrients supply contributing to several pathophysiologies (e.g. myocardial infarction, peripheral vascular insufficiency, stroke and hypovolemic shock). The restoration of blood flow to an ischemic organ is essential to prevent irreversible injury. Paradoxically, reperfusion induces more damage to the tissue than ischemia alone. During heart transplantation, various stressful conditions occur which include long periods of ischemic organ storage in cold cardioplegic solutions followed by transplant and reperfusion. Since ATP production in mitochondria is essential to the heart in order to maintain contractile activity, mitochondrial function may be the main mediator of ischemia and ischemia-reperfusion injury.

    We focused on two selected cardioplegic solutions: Histidine Buffer Solution (HBS) and Celsior Solution (CS) and their ability to protect cardiac mitochondrial function. A standard Krebs-Henseleit (K-H) Solution was used as negative control. Male and Female Wistar rats (6-8 weeks) were each divided in 2 sets of groups. Isolated hearts from both sets were subjected to different ischemia periods (0, 4 and 6 h) and immersed in the different preservation solutions at 4 ºC. Subsequently, heart mitochondria (HM) of one experimental group were immediately isolated using standard procedures, while the hearts from another group were subjected to 30 minutes reperfusion in a Langendorf column prior to HM isolation.  Two different control groups were used. In one of them HM were immediately isolated after animal sacrifice. In the second group (reperfusion control), hearts were excised and reperfused during 30 minutes. A Clark-type oxygen electrode was used to measure mitochondrial respiration parameters. HM transmembrane electric potential was measured with a TPP⁺- selective electrode.

    Ischemia, particularly for 6 h, induced damage in some mitochondrial parameters (e.g. ADP-induced depolarization and lag phase), although the majority of indexes evaluated were unchanged for both 4 and 6 h. Reperfusion particularly induced damage to some mitochondrial parameters (e.g. lag phase, state 3 and FCCP respiration or maximum potential attained). Some parameters such as the Respiratory Control Rate, FCCP-stimulated respiration or Potential Recuperation recovery after ADP phosphorylation with complex I substrates were especially affected by reperfusion, which suggests that this complex is particularly affected by reperfusion.

    The data confirms reperfusion as the main damage generator to HM. Most importantly, the results suggest that the CS is more effective in preventing HM damage.

     Supported by FCT: SFRH/BD/31655/2006; POCI/SAU-OBS/55802/2004

1P-02.   Study of the mitochondrial respiration in the equine muscle with high-resolution respirometry: Feasibility, preliminary results and potential applications.

               Dominique-Marie Votion^1a,b, Hélène Lemieux², Ange Mouithys-Mickalad³,    Didier Serteyn^1a, Erich Gnaiger²

^1a Equine Teaching Hospital, Department of Clinical Sciences, Faculty of Veterinary Medicine and ^1bEquine European Centre of Mont-le-Soie, University of Liège, Sart Tilman, B-4000 Liège, Belgium

²  Medical University of Innsbruck, Dept. General and Transplant Surgery, D. Swarovski Research Laboratory, A-6020 Innsbruck, Austria.

³  Centre of Oxygen: Research and Development (C.O.R.D.), University of Liège, Sart Tilman, B-4000 Liège, Belgium - dominique.votion@ulg.ac.be

    Equines have been bred since time in memoriam to a large variety of uses of high specificity.  Regardless of their intended use, horses are able to perform physical activities at a level that surpasses other animals of similar body size.  The athletic potential of an individual relates to oxygen (O₂) transport and utilisation¹.  As regard to the maximal oxygen consumption (VO_2max) which is considered as an index of exercise’s capacity², the horse is an amazing athletic animal.  For example, when expressed on a mass specific basis, race horses have values for VO_2max twice those of elite human athletes^3,4. During high-intensity exercise, the large VO_2max is achieved as a result of remarkable cardiopulmonary adaptations.  It might be expected that the respiratory capacity of equine muscle would be also proportionately higher than the one of human.  Measurements of the maximal respiratory capacity of the equine skeletal muscle are scarce⁵.  However, it has always been the ambition of physiologists to determine athletic suitability for specific disciplines and to predict athletic performance. 

Up to now, cellular energetics of muscles has been based upon the histochemical and biochemical analyses of large biopsy samples⁶ (200 to 300 mg of muscle).  Given the little practicality of performing muscle biopsy in performance horses, the technique has been limited to scientific protocols or to the evaluation of horses with suspected myopathy⁷.

