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Symposium Reports |
1 Departments of Radiology2 Physiology & Biophysics3 Bioengineering, University of Washington Medical Center, Seattle, WA 98195, USA
Abstract
Mitochondrial changes are at the centre of a wide range of maladies, including diabetes, neurodegeneration and ageing-related dysfunctions. Here we describe innovative optical and magnetic resonance spectroscopic methods that non-invasively measure key mitochondrial fluxes, ATP synthesis and O2 uptake, to permit the determination of mitochondrial coupling efficiency in vivo (P/O: half the ratio of ATP flux to O2 uptake). Three new insights result. First, mitochondrial coupling can be measured in vivo with the rigor of a biochemical determination and provides a gold standard to define well-coupled mitochondria (P/O 
2.5). Second, mitochondrial coupling differs substantially among muscles in healthy adults, from values reflective of well-coupled oxidative phosphorylation in a hand muscle (P/O = 2.7) to mild uncoupling in a leg muscle (P/O = 2.0). Third, these coupling differences have an important impact on cell ageing. We found substantial uncoupling and loss of cellular [ATP] in a hand muscle indicative of mitochondrial dysfunction with age. In contrast, stable mitochondrial function was found in a leg muscle, which supports the notion that mild uncoupling is protective against mitochondrial damage with age. Thus, greater mitochondrial dysfunction is evident in muscles with higher type II muscle fibre content, which may be at the root of the preferential loss of type II fibres found in the elderly. Our results demonstrate that mitochondrial function and the tempo of ageing varies among human muscles in the same individual. These technical advances, in combination with the range of mitochondrial properties available in human muscles, provide an ideal system for studying mitochondrial function in normal tissue and the link between mitochondrial defects and cell pathology in disease.
(Received 6 November 2006;
accepted after revision 13 December 2006; first published online 14 December 2006)
Corresponding author K. E. Conley: Department of Radiology, Box 357115, University of Washington Medical Center, Seattle, WA 98195-7115, USA. Email: kconley{at}u.washington.edu
Coupling and pathology
Mitochondria are central to the conversion of energy by oxidizing substrates and generating the cell fuel, ATP. Defects in this conversion process are emerging as central to cell pathology and may be a critical part of cellular ageing. Despite the extensive research into mitochondrial pathology (over 7500 hits for ‘mitochondrial dysfunction’ in PubMed), we know little about the nature and extent of mitochondrial dysfunction in vivo, especially in human tissues, and we are still unclear as to the underlying cause of the defects that lead to dysfunction. One reason for this has been the lack of experimental tools to study mitochondrial function in vivo. Here we show that new technical advances make these measurements possible and highlight human muscle as an ideal system to evaluate the factors responsible for mitochondrial dysfunction. New spectroscopic innovations from our laboratory permit quantitative measurement of mitochondrial O2 and ATP fluxes to yield mitochondrial capacity and coupling efficiency in vivo [half the ratio of ATP flux to O2 uptake (P/O: half the ratio of ATP flux to O2 uptake); Marcinek, 2004]. These innovations permit us to extend our studies beyond the traditional focus of human studies (i.e., the vastus lateralis muscle) to take advantage of a wide-range of human muscles (and muscle properties) in exploring the cellular factors leading to dysfunction. These methods and capabilities provide a number of new findings and insights: (1) mitochondrial properties can be measured in vivo with the rigor of a biochemical determination; (2) mitochondrial coupling differs substantially among muscles; and (3) these coupling differences have an important impact on cell ageing.
