Chronic hypoxia-induced alterations in mitochondrial ...

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Chronic hypoxia-induced alterations in mitochondrial energy metabolism are not reversible in rat heart ventricles Karine Nouette-Gaulain, Matthieu Biais, Jean-Pierre Savineau, Roger Marthan, Jean-Pierre Mazat, Thierry Letellier, and François Sztark

Abstract: Chronic hypoxia alters mitochondrial energy metabolism. In the heart, oxidative capacity of both ventricles is decreased after 3 weeks of chronic hypoxia. The aim of this study was to evaluate the reversal of these metabolic changes upon normoxia recovery. Rats were exposed to a hypobaric environment for 3 weeks and then subjected to a normoxic environment for 3 weeks (normoxia-recovery group) and compared with rats maintained in a normoxic environment (control group). Mitochondrial energy metabolism was differentially examined in both left and right ventricles. Oxidative capacity (oxygen consumption and ATP synthesis) was measured in saponin-skinned fibers. Activities of mitochondrial respiratory chain complexes and antioxidant enzymes were measured on ventricle homogenates. Morphometric analysis of mitochondria was performed on electron micrographs. In normoxia-recovery rats, oxidative capacities of right ventricles were decreased in the presence of glutamate or palmitoyl carnitine as substrates. In contrast, oxidation of palmitoyl carnitine was maintained in the left ventricle. Enzyme activities of complexes III and IV were significantly decreased in both ventricles. These functional alterations were associated with a decrease in numerical density and an increase in size of mitochondria. Finally, in the normoxia-recovery group, the antioxidant enzyme activities (catalase and glutathione peroxidase) increased. In conclusion, alterations of mitochondrial energy metabolism induced by chronic hypoxia are not totally reversible. Reactive oxygen species could be involved and should be investigated under such conditions, since they may represent a therapeutic target. Key words: chronic hypoxia, reoxygenation, rat heart, mitochondria, energy metabolism, oxidative phosphorylation, antioxidants. Re´sume´ : L’hypoxie chronique modifie le me´tabolisme eńerge´tique des mitochondries. Dans le cœur, la capacite´ oxydative des 2 ventricules diminue apre`s 3 semaines d’hypoxie chronique. La pre´sente e´tude a eu pour but d’e´valuer la re´versibilite´ des modifications me´taboliques lors du retour en normoxie. On a expose´ des rats a` un environnement hypobare pendant 3 semaines, puis a` un environnement normoxique pendant 3 semaines (groupe retour en normoxie), et on les a ensuite compare´s avec des rats maintenus dans un environnement normoxique (groupe te´moin). On a examine´ le me´tabolisme eńerge´tique des mitochondries dans les ventricules gauche et droit. On a mesure´ la capacite´ oxydative (consommation d’oxyge`ne et synthe`se d’ATP) dans des fibres peleés a` l’aide de saponine. On a mesure´ les activite´s des complexes de la chaıˆne respiratoire mitochondriale et d’enzymes antioxydantes sur des homogeńats de ventricules. On a effectue´ une analyse morphome´trique des mitochondries en microscopie e´lectronique. Chez les rats du groupe retour en normoxie, les capacite´s oxydatives du ventricule droit ont diminue´ lorsque le glutamate ou la palmitoyl carnitine ont e´te´ ` l’oppose´, l’oxydation de la palmitoyl carnitine a e´te´ maintenue dans le ventricule gauche. Les utilise´s comme substrats. A activite´s enzymatiques des complexes III et IV ont diminue´ de manie`re significative dans les 2 ventricules. Ces modifications fonctionnelles ont e´te´ associeés a` une diminution de la densite´ nume´rique et a` une augmentation de la taille des mitochondries. Enfin, chez le groupe retour en normoxie, les activite´s des enzymes antioxydantes (catalase et glutathion peroxydase) ont augmente´. Ainsi, les modifications du me´tabolisme eńerge´tique des mitochondries induites par l’hypoxie chronique ne sont pas totalement re´versibles. Les ROS pourraient eˆtre en cause et devraient eˆtre examine´s dans ces conditions puisqu’elles pourraient repre´senter une cible the´rapeutique. Mots-cle´s : hypoxie chronique, reóxygeńation, cœur de rat, mitochondrie, me´tabolisme eńerge´tique, phosphorylation oxydative, antioxydants. Received 11 August 2010. Accepted 27 October 2010. Published on the NRC Research Press Web site at cjpp.nrc.ca on 24 December 2010. K. Nouette-Gaulain, M. Biais, and F. Sztark.1 Department of Anesthesiology and Intensive Care Medicine and Laboratoire de physiopathologie mitochondriale (INSERM U688), Centre Hospitalier Universitaire de Bordeaux and Universite´ Victor Segalen Bordeaux 2, 33076 Bordeaux, France. J.-P. Savineau and R. Marthan. Laboratoire de physiologie cellulaire respiratoire (INSERM U885), Universite´ Victor Segalen Bordeaux 2, 33076 Bordeaux, France. J.-P. Mazat and T. Letellier. Laboratoire de physiopathologie mitochondriale (INSERM U688), Universite´ Victor Segalen Bordeaux 2, 33076 Bordeaux, France. 1Corresponding