High-resolution respirometry offers the opportunity to perform bioenergetics’ studies with a minimal amount of sample using permeabilised tissue⁸.  This technique precludes the use of time-consuming preparation of isolated mitochondria and enables the study of mitochondrial function within a preserved intramitochondrial environment.  We aimed at studying the feasibility of high-resolution respirometry for investigating the mitochondrial respiratory function of the equine muscles with permeabilised fibres obtained by microsampling.

Microbiopsies were performed in the triceps brachii of 3 horses with a 14 G biopsy needle that enables the sampling of 20 to 40 mg of muscle.  The specimens were prepared according to the Oroboros Oxygraph-2k manual.  From 1 to 4 mg wet weight of sample were put in Oxygraph-2k chambers filled with media warmed at 37°C in presence of malate plus glutamate.  Oxygen was injected into the chambers in order to reach a 500 nmol/ml O₂ concentration. Then, the experiment starts with the measurement of State 2 respiration after steady-state respiratory flux.  State 3 of respiration was initiated by adding ADP in excess.  Integrity of mitochondria was estimated by assaying the mixture with Cyt c.  A further increase of respiration was stimulated by adding succinate.  After that, uncoupling of oxidative phosphorylation by stepwise titration of FCCP was used to obtain the maximal stimulation of flux.  Finally, the respiration was inhibited by the addition of rotenone (inhibition of complex I) and antimycin A (inhibition of complex III).  All measurements were performed at steady-state.

A single biopsy enabled to perform at least 4 titration protocols that was completed within 1.5 hour.  The O₂ concentration remained above 240 nmol/ml for all the procedure which may of importance to avoid O₂ dependence of results.  Integrity of mitochondrial function in samples preserved for 4 days (at 4°C in BIOPS) did not demonstrate any alteration thus enabling to delay analysis (and open many perspectives such as sampling during field trials).  These preliminary results demonstrated the feasibility of studying mitochondrial respiration in the equine muscle based on microsamples which may, with no doubt, be performed on performance horses without consequence (no scar, no pain, no sedation required).  It is now necessary to evaluate the variability between runs, to determine reference ranges taking into account all the parameters that may influence results (age, gender, breed, level of training etc.) and to compare mitochondrial physiology with the one of other athletic and non athletic species.

Such a database will offer the possibility to study the relationship between mitochondrial function and parameters of athletic capacity as well as to the pathophysiological mechanism underlying equine myopathic disorders.

     Our grateful thanks are due to the “Ministre de l’Agriculture et de la Ruralité de la Région   wallonne” of Belgium who has funded this study

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2.         Bassett DR Jr, Howley ET. Limiting factors for maximum oxygen uptake and determinants of endurance performance. Med Sci Sports Exerc. 2000; 32:70-84.

3.  Hörnicke H, Meixner R, Pollmann U. Respiration in exercising horses. In: Snow DH, Persson SGB, Rose RJ, eds. Equine Exercise Physiology. (Proceedings of the first International Conference on Equine Exercise Physiology, Oxford: Great Britain), Cambridge: Granta Editions. 1982; 7-16.

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5.  Gollnick PD, Bertocci LA, Kelso TB, Witt EH, Hodgson DR.  The effect of high-intensity exercise on the respiratory capacity of skeletal muscle.  Pflugers Arch. 1990; 415:407-13.

6.  Votion DM, Navet R, Lacombe VA, Sluse F, Essén-Gustavsson B, Hinchcliff KW, Rivero JLL, Serteyn D, Valberg SJ. Muscle energetics in exercising horses. Equine and Comparative Exercise Physiology.  2007; In Press.

7.  Ledwith A, McGowan CM.  Muscle biopsy: a routine diagnostic procedure. Equine Vet Ed. 2004; 16:62-67.

8.  Kuznetsov AV, Strobl D, Ruttmann E, Königsrainer A, Margreiter R., Gnaiger E. Evaluation of mitochondrial respiratory function in small biopsies of liver. Analyt Biochem. 2002; 305:186-194.

9.  Valberg SJ, Carlson GP, Cardinet GH, Birks EK, Jones JH, Chomyn A, DiMauro S. Skeletal muscle mitochondrial myopathy as a cause of exercise intolerance in a horse. Muscle Nerve. 1994; 17:305-312.