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Am J Physiol Heart Circ Physiol 292: H224-H230, 2007. First published August 25, 2006; doi:10.1152/ajpheart.00689.2006
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Role of oxidative stress in PKC-{delta} upregulation and cardioprotection induced by chronic intermittent hypoxia

Frantisek Kolár,1,4 Jana Jezková,2,4 Patricie Balková,2 Jirí Breh,2 Jan Neckár,1,4 Frantisek Novák,2 Olga Nováková,2,4 Helena Tomásová,3,4 Martina Srbová,3,4 Bohuslav Ost’ádal,1,4 Jirí Wilhelm,3,4 and Jan Herget3,4

1Institute of Physiology, Academy of Sciences of the Czech Republic; 2Departments of Animal Physiology and Biochemistry, Faculty of Science; 3Second Faculty of Medicine, Charles University; and 4Centre for Cardiovascular Research, Prague, Czech Republic

Submitted 29 June 2006 ; accepted in final form 16 August 2006


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The aim was to determine whether increased oxidative stress during the adaptation to chronic intermittent hypoxia (CIH) plays a role in the induction of improved cardiac ischemic tolerance. Adult male Wistar rats were exposed to CIH in a hypobaric chamber (7,000 m, 8 h/day, 5 days/wk, 24–30 exposures). Half of the animals received antioxidant N-acetylcysteine (NAC; 100 mg/kg) daily before the exposure; the remaining rats received saline. Control rats were kept under normoxia and treated in a corresponding manner. One day after the last exposure (and/or NAC injection), anesthetized animals were subject to 20 min of coronary artery occlusion and 3 h of reperfusion for determination of infarct size. In parallel subgroups, biochemical analyses of the left ventricular myocardium were performed. Adaptation to CIH reduced infarct size from 56.7 ± 4.5% of the area at risk in the normoxic controls to 27.7 ± 4.9%. NAC treatment decreased the infarct size in the controls to 42.0 ± 3.4%, but it abolished the protection provided by CIH (to 41.1 ± 4.9%). CIH decreased the reduced-to-oxidized glutathione ratio and increased the relative amount of PKC isoform-{delta} in the particulate fraction; NAC prevented these effects. The expression of PKC-{epsilon} was decreased by CIH and not affected by NAC. Activities of superoxide dismutase, catalase, and glutathione peroxidase were affected by neither CIH nor NAC treatment. It is concluded that oxidative stress associated with CIH plays a role in the development of increased cardiac ischemic tolerance. The infarct size-limiting mechanism of CIH seems to involve the PKC-{delta}-dependent pathway but apparently not the increased capacity of major antioxidant enzymes.

ischemia-reperfusion; oxygen radicals; infarct size; protein kinase C

ADAPTATION OF RATS to chronic intermittent hypoxia (CIH) increases cardiac tolerance to acute ischemia-reperfusion injury, as evidenced by reduced myocardial infarction, improved recovery of contractile function, and limitation of ventricular arrhythmias (24, 25). Although the mechanism of this long-lasting protective phenomenon is not precisely understood, it has been shown that a key role is played by mitochondrial ATP-sensitive potassium channels (mitoKATP) (3, 31, 48) and PKC (13, 28). Emerging evidence based on studies of preconditioning suggests that the link between PKC activation and mitoKATP opening is reactive oxygen species (ROS), formed during the trigger phase in various forms of protection (6, 26, 35, 47), although their source and the sequence of signaling events are a matter of debate. CIH is also associated with oxidative stress (7, 8), and increased ROS generation may be implicated in the induction of its cardioprotective mechanism. We have shown previously that in rats exposed simultaneously to hypoxia and hypercapnia, which is known to reduce oxidative stress (27), the infarct size-limiting effect of hypoxic adaptation was blunted (30). Increased ROS production also appears to be involved in the adverse effects of chronic hypoxia, such as the structural remodeling of pulmonary vessels and resulting pulmonary hypertension, because hypercapnia or antioxidants attenuated these pathological manifestations (22, 25, 32).