author (e-mail: [email protected]).

Can. J. Physiol. Pharmacol. 89: 58–66 (2011)

doi:10.1139/Y10-105

Published by NRC Research Press

Nouette-Gaulain et al.

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[Traduit par la Re´daction]

_______________________________________________________________________________________ Introduction Chronic hypoxia (CH) occurs under certain physiological (high altitude) or pathological conditions (chronic pulmonary diseases). Adaptation to CH has been extensively studied in animals and humans (Leverve 1998). CH is characterized by a reduction in oxygen supply, and one of the initial adaptive mechanisms is the decrease in energyrequiring reactions with a marked suppression of adenosine 5’-triphosphate (ATP) demand and supply pathways (Hochachka et al. 1996). Mitochondrial metabolism is involved in CH adaptation via energy regulation, generation of reactive oxygen species (ROS), and apoptosis (Nouette-Gaulain et al. 2005). In the heart, mitochondria provide, by means of oxidative phosphorylation, more than 95% of the energy supply in the form of ATP. Whereas CH is characterized by a reduction in oxygen supply, it selectively induces a chronic functional overload of the right ventricle (RV) due to pulmonary hypertension that requires additional energy to overcome this increase in pulmonary vascular resistance. Very few studies have indicated that the adaptive mechanism to CH is different in the left ventricle (LV) versus the RV. LV appeared very sensitive to CH and exhibits a rapid decrease in oxidative capacity (Rumsey et al. 1999; NouetteGaulain et al. 2002). We have previously shown that CH decreases ATP synthesis as a consequence of an alteration in mitochondrial function in both ventricles, but that this effect is delayed in the RV (Nouette-Gaulain et al. 2005). This observation suggests some specific adaptive processes at the onset of RV hypertrophy. However, when RV hypertrophy was fully developed after 3 weeks of CH, mitochondrial energy metabolism was decreased in both ventricles. Several aspects of CH-induced pulmonary artery hypertension are reversed upon recovery under normoxic conditions. For example, return to normoxia normalizes pulmonary pressure and reverses RV hypertrophy, although RV compliance remains decreased in normoxia recovery (Bonnet et al. 2002, 2004). Moreover, fibrous bands with metaplasia were observed on histological sections of the RV free wall after normoxia recovery. In the same way, no study has yet investigated the evolution of mitochondrial metabolism of RV and LV after the recovery period. The aim of this study was to examine the reversibility in mitochondrial energy metabolism alterations induced by CH. We studied, in the rat heart, the effect of 3 weeks of normoxia recovery on the mitochondrial energy metabolism changes induced by 3 weeks of CH exposure. Our results in normoxia-recovery rats indicate that cardiac metabolism remains altered, with a decrease in oxidative capacities in both ventricles.

