1Institute of Physiology and Center for Integrated Genomics, Academy of Sciences of the Czech Republic, 142 20 Prague; 3Department of Pediatrics, 1st Medical Faculty, Charles University, 110 00 Prague, Czech Republic; 2Department of Transplant Surgery, D. Swarovski Research Laboratory, University Hospital Innsbruck, A-6020 Innsbruck, Austria; and 4Department of Metabolic Diseases, Children's Memorial Health Hospital, 04-730 Warsaw, Poland
Submitted 16 June 2004 ; accepted in final form 13 July 2004
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ABSTRACT |
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oxygen kinetics; mitochondrial disease
Mammalian COX is composed of 13 subunits: mitochondrial (mt)DNA genes encode the 3 largest subunits forming the catalytic core of the enzyme, and the other 10 subunits are encoded by the nuclear genome (27). In addition, numerous nucleus-encoded factors are required for efficient assembly and maintenance of the COX holoenzyme that are similar in yeast and humans (11, 18). Mutations in genes encoding these COX assembly factors are a frequent cause of COX deficiencies; in fact, they are much more common than mutations in genes encoding the COX subunits themselves (25). One of these assembly proteins is encoded by the SURF1 gene. Two groups identified mutations in this gene to be responsible for the autosomal recessive form of Leigh syndrome (LSCOX) (29, 33). Leigh syndrome, a subacute necrotizing encephalomyopathy, is a progressive neurodegenerative disease. The severe symptoms usually have onset within the first year of life and are characterized by general psychomotor retardation and bilaterally symmetrical lesions in the basal ganglia region. An increased level of lactate in both blood and cerebrospinal fluid is observed. The disease is fatal in the vast majority of cases; the patients usually die before 5 years of age (21). Leigh syndrome is the most common form of COX disorders and one of the most frequently occurring respiratory chain defects in infancy and childhood (23). To date, more than 30 different pathogenic mutations have been described in SURF1 (16). Most of these are nonsense mutations inducing the formation of a premature stop codon; missense and splicing site mutations are less common. It is generally assumed that the severe, isolated COX defect in patients harboring SURF1 mutations results from impaired assembly of the complex (3, 28). In a previous study (15), researchers at our laboratory further demonstrated that there exist several functional forms of COX in mitochondria of LSCOX cells that differ in subunit composition, electron transport, and proton pumping properties.
Because all previous studies with LSCOX cells were performed at much higher PO2 than is actually present in cells in vivo, we focused on low oxygen levels in our respirometric investigation of the functional impact of SURF1 mutations. We analyzed the oxygen kinetics of COX in fibroblasts from five Leigh syndrome patients by performing high-resolution respirometry. Although respiration at high oxygen levels was indistinguishable from control cells in these patients' cells, we observed a highly significant decrease of COX oxygen affinity in fibroblasts from all patients studied. These findings are discussed in the context of cell energetics in these pathological states.
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METHODS |
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High-resolution respirometry and oxygen kinetics. The Oxygraph-2k (Oroboros) was used for measurements of oxygen consumption. This instrument provides sufficient sensitivity and time resolution for analysis of the oxygen kinetics of mitochondrial and cellular respiration (5). Before all measurements, the chambers were sterilized with 70% ethanol for 20 min. For calibration of the oxygen sensor, a constant signal was obtained in the opened chamber with medium in equilibrium with the air in the gas phase. Internal zero-oxygen calibration was performed with Datlab software (Oroboros) as described previously (10). All measurements were performed at 30°C in a 2-ml chamber volume.
For measurements with intact fibroblasts, an extracellular buffer (32) was used that contained (in mM) 137 NaCl, 5 KCl, 0.7 sodium phosphate, and 25 Tris·HCl (pH 7.4). A mitochondrial KCl medium (13) containing (in mM) 80 KCl, 10 Tris·HCl, 3 MgCl2, 1 EDTA, and 5 potassium phosphate (pH 7.4) was used for experiments when the cell membrane of the fibroblasts was permeabilized with digitonin (0.1 g/g protein). In this case, 10 mM succinate was added as a substrate and 1 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used to uncouple and maximally stimulate respiration (state 3u). The volume-specific rate of oxygen consumption (oxygen flux) was calculated as the negative slope of oxygen concentration recorded at 2-s time intervals. The signal was deconvoluted with the exponential time constant of the oxygen sensor (35 s). Cellular oxygen flux (J02) was corrected for instrumental background, which is a linear function of experimental PO2 and results from oxygen consumption by the sensor and oxygen back-diffusion from low-capacity oxygen reservoirs. P50 is the PO2 at which the cellular respiratory rate is half-maximal. This parameter was obtained from the hyperbolic function J02 = (Jmax·PO2)/(P50 + PO2) fitted over the low-oxygen range of 01.1 kPa, where Jmax is maximal flux. All calculations were performed with routine functions of the Datlab software (10).
Protein determination. Protein content was measured in 10-µl and 20-µl aliquots withdrawn from the Oxygraph chamber by the method of Bradford (2) with bovine serum albumin as a standard. Samples were sonicated for 20 s before protein determination.