New methods measure mitochondrial function in vivo
Figure 1 is a diagram of the sites of the key mitochondrial fluxes on the inner membrane that can now be measured in vivo: O2 uptake at the end of the electron transport chain and ATP generation via the ATP synthase. Also shown are the sites of H+ leak that dissipate the electrochemical gradient and uncouple O2 uptake from ATP synthesis. Measurement of these mitochondrial fluxes that underlie energy coupling is made possible by a combination of non-invasive spectroscopic techniques (Marcinek et al. 2004). Recently developed magnetic resonance spectroscopy (MRS) tools have provided evidence of mitochondrial uncoupling in vivo in human muscle with thyroid hormone treatment (Lebon et al. 2001) and in normal mouse muscle in comparison with mice without uncoupling protein (Cline et al. 2001). A pairing of optical and MR spectroscopies extends these indices of mitochondrial function to permit a quantitative measure of P/O that has revealed substantial uncoupling in mouse hindlimb muscles in vivo when treated with a chemical uncoupler (Marcinek et al. 2004) and with age (Marcinek et al. 2005). Figure 2 shows the results of new optical methods that permit separation of the dynamics of the key oxygen carriers, myoglobin (Mb) and haemoglobin (Hb) in human muscles (Schenkman et al. 1999). The MbO2 and HbO2 deoxygenation time courses seen in Fig. 2 result from cellular O2 uptake in the absence of O2 delivery when blood flow is blocked by a tourniquet. Conventional near-infrared (NIR) optical tools do not separate these spectroscopic signals and therefore do not distinguish blood from cellular oxygenation (Richards-Kortum & Sevick-Muraca, 1996). However, analytical methods adopted from the chemical industry to separate optically similar compounds in solvent mixtures (Martens & Naes, 1989) achieve this separation in vivo. The result is an ability to measure the change in relative oxygen saturations of these compounds which, in combination with methods that determine Mb and Hb concentration in vivo, permits the determination of O2 uptake non-invasively in resting muscle for the first time (Marcinek, 2004; Marcinek et al. 2004; Amara et al. 2007).
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PCr) reflects the difference between glycolytic ATP synthesis (< 8% of total ATP supply; Amara et al. 2007) and the ATP turnover by the cell (PCr = total ATP turnover minus glycolytic ATP supply). This net ATP turnover represents the flux that must be supplied by the mitochondria to meet the ATP demands of the cell under aerobic conditions. We have validated this approach in vivo by comparing these spectroscopic flux determinations with direct measurements of O2 uptake and lactate flux into blood circulating through the rattlesnake tailshaker muscles during rattling (Kemper et al. 2001). These new non-invasive, in vivo methods allow us to measure mitochondrial oxidation (O2 uptake) and phosphorylation (ATP flux) and thereby evaluate the presence and extent of uncoupling in human muscles not typically studied by invasive methods. Muscles differ in energetics
An important insight from the new methods is that mitochondrial energetics differ among resting muscles of the same individual. Figure 3 shows ATP flux, O2 uptake and P/O in two human muscles, the first dorsal interosseus (FDI) of the hand and the tibialis anterior muscle (TA) of the leg from the same individuals (Amara et al. 2007). Both muscles had the same ATP flux (Fig. 3), but differed in O2 uptake. For the FDI, the O2 uptake rate (1.4 ± 0.1 µM s–1) was in good agreement with traditional, invasive measurements of O2 uptake from blood using the Fick method reported for resting forearm muscles (1.0 ± 0.4 (S.D.) µM s–1; Zurlo et al. 1990; Van Beekvelt et al. 2001). However, the O2 uptake of the TA was significantly higher on average than in the FDI. This disparity in O2 uptake (but similar ATP flux) was also found in mouse hindlimb muscles in vivo before and after treatment with the mitochondrial uncoupler dinitrophenol (DNP) (Marcinek et al. 2004). Thus, the greater O2 uptake in the human TA versus FDI at the same ATP flux is similar to the effect of an uncoupler on mitochondrial function and points to differences in mitochondrial coupling between the two muscles at rest.
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The level of mitochondrial coupling in the two muscles can be assessed using a quantitative measure of P/O referenced to the theoretical value for coupling of oxidative phosphorylation (P/O 2.3–2.5; Nicholls & Ferguson, 2002; Brand, 2005). Combination of the measurement of O2 flux with ATP flux yields a direct measure of energy coupling or P/O. A P/O of 2.7 is found in the FDI and is similar to the average of P/O = 3.0 reported for several studies of mitochondria isolated from human quadriceps muscle, as summarized by Tonkonogi & Sahlin (1997). This value also falls between the traditional value for well-coupled oxidative phosphorylation (P/O = 3.0) and the revised value from recent studies in rodents (P/O 2.3–2.5). Thus, our measurement of P/O in the FDI falls in the range of maximum values for the stoichiometry of oxidative phosphorylation, which indicates that the mitochondria are well coupled in the FDI in subjects up to 50 years old.