Materials and methods The investigations in this study were approved by the local Animal Care and Use Committee and conformed to the Guide for the care and use of laboratory animals published by the US National Institutes of Health (Institute of Labora-

tory Animal Resources 1996) and European Directives (86/ 609/CEE). Animals Adult male Wistar rats (aged 8–10 weeks, weighing 220– 240 g) were separated into 2 groups. One group (normoxiarecovery rats) was exposed to a simulated altitude of 5000 m (barometric pressure 50.5 kPa) in a well-ventilated, temperature-controlled hypobaric chamber for 3 weeks and was then returned to a normoxic environment for 3 weeks. The hypobaric chamber was opened for 15–30 min twice a week. A duration of 3 weeks for CH exposure as well as for normoxia recovery was chosen on the basis of previous experiments showing that (i) RV hypertrophy and pulmonary artery hypertension are maximum after 21 days of CH, with altered oxidative capacity in both ventricles (Bonnet et al. 2001; Nouette-Gaulain et al. 2005), and that (ii) structural changes regress in relation to the duration of exposure to hypoxia (Liu 1997). The other group (control rats) was kept in the same room but not in the hypobaric chamber, with the same 12 h light : 12 h dark cycle. Free access to a standard rat diet and water was allowed throughout the experimental period. Heart preparation Animals were sacrificed by cervical dislocation, and the heart was quickly removed in a normoxic (i.e., equilibrated with air) cooled relaxing solution (solution 1:10 mmol/L EGTA, 3 mmol/L Mg2+, 20 mmol/L taurine, 0.5 mmol/L dithiothreitol, 5 mmol/L ATP, 15 mmol/L phosphocreatine, 20 mmol/L imidazole, and 0.1 mol/L 2-(N-morpholino) ethanesulfonic acid potassium salt, pH 7.2); chemicals were from Sigma Chemical Company (St. Louis, Mo.). The heart was then dissected and weighed. Pulmonary hypertension was assessed by measuring the ratio of RV free wall weight to the sum of septum plus LV free wall (LVS) weight (Bonnet et al. 2002; Nouette-Gaulain et al. 2005). Oxidative phosphorylation Bundles of fibers (nonisolated cardiomyocytes) between 5 and 10 mg were excised from the endocardial surface of both LV and RV, then permeabilized in solution 1 with 50 mg/mL saponin added. The bundle was then washed twice for 10 min, each time in solution 2 (10 mmol/L EGTA, 3 mmol/L Mg2+, 20 mmol/L taurine, 0.5 mmol/L dithiothreitol, 3 mmol/L phosphate, 1 mg/mL fatty acid-free bovine serum albumin, 20 mmol/L imidazole, and 0.1 mol/ L 2-(N-morpholino)ethanesulfonic acid potassium salt, pH 7.2) to remove saponin. All procedures were carried out at 4 8C with extensive stirring. The success of the permeabilization procedure was estimated by determining the activity of the cytosolic lactate dehydrogenase and the mitochondrial citrate synthase in the medium. After 15–20 min of permeabilization, more than 60% of the cytosolic lactate dehydrogenase was found in the external medium, and the mitochondrial citrate synthase activity in the medium remained below 5% (Sztark et al. 1998). The oxygen consumption rate Published by NRC Research Press