It is well known that ROS-induced acute preconditioning is mediated by activation of PKC (4, 43). Therefore, the present study was designed to verify the hypothesis that oxidative stress acting during the long-term adaptation to hypoxia contributes to the development of increased cardiac ischemic tolerance in a PKC-dependent manner. Effects of chronic treatment with the antioxidant N-acetylcysteine (NAC) on myocardial abundance and subcellular distribution of PKC isoforms-{delta} and -{epsilon} and the size of myocardial infarction induced by acute coronary artery occlusion were compared in rats adapted to CIH and in normoxic controls. Moreover, activities of major antioxidant enzymes in the myocardium were determined.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Adult male Wistar rats (250–280 g body wt) were exposed to intermittent hypobaric hypoxia corresponding to the altitude of 7,000 m for 8 h/day, 5 days/wk (Fig. 1). Barometric pressure (Pb) was lowered stepwise, so that the level equivalent to an altitude of 7,000 m (Pb = 308 mmHg, 41 kPa; and PO2 = 65 mmHg, 8.6 kPa) was reached after 13 exposures. The total number of exposures was 24–30 to allow for successive processing of animals in physiological experiments; no appreciable changes of hypoxia-induced responses occurred within this interval. A subgroup of the animals received NAC by subcutaneous injections in a dose of 100 mg/kg daily before the hypoxic exposure; the remaining rats received the same volume (2 ml/kg) of saline. The control group of animals was kept for the same period of time at Pb and PO2 equivalent to an altitude of 200 m (Pb = 742 mmHg, 99 kPa; and PO2 = 155 mmHg, 20.7 kPa); a subgroup was treated with NAC or saline in a corresponding manner. All animals had free access to water and a standard laboratory diet. The study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). Experimental protocols were approved by the Animal Care and Use Committee of the Institute of Physiology, Academy of Sciences of the Czech Republic.


Figure 1
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  		Fig. 1. Model of chronic intermittent hypoxia and the experimental protocol. Hypoxic animals were subject to hypobaric hypoxia starting at PO2 = 119 mmHg (equivalent to an altitude of 2,400 m) and decreasing stepwise up to PO2 = 65 mmHg (equivalent to an altitude of 7,000 m) during the first 13 exposures; this level of hypoxia was maintained for additional 11–17 exposures. Filled squares indicate daily exposures lasting 8 h. For the remaining period of each day and for 2 days after each 5-day series of hypoxic exposures, the animals were kept at normoxia (PO2 = 155 mmHg, equivalent to an altitude of 200 m). Vertical lines at the bottom of the graph indicate N-acetylcysteine (NAC) or saline injections given before each hypoxic exposure. Normoxic animals were kept at PO2 = 155 mmHg during the whole experiment (indicated by a continuous line) and treated with NAC or saline in a corresponding manner. All animals were employed on the next day following the last hypoxic exposure and NAC or saline injection.

 
All animals were employed on the next day following the last hypoxic exposure and/or NAC injection. Hematocrit was measured in the tail blood. The animals assigned to biochemical analyses were euthanized by decapitation, and their hearts were rapidly excised, washed in cold (0°C) saline, and dissected into the right and left (LV) free ventricular walls and the septum. All parts were weighed, and the LVs were frozen in liquid nitrogen and stored at –80°C until use. All of the chemicals were purchased from Sigma, unless otherwise indicated.

Infarct size determination. Animals were subjected to myocardial ischemia-reperfusion as described previously (29). Anesthetized (pentobarbital sodium, 60 mg/kg ip, Sanofi) rats were ventilated (Columbus Instruments) with room air at 68 strokes/min (tidal volume, 1.2 ml/100 g body wt). Blood pressure in the left carotid artery was measured (Gould P23Gb) and subsequently analyzed by our custom-designed software. The rectal temperature was maintained between 36.5° and 37.5°C by a heated table throughout the experiment.

A left thoracotomy was performed, and a polyester suture 6-0 (Ethibond-Ethicon) was placed around the left anterior descending coronary artery about ~1–2 mm distal to its origin. After a 10-min stabilization, regional myocardial ischemia was induced by the tightening of the suture threaded through a polyethylene tube. After a 20-min occlusion period, the ligature was released and reperfusion of previously ischemic tissue continued for 3 h. The hearts were then excised and washed with saline through the aorta. The infarct area and the area at risk (AR) were delineated by perfusion with 2,3,5-triphenyltetrazolium chloride and potassium permanganate (after coronary artery occlusion), respectively. The hearts were cut into slices 1 mm thick and fixed in formaldehyde solution. The size of the infarct area (IA), the size of the AR, and the size of the LV were determined by computerized planimetry. The IA was normalized to the AR (IA/AR), and the AR was normalized to the LV (AR/LV).