Coupling differs between muscles
Many of the TA muscles of the same subjects showed a lower P/O (mean P/O = 2.0; Fig. 3) than found in the FDI, which is suggestive of mild uncoupling. Evidence of uncoupling has been reported in isolated human mitochondria, as measured by non-phosphorylating respiration in muscle biopsies (Fernstrom et al. 2004). These lower P/O values in many of the TAs are similar to that measured in the mouse hindlimb (P/O = 2.2; Marcinek et al. 2004) using the same methods. Mitochondria in small animals have been shown to have a significant level of H+ leak across the inner mitochondrial membrane that is thought to uncouple O2 uptake from ATP synthesis, resulting in a reduction in P/O (Porter & Brand, 1993).
Two mitochondrial properties in rodents have been suggested to be responsible for the high H+ leak and respiration that does not generate ATP (so-called non-phosphorylating respiration). The first property is a change in membrane composition that results in greater permeability to H+ (Else & Hulbert, 1987) and the second is activation of uncoupling proteins (UCP) and the adenine nucleotide transporter (ANT; Speakman et al. 2004). Figure 1 shows that these mechanisms dissipate the H+ gradient and short-circuit oxidative phosphorylation. The regulation of UCP and ANT to protect the mitochondria from damage by reactive oxygen species (ROS) has been proposed to underlie mitochondrial uncoupling (Echtay et al. 2005). One difficulty in sorting out the role of these H+ translocators is that they are regulated, so their content indicates the capacity for H+ translocation but not the level of uncoupling (Esteves et al. 2004). This uncoupling is thought to lessen ROS production by reducing the mitochondrial membrane potential and may be a survival tactic for minimizing oxidative damage in tissue with age (Brand, 2000). Thus, physiological control of UCP and ANT may well regulate coupling in vivo and has the potential to account for the uncoupling observed in adult human TA muscle.
Importance to ageing
Two leading theories of ageing predict that a disparity in cellular energetics is responsible for the difference in cell longevity evident in the preferential loss of type II muscle fibres with age (Lexell, 1995). The rate of living hypothesis proposes that higher O2 uptake rates will produce greater levels of ROS (Beckman & Ames, 1998), leading to oxidative damage and mitochondrial dysfunction in aged tissue. An alternative hypothesis is that mild uncoupling impacts cell ageing by reducing ROS production and minimizing oxidative damage (Echtay et al. 2002). We first tested for differences in cellular ageing using two measures reflective of mitochondrial dysfunction: reduced P/O and depletion of [ATP]. Both uncoupling and loss of [ATP] are thought to be indicative of the processes that trigger apoptotic and necrotic pathways responsible for muscle cell death (sarcopenia) and are therefore good measures of cellular ageing (Skulachev, 2006). Figure 4 shows that the change in both of these factors with age differed among human muscles (shown as adult minus elderly values). Between adult and elderly subjects, no significant change in P/O or [ATP] was found in the TA, but significant changes with age in coupling and depletion of ATP (as measured by (ATP/PCr)) were evident in the FDI and the vastus lateralis (VL). Thus, striking differences in the extent of ageing are apparent among muscles, as is especially clear between muscles sampled from the same individuals: TA and FDI.