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was measured polarographically at 30 8C using a Clark-type electrode (Hansatec, Norfolk, UK) connected to a computer that displayed online the respiration rate value (original software developed in the laboratory). Solubility of oxygen in the medium was considered to be equal to 450 nmol O/mL. Respiratory rates were determined in a 1 mL oxygraph cuvette containing one bundle of fibers in solution 2 with 10 mmol/L malate plus 10 mmol/L glutamate or 20 mmol/L palmitoyl carnitine as substrates; 50 mmol/L di(adenosine 5’)-pentaphosphate, 20 mmol/L EDTA, and 1 mmol/L iodoacetate were also added to the cuvette to inhibit extramitochondrial ATP synthesis (via the glycolysis or the adenylate kinase) and ATP hydrolysis (Ouhabi et al. 1998). ADP-stimulated respiration, associated with ATP synthesis, was determined in the presence of 1 mmol/L ADP. Basal respiration without ATP synthesis was measured after addition of 70 mmol/L atractyloside and 1 mmol/L oligomycin. After measurements, fibers were removed, dried on a precision wipe, and weighed. Results were expressed in nanomoles of atom oxygen consumed per minute per milligram wet weight of fiber. Under identical conditions, the mitochondrial ATP synthesis rate in skinned fibers was determined by bioluminescence measurement (luciferine–luciferase system) of the ATP produced after addition of 1 mmol/L ADP (Sztark et al. 2000). The ATP Bioluminescence Assay Kit HS II from Roche Diagnostics GmbH (Mannheim, Germany) was used. At various time intervals after addition of ADP, 10 mL aliquots were withdrawn from the oxygraph chamber, quenched in 100 mL DMSO, and diluted in 5 mL ice-cold distilled water. Standardization was performed with known quantities of ATP measured under the same conditions. ATP synthesis rate was expressed in nanomoles ATP produced per minute per milligram wet weight of fiber. The efficiency of oxidative phosphorylation was taken as the ratio of ATP synthesis rate to oxygen consumption rate (ATP/O) (Ouhabi et al. 1998). Enzyme activities of the respiratory chain About 100 mg of LV or RV was minced and homogenized with a glass Potter homogenizer in an ice-cold medium (10%, w/v) containing 225 mmol/L mannitol, 75 mmol/L sucrose, 10 mmol/L Tris–HCl, and 0.10 mmol/L EDTA, pH 7.2. The homogenate was then centrifuged for 20 min at 650g. The supernatant was collected and the protein concentration determined (Lowry et al. 1951). Enzymatic activity was assessed using previously described spectrophotometric procedures (model UVIKON 940, KONTRON) and expressed in nanomoles of substrate transformed per minute per milligram of protein. The enzyme activity of citrate synthase was measured as described by Srere in the presence of 4% (v/v) Triton by monitoring at a wavelength of 412 nm at 30 8C the formation of thionitrobenzoate dianion from the reaction of coenzyme A and 5,5’-dithiobis(2-nitrobenzoic acid) (Srere 1969). The enzyme activity of complex I, reduced nicotinamide adenine dinucleotide (NADH) ubiquinone reductase, was measured as described by Birch-Machin et al. (1989). The oxidation of NADH by complex I was recorded using the ubiquinone analog decylubiquinone as the electron acceptor. The decrease in absorption resulting from NADH oxidation was measured at 340 nm at 30 8C. Complex I activity was calculated from the difference in the rate before and after the addition of rotenone (2 mmol/L), a specific inhibitor of complex I. The complex II (succinate dehy-

Can. J. Physiol. Pharmacol. Vol. 89, 2011

drogenase) specific activity was measured by monitoring the reduction of 2,6-dichlorophenol indophenol at 600 nm at 30 8C in the presence of phenazine methosulphate (Trijbels et al. 1988). The oxidation of ubiquinol (UQ1H2) by complex III (ubiquinol cytochrome c reductase) was determined using cytochrome c (III) as the electron acceptor (Birch-Machin et al. 1989). The reduction of cytochrome c (III) was recorded at 550 nm at 30 8C. Complex IV (cytochrome c oxidase) was measured by the method described by Wharton and Tzagoloff using cytochrome c (II) as the substrate (Wharton and Tzagoloff 1967). The oxidation of cytochrome c was monitored at 550 nm at 30 8C. Morphometric analysis Three blocks were taken from each heart, and 10–20 electron micrographs of randomly chosen fields were obtained from each block. Myocardial fibers for electron microscopic examination were fixed using 2.5% glutaraldehyde and postfixed in osmium tetroxide; they were then embedded in epoxy resin. Ultrathin sections were stained with saturated uranyl acetate and lead citrate. Mitochondrial volume density (Vv), numerical density (Nv), and mean volume (V) were determined in final prints of 161 cm2 at a magnification of 17 250, according to the method of Weibel et al. (1969), as follows: Vv is the relative volume fraction of the mitochondria, determined as the relative surface fraction of the unit area consisting of all mitochondrion slices. Nv depends on mitochondria sections (NA) counted per standard measuring surface, on the size distribution (K), and finally on the shape of the mitochondria, expressed as Nv = K NA3/2 / bVv1/2. The form coefficient b for ellipsoidal structures is a function of the longitudinal and transverse diameter. On the basis of previous studies it was assumed to be b = 2 in the heart (Cervo´s Navarro et al. 1999). K is determined by the size distribution of the objects. According to previous studies in chronic hypoxic heart, K was assumed to be 1.1 in the present study (Cervo´s Navarro et al. 1999). V is the mean volume per mitochondria obtained by simple division of Vv by Nv. Antioxidant enzyme activities A 100 mg sample of LV or RV was homogenized (10%, w/v) in an ice-cold isotonic buffer (0.1 mmol/L EDTA, 50 mmol/L Tris–HCl, pH 7.6) (Germansky and Jamall 1988). Cytosol extracts were prepared by centrifuging at 105 000g for 90 min at 4 8C, and the protein concentration was measured (Lowry et al. 1951). Enzymatic activities were assessed using spectophotometric procedures (model UVIKON 940, KONTRON). Catalase activity was measured by the decrease in absorbance of H2O2 at 240 nm, according to the method of Aebi (Aebi 1984). Glutathione peroxidase activity was measured by the method of Paglia et al., in which glutathione peroxidase activity was coupled to the oxidation of 0.12 mmol/L NADPH at 340 nm by glutathione reductase (Paglia and Valentine 1967). Glutathione reductase was measured by the oxidation of NADPH at 340 nm according to the method of Tayrani et al. (Tayarani et al. 1987). Copper–zinc superoxide dismutase was assayed by the method of Flohe´ and Otting (1984). This method is Published by NRC Research Press