Statistics. Data are presented as means ± SD from several measurements of each individual cell culture. The statistical significance between control and patient cells was evaluated with standard t-test and Kruskal-Wallis one-way ANOVA on ranks and by Tukey-Kramer multiple-comparison test with a freeware version of NCSS software.
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RESULTS |
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Typical experimental records of the simultaneous decrease of oxygen concentration and flux are shown in Fig. 1, A and B, for a control and a patient (patient 1) fibroblast culture, respectively. Oxygen flux and the corresponding hyperbolic fits are plotted as a function of oxygen concentration in Fig. 1C. In intact cells the P50 of control fibroblasts ranged from 0.03 to 0.05 kPa (0.039 ± 0.010 kPa), whereas in patient cells it increased 2.1-fold to a range of 0.080.10 kPa (0.082 ± 0.015 kPa) (Fig. 2A). No statistically significant differences were found between the average P50 of fibroblast cultures of individual patients (not shown). The P50 of cells for each patient (patients 14), however, was significantly higher compared with control subjects (Fig. 2A).
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In contrast to P50, no statistical differences were found in respiratory rates at kinetic oxygen saturation normalized on protein content (Jmax) between control and patient cells, in agreement with a previous study (15). In intact control cells, Jmax ranged from 42.8 to 99.6 pmol O2·s1·mg1 (mean 60.5 ± 17.9 pmol O2·s1·mg1); in patient fibroblasts it ranged from 55.0 to 118.6 pmol O2·s1·mg1 (mean 78.8 ± 25.4 pmol O2·s1·mg1). In state 3u, Jmax increased to a range of 60.2215.5 pmol O2·s1·mg1 (mean 108.1 ± 44.5 pmol O2·s1·mg1) in control fibroblasts compared with 68.1146.0 pmol O2·s1·mg1 (mean 95.0 ± 24.6 pmol O2·s1·mg1) in patient fibroblasts.
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DISCUSSION |
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We analyzed the COX oxygen kinetics by high-resolution respirometry (10). This method yields the P50 parameter, a measure of the sensitivity of cellular respiration to oxygen availability. Such a "macroscopic" approach is advantageous for several reasons: the method is relatively simple compared with, e.g., the flow-flash kinetic approach, which can yield microscopic oxygen binding constants (31). The P50 can be used to assess the oxygen dependence of respiration in mitochondria and even in small cells with relatively low aerobic activity such as fibroblasts (12) and human umbilical vein endothelial cells (26), where intracellular oxygen gradients are small and the P50 of cellular respiration is close to the P50 of isolated mitochondria. Possible changes of intracellular diffusion gradients to mitochondria in pathological cells, therefore, can be ignored in these cells but might be considerable in more active and larger cells (6). This is supported by our present study, in which P50 values of control fibroblasts are similar to P50 of mitochondria isolated from rat heart (7). The P50, therefore, can be conveniently analyzed in whole cells under conditions of intact mitochondrial membranes, where COX function is integrated in the respiratory chain. Results of such analyses might thus be more relevant in the context of mitochondrial and cellular physiology.
Our results clearly show that P50 is increased in patient cells compared with control cells. Basically, two reasons for the P50 increase can be considered: the P50 increase may be due to a decrease of the COX excess capacity or an increased apparent Michaelis-Menten constant (K'm) of COX for oxygen. A lower excess capacity of COX relative to the flux through the respiratory chain yields a corresponding increase of COX turnover, which then causes the P50 of oxygen flux through the respiratory chain to increase (8). To find out whether the observed increase of P50 in patient cells is due to a decrease of COX capacity, we plotted the P50 values versus the specific oxygen flux normalized on protein content (Fig. 3). P50 increased as a function of oxygen consumption of intact cells respiring on endogenous substrates (i.e., as COX turnover increased). In patient cells, P50 values increased without a corresponding increase of oxygen flux, indicating that the difference in P50 cannot be explained by the change of protein-specific flux observed in control and patient cells (Fig. 3A). Although the dependence of P50 on oxygen flux is more complex under uncoupled conditions (26), an analogous picture appeared when we analyzed P50 measurements in FCCP-treated cells (Fig. 3B). These data, therefore, provide strong evidence that the P50 increase in patient fibroblasts was due to changes in COX affinity for oxygen.
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In addition, a recent study shows that the decrease of COX affinity for oxygen caused by nitric oxide results in decreased levels of hypoxia-induced factor 1- and thus lower expression of "hypoxic genes" (14). If an analogous situation occurs when COX oxygen affinity is decreased because of SURF1 mutations, the patients not only would have impaired oxidative energy production due to oxygen limitation but would also suffer from impairment of glycolytic energy production due to lower expression of hypoxic genes.
The observed phenomenon of increased P50 in LSCOX fibroblasts could be one of the key mechanisms that translate the COX assembly defect triggered by SURF1 mutation to deleterious changes of cell energetics that finally result in the fatal symptoms of Leigh syndrome. In general, experimental and clinical investigations of cells and isolated mitochondria should be extended into the low physiological oxygen range, including evaluation of COX oxygen kinetics, to study the etiopathogenic mechanisms of COX deficiencies.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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