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Impact of fibre type on mitochondrial ageing
The disparity in the age-related [ATP] depletion and uncoupling between the FDI (45–50% type II fibre content) and TA (25% type II content) suggests that mitochondrial dysfunction with age is related to muscle fibre type (muscle fibre type compositions from Johnson et al. (1973)). Supporting this fibre type dependence is a lack of age-related uncoupling reported in the predominantly type I human soleus muscle in vivo (SOL, 10% type II; Petersen et al. 2003), but substantial mitochondrial dysfunction reported in the predominantly type II human vastus lateralis (VL, 60% type II; Conley et al. 2000). Additional evidence for this fibre type dependence of mitochondrial dysfunction is that lipid peroxidation in elderly muscles is greater in muscles with higher type II fibre content (Pansarasa et al. 1999). This peroxidation is thought to be a factor responsible for membrane damage and reduced mitochondrial efficiency (Schrauwen & Hesselink, 2004). Variation in mitochondrial function with the fibre type composition of muscle is likely to reflect the metabolic properties associated with muscle fibre types (e.g. type I = slow-twitch, oxidative). Higher intracellular lipid in type I fibres (Howald et al. 1985) is an example of a factor that could be responsible for mild uncoupling in adults that leads to preservation of mitochondrial function with age in type I fibres. An alternative possibility is that chronic muscle use or activity sets the pace of ageing, resulting in the extended longevity of the chronically active type I fibres and faster ageing in type II fibres. However, the similar degree of cell ageing in a leg (VL) and a hand (FDI) muscle with clearly different use patterns but high type II content argues against activity or training state as the determining factor in the tempo of ageing. Muscle use may interact with other factors to impact cellular ageing, but the results reported here indicate that mitochondrial defects in elderly human muscle vary primarily with fibre type content (or the associated metabolic properties), just as has been found for ROS generation and the longevity of muscle fibres (Anderson & Neufer, 2006).
The greater mitochondrial dysfunction in muscles with high type II content provides insight into the preferential loss of type II fibres with age. Recent results suggest that mitochondrial defects may trigger the cascade of events that leads to cell death and muscle fibre loss (i.e. apoptosis; Skulachev, 2006). Such cell loss is irreversible and may be responsible for up to half the decline in muscle size with age (i.e. sarcopenia; Deschenes, 2004). Clearly, the extent of mitochondrial dysfunction with age is not uniform among muscles of the same subject, as shown by the greater age-related [ATP] depletion and mitochondrial uncoupling in muscles with more type II fibres. The mitochondrial dysfunction evident in the FDI and VL with age may well reflect the higher ROS generation reported in type II muscle fibres (Anderson & Neufer, 2006) and may be responsible for the reduced longevity characteristic of this fibre type (Lexell, 1995). The fibre-type dependence of mitochondrial dysfunction discussed here may therefore be at the root of the preferential loss of type II fibres found in the elderly. Thus, mitochondrial dysfunction is not only key to many age-related maladies but also has an important impact on cell fate, resulting in sarcopenia, which is a leading cause of disability in the elderly.
Human muscles as model systems
The standard for studies of mitochondrial function has been in vitro biochemical assays of rodent tissue. The data presented here show the great potential for studying mitochondrial function in vivo in humans. Evaluation of mitochondrial dysfunction and its reversibility in vivo is now possible with direct measurement of mitochondrial capacity, coupling (P/O) and [ATP] using the combination of non-invasive spectroscopic techniques described here. Human muscles have a number of advantages for these studies, including large fibre type differences between muscles and the ability to sample tissue, both non-invasively and by biopsy, to evaluate the factors leading to mitochondrial dysfunction with age in humans. Other important factors in favour of human studies in vivo are the avoidance of artifacts associated with mitochondrial isolation, inhibition of mitochondrial function by anaesthetics, and species differences inherent in animal studies. Thus, technical advances now permit the study of cellular energetics non-invasively and in vivo in human muscles. These technical advances, combined with the range of mitochondrial properties available in humans, provide an ideal system for studying the link between mitochondria and cell energetics in normal tissue, and mitochondrial defects and cell pathology in disease.
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Acknowledgements
Thanks go to Lori Arakaki, Wayne Ciesielski, Martin J. Kushmerick, David Niles and Ken A. Schenkman for their contributions. This work was supported by National Institutes of Health R01 grants AR 41928, AR 45184 and AR 36281, AG-022385, as well as training grant AG00057.
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35 years old) minus the average for the elderly group (mean age,