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based on the reduction of cytochrome c by superoxide, which is produced by the xanthine oxidase system. One unit of copper–zinc superoxide dismutase is defined as the amount of enzyme that inhibits the rate of cytochrome c reduction by 50%. Enzyme activity was expressed in nanomoles of substrate transformed per minute per milligram of protein, except for superoxide dismutase, which was expressed in units per milligram of protein.

Table 1. Physical characteristics of heart in control and normoxia-recovery rats.

Data analysis Results were expressed as means ± SE. Data were plotted and analyzed using SigmaPlot 8.0 and Systat 10.0 (SPSS Inc., Chicago, Ill.). Comparison of 2 means was performed using paired or unpaired Student’s t tests, as appropriate. All p values were two-tailed, and p < 0.05 was required to reject the null hypothesis.

Note: Values are means ± SE (n = 10 in each group). BW, body weight; RVW, right ventricular free wall weight; LVSW, left ventricular free wall plus septum weight. *, p < 0.05 versus control animals.

Results Physical characteristics At the end of the experiment, cardiac weight was significantly increased in normoxia-recovery animals (1.69 ± 0.04 and 1.25 ± 0.03 g, in normoxia-recovery and control animals respectively, p < 0.05), whereas body weight of normoxiarecovery rats significantly decreased. A moderate but significant elevation of the RV weight / LV plus septum ratio was observed after the recovery period (Table 1). Oxidative capacity In control rats, no difference was found in any respiratory parameters between RV and LV with either glutamate or palmitoyl carnitine as substrates. In normoxia-recovery rats, mitochondrial oxygen consumption and ATP synthesis significantly decreased in the RV with both substrates (Tables 2 2 and 3). In the same way, oxidative capacity of the LV was decreased with glutamate as substrate, but not with palmytoil carnitine (Table 3). In normoxia-recovery rats, enzyme activities of the respiratory chain complexes were changed in both ventricles. Activities of complexes III and IV, as well as citrate synthase, were significantly decreased in RV and LV in normoxia-recovery rats. In contrast, absolute activity of complex I significantly increased in both ventricles, whereas that of complex II did not change (Table 4). Ratios of the enzyme activities of the 4 respiratory chain complexes to citrate synthase activity are reported in Fig. 1 and showed a relative increase in complexes I and II in both ventricles of normoxia-recovery rats. Morphometric analysis In both ventricles of normoxia-recovery rat hearts, mitochondrial Nv sharply decreased and V of mitochondria significantly increased without a change in mitochondrial Vv, that is, the relative mitochondrial fraction of sarcoplasm (Table 5). In the same way, there was a slight expression of qualitative ultrastructural modifications that consisted of altered z-line and partially cellular œdema (Fig. 2). Antioxidant enzymes In the RV and LV, activity of catalase in the normoxiarecovery group (21.4 ± 0.8 and 18.9 ± 0.5 nmol H2O2 transformedmin–1(mg protein)–1 in RV and LV, respectively)

BW (g) RVW/BW (mg/g) LVSW/BW (mg/g) RVW/LVSW

Control group 498±6 0.41±0.01 1.66±0.03 0.25±0.01

Normoxia-recovery group 358±8* 0.78±0.03* 2.01±0.11* 0.39±0.02*

Table 2. Mitochondrial oxidative phosphorylation with glutamate as substrate. Oxygen consumption (nmolmin–1(mg wet weight)–1)

– ADP Control group RV 3.0±0.2 LV 3.5±0.2

+ ADP

ATP synthesis (nmolmin–1(mg wet weight)–1)

ATP/O

13.3±1.1 14.4±1.0

27.9±2.5 30.0±2.5

2.1±0.1 2.1±0.1

14.5±1.8* 20.8±2.2*,{

1.9±0.1 2.3±0.2

Normoxia-recovery group RV 2.3±0.2* 7.5±0.9* LV 2.6±0.4* 9.1±1.1*

Note: Values are means ± SE (n = 10 in each group). RV, right ventricular free wall; LV, left ventricular free wall. Experimental conditions are described in the Materials and methods. Basal, without ADP (–ADP), and ADP-stimulated (+ ADP) oxygen consumption rates supported by glutamate were measured in the presence of malate. ATP to oxygen ratio (ATP/ O) is calculated as the ratio of the rate of ATP synthesis to the rate of the concomitant respiration in the presence of ADP. *, p < 0.05 versus control animals; {, p < 0.05 versus right ventricle under the same conditions.

Table 3. Mitochondrial oxidative phosphorylation with palmitoyl carnitine as substrate. Oxygen consumption (nmolmin–1(mg wet weight)–1)

– ADP Control group RV 3.3±0.3 LV 3.3±0.2

+ ADP

ATP synthesis (nmolmin–1(mg wet weight)–1)

ATP/O

7.6±0.2 6.8±0.3

16.4±1.1 14.3±1.6

2.1±0.1 2.1±0.2

9.3±1.1* 16.2±1.4{

1.6±0.2 2.0±0.2

Normoxia-recovery group RV 2.0±0.2* 5.7±0.4* 8.4±0.8{ LV 3.1±0.3{

Note: Values are means ± SE (n = 10 in each group). RV, right ventricular free wall; LV, left ventricular free wall. Experimental conditions are described in the Materials and methods. Basal, without ADP (–ADP), and ADP-stimulated (+ ADP) oxygen consumption rates supported by palmitoyl carnitine were measured in the presence of malate. ATP to oxygen ratio (ATP/O) is calculated as the ratio of the rate of ATP synthesis to the rate of the concomitant respiration in the presence of ADP. *, p < 0.05 versus control animals; {, p < 0.05 versus right ventricle under the same conditions.


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Can. J. Physiol. Pharmacol. Vol. 89, 2011 Table 4. Enzymatic activities of the respiratory chain complexes and citrate synthase. Enzymatic activity (nmol substratemin–1(mg protein)–1) Complex I Control group RV 272±30 LV 241±21

Complex II

Complex III

Complex IV

Citrate synthase

284±37 259±36

965±55 905±65

2160±218 2087±194

996±64 964±63

597±44* 479±28*,{

1483±75* 1310±59*,{

664±24* 675±33*

Normoxia-recovery group RV 372±33* 312±47 LV 360±27* 242±16

Note: Values are means ± SE (n = 10 in each group). RV, right ventricular free wall; LV, left ventricular free wall. Experimental conditions are described in the Materials and methods. *, p < 0.05 versus control animals; {, p < 0.05 versus right ventricle under the same conditions.

Fig. 1. Ratio of the enzyme activities of the respiratory chain complexes to citrate synthase activity. Experimental conditions are described in the Materials and methods. Enzyme activities are reported in Table 4. Values are means ± SE (n = 10 in each group).

was significantly higher than in controls (15.8 ± 0.9 and 15.9 ± 0.4 nmolmin–1(mg protein)–1 in RV and LV, respectively) (Fig. 3). The glutathione peroxidase was also increased in the ventricles of normoxia-recovery rats (928 ± 34 and 964 ± 20 nmol NADPH oxidizedmin–1(mg protein)–1 in RV and LV, respectively) compared with that of the normoxic group (825 ± 33 and 832 ± 49 nmolmin–1(mg protein)–1 in RV and LV, respectively). Following 21 days of normoxia recovery, superoxide dismutase activity in-

creased significantly in RV. Finally, glutathione reductase activity did not differ significantly between normoxiarecovery and normoxic groups.

Discussion The purpose of the present investigation was to examine the reversibility in mitochondrial energy metabolism alterations induced by 3 weeks of CH. This duration of CH was Published by NRC Research Press


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Table 5. Morphometric analysis of heart mitochondria. Control group

Normoxia-recovery group

Right ventricle V (mm3) Nv ( 10–2/mm3) Vv (%)

0.93±0.03 40.34±2.53 32.29±2.09

2.43±0.31* 14.81±1.39* 33.28±1.03

Left ventricle V (mm3) Nv ( 10–2/mm3) Vv (%)

0.99±0.02 37.30±1.84 35.30±1.24

2.50±0.30* 15.74±2.39* 35.59±2.44

Note: Values are means ± SE (n = 5 in each group). Experimental conditions are described in the Materials and methods. V, mean volume of mitochondrion; Nv, numerical density; Vv, volume density.*, p < 0.05 versus control animals.

chosen on the basis of our previous studies to have a full and stable development of RV hypertrophy and a well-characterized modification in mitochondrial metabolism (Bonnet et al. 2001; Nouette-Gaulain et al. 2005). In agreement with previous observations, pulmonary hypertension due to CH was fully reversed after normoxia recovery (Novotna´ et al. 2001; Bonnet et al. 2002), whereas oxidative capacities in both ventricles remained decreased. This decrease in oxidative phosphorylation might be a consequence of either alterations in mitochondrial structure and function or permanent oxidative stress. Our results in normoxia-recovery rats thus indicate that cardiac metabolism remains altered and suggest that further investigations targeting oxidative stress should be evaluated to prevent CH-induced alteration in mitochondrial energy metabolism. Oxidative capacity Oxidation of glutamate and most enzyme activities of the respiratory chain decreased in both ventricles following 21 days of chronic hypoxia (Nouette-Gaulain et al. 2005). On glutamate, oxygen consumption and ATP synthesis remained decreased in both ventricles following 3 weeks of normoxia recovery. The changes in oxidative capacity were characterized by a similar decline in ATP synthesis so that the ATP/O ratio remained normal. After 3 weeks of normoxia recovery, complex I and II activities were restored and even increased, but could not compensate for the decrease in the activities of complexes III and IV, which thus appear to be the main controlling steps under these conditions. In contrast, a slight decrease in oxygen consumption and ATP synthesis with palmitoyl carnitine as a substrate was observed in RV, but not in LV. This difference with glutamate can be explained by a slower rate of oxygen consumption on palmitoyl carnitine (one half the rate with glutamate). At this lower flux, the decrease in activities of complexes III and IV can be compensated for, these steps being no longer limiting. In addition, the increase in complex I and II activities could facilitate the return to normality with palmitoyl carnitine. While no studies report oxidative capacity in the heart during a long period of CH followed by normoxia recovery, many studies have shown the effects of reoxygenation or reperfusion during shorter periods. Oxygen consumption and ATP synthesis decreased in heart mitochondria submitted to anoxia–reoxygenation or

during ischemia–reperfusion injury in perfused hearts (Iwai et al. 2002; Ozcan et al. 2002). Moreover, similar results concerning enzyme activities have been found when hypoxia or anoxia was followed by a normoxic period in rat heart mitochondria: (i) a decrease in complex III activity has been found in both conditions (Petrosillo et al. 2003) and (ii) a decreased activity of complex I is restored by normoxic perfusion (Maklashina et al. 2002). These metabolic alterations are associated with a postischemic contractile dysfunction. Previous hemodynamic investigations in our normoxic-recovery rats have shown longer ejection and preejection time of RV (Bonnet et al. 2004). Collectively, these results indicate that a decrease in oxidative capacities, considered an adaptive mechanism to CH, were persistent in spite of normoxia recovery. Thus, we suggest that the posthypoxic dysfunction could partly result from an inability of the RV to synthesize enough adenine high-energy phosphates to sustain contractile function. Morphometric analysis Activity of citrate synthase, an enzyme of the mitochondrial matrix often used as an index of the mitochondrial mass, was significantly decreased in normoxia recovery compared with the control group. In the same way, mitochondrial numerical density was significantly decreased, but mitochondria mean volume was clearly increased in the normoxia-recovery group. While a decrease in citrate synthase activity was linked to small mitochondria under hypoxic conditions, the structural changes observed in normoxia recovery suggest the presence of swollen mitochondria, likely along with the formation of megamitochondria (Wakabayashi and Karbowski 2001). Indeed, the presence of swollen mitochondria is frequently associated with mitochondrial myopathy (skeletal muscle, liver) or with hypoxia or ischemia injury in the heart (Wakabayashi 2002; Halestrap et al. 2007). In cardiomyopathic hamsters submitted to acute hypoxia, similar mitochondrial morphometrical disorders have been described (Fitzl et al. 1998). In the same way, Oczan et al. (2002) showed that mitochondria were damaged or swollen with a decrease in oxidative capacity after anoxia– reoxygenation. Both metabolic mitochondrial alteration and morphometric changes have also been reported in the case of neuromuscular disorders (Gimeno et al. 1973; Worsfold et al. 1973). Not only oxidative phosphorylation, but also ROS production may be associated with swollen mitochondria formation as described in the heart or liver (Ishikawa et al. 2005). For instance, in rats chronically treated with ethanol, the overproduction of free radicals decreases ATP synthesis by swollen mitochondria in hepatocytes (Adachi et al. 1995). After removal of free radicals from the cell, both structure and function of mitochondria are normalized. However, if cells are further exposed to an excess of free radicals, mitochondria again swell, leading to further mitochondrial metabolism alterations. Antioxidant system Mitochondria are the main site of ROS production in the cell (Papa and Skulachev 1997; Cadenas and Davies 2000). Whereas under physiological conditions, mitochondrial respiration accounts for about 90% of cellular uptake, 1%–2% of the oxygen consumed is converted to ROS. Complexes I Published by NRC Research Press

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Fig. 2. Electron micrographs of mitochondria in rat heart (17 250): mitochondria in control left ventricle (A) and in left ventricle from normoxia-recovery rat (B).

Fig. 3. Antioxidant enzyme activities in control and normoxia-recovery rat hearts. Values are means ± SE (n = 10 in each group). Experimental conditions are described in the Materials and methods. Enzyme activity was expressed in nanomoles substrate per minute per milligram of protein, except for copper–zinc superoxide dismutase (Cu,Zn-SOD), which was expressed in units per milligram of protein.*, p < 0.05 versus control animals; {, p < 0.05 versus right ventricle under the same conditions.

and III of the electron transport chain are the major sites for ROS production (Chen et al. 2003). In this study, antioxidant enzymes were investigated to highlight a deregulation in oxidative stress metabolism. Glutathione peroxidase and catalase were significantly higher in ventricles of normoxia-

recovery rats than in those of the control group. Catalase is one of the most efficient enzymes known (Lledıás et al. 1998). The transcript levels and activities of antioxidant enzymes (glutathione peroxidase, catalase) are up-regulated in response to oxidative stress, as in the case of ischemia–rePublished by NRC Research Press


perfusion (Franco et al. 1999; Sharma et al. 2006). Overexpression of catalase in the cytosolic compartment contributes to protecting cells against oxidative injury (Bai et al. 1999). However, when the rate of ROS formation is excessive, it can overcome antioxidant capacities, creating oxidative stress. The persistence of swollen mitochondria suggests that these protective mechanisms might be insufficient under the present experimental conditions. In this case, swollen mitochondria could be regarded as an adaptive process to an unfavorable environment. Further studies are required to investigate the underlying mechanisms and the benefits of scavengers for free radical administration, such as the evaluation of a-tocopherol. Clinically, such situations of chronic hypoxia followed by normoxia (even hyperoxia) could be encountered in chronic obstructive pulmonary diseases or during the course of acute lung injury. Our data should be considered in clinical studies on the long-term outcome of these conditions (Herridge et al. 2003). In conclusion, unlike pulmonary hypertension or hypertrophy, alterations in cardiac mitochondrial energy metabolism induced by chronic hypoxia are not totally restored after normoxia recovery. The mechanisms involved in these persistent alterations require further investigation, although a deregulation in oxidative stress may play a role. In this way, oxidative stress target drugs should be evaluated to prevent CH-induced alteration in mitochondrial energy metabolism.

Acknowledgements The authors gratefully thank Ray Cooke for revising the English. This work was supported by a grant from Conseil Re´gional d’Aquitaine and Agence Nationale de la Recherche (ANR), France.

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