Tissue fractionation and Western blot analysis of PKC isoforms. Frozen LV myocardium was pulverized to a fine powder at the temperature of liquid nitrogen, followed by Potter-Elvehjem homogenization in eight volumes of ice-cold buffer composed of (in mmol/l) 12.5 Tris·HCl (pH 7.4), 250 sucrose, 2.5 EGTA, 1 EDTA, 100 NaF, 5 DTT, 0.3 phenylmethylsulfonyl fluoride, 0.2 leupeptin, and 0.02 aprotinin. The homogenate was centrifuged at 100,000 g for 90 min. The resulting pellet represented the particulate fraction; the supernatant was the cytosolic fraction. The homogenate and pellet of the particulate fraction were resuspended in homogenization buffer containing 1% Triton X-100, held on ice for 90 min, and then centrifuged at 100,000 g for a further 90 min. The resulting detergent-treated supernatants were used for immunoblot analyses. Triton X-100 was added to the cytosolic fraction to reach the final concentration of 1%. Protein content was determined according to Lowry's assay modified by Peterson (37).

Detergent-treated extracts of subcellular fractions were subjected to SDS-PAGE electrophoresis on 8% bis-acrylamide polyacrylamide gel at 20 mA/gel for 90 min on a Mini-Protean II apparatus (Bio-Rad). After electrophoresis, the resolved proteins were transferred to a nitrocellulose membrane (Amersham). Equal protein transfer efficiency was verified by staining with Ponceau S. After being blocked with 5% dry low-fat milk in Tris-buffered saline with Tween 20, the membranes were immunoblotted using the enhanced chemiluminescence detection system (Amersham) as previously described (28). Samples from all experimental groups compared were run on the same gel and quantified on the same membrane. To ensure the specificity of PKC-{delta} and PKC-{epsilon} immunoreactive proteins, prestained molecular mass protein standards (Fluka), recombinant human PKC-{delta} and PKC-{epsilon} standards (Sigma), rat brain extract, and the respective blocking immunizing peptides (Sigma) were used.

Measurement of antioxidant enzyme activities. Myocardium was pulverized and homogenized as described in Tissue fractionation and Western blot analysis of PKC isoforms. The homogenate was clarified by centrifugation at 5,000 g for 10 min. Catalase activity was measured by the method of Aebi (1). The rate of hydrogen peroxide decomposition was monitored spectrophotometrically at 240 nm in 50 mM phosphate buffer (pH 7.0) containing 10 mM hydrogen peroxide at 28°C.

Glutathione peroxidase (GPX) activity was determined by the indirect procedure described by Paglia and Valentine (34). GSSG was produced by GPX reaction and immediately reduced by NADPH in the presence of glutathione reductase. The rate of NADPH consumption was recorded at 340 nm as a measure of GSSG formation. The reaction was conducted in 1 M Tris·HCl buffer containing 5 mM Na2EDTA, 2 mM NADPH, 20 mM GSH, and 10 U/ml glutathione reductase and started by the addition of t-butyl hydroperoxide. Consumption of NADPH was calculated by using millimolar extinction coefficient for NADPH of 6.22.

Total SOD activity was determined by the modified nitroblue tetrazolium method (14). Xanthine-xanthine oxidase reaction was utilized to generate a superoxide flux. Nitroblue tetrazolium reduction by superoxide anion to blue formazan was measured spectrophotometrically at 540 nm (28°C). Chloroform-ethanol extracts of homogenates were then used to determine SOD activity. The assay contained the following reagents: 0.1 mM phosphate buffer (pH 7.8), 4 g/l bovine serum albumin, 2 mg/ml nitroblue tetrazolium, and 1 mM xanthine. Manganese SOD (Mn SOD) activity was quantified in the presence of 5 mM NaCN, the selective inhibitor of copper-zinc SOD (41).

Measurement of glutathione concentration. Myocardium was homogenized in 1% picric acid using a glass Teflon device, and the homogenate was centrifuged at 10,000 g for 10 min. Concentration of total glutathione in supernatant was determined spectrophotometrically at 412 nM using glutathione reductase-coupled enzymic assay at 30°C (16). The assay contained the following reagents: 0.2 mmol/l NADPH, 100 mmol/l phosphate, 5 mmol/l EDTA, 0.6 mmol/l 5,5-dithio-bis(2-nitrobenzoic acid), and 1 U/ml glutathione reductase. GSSG (glutathione disulfide) was measured by masking the reduced GSH with 2-vinylpyridine. The ratio of GSH to GSSG was taken as a measure of tissue oxidative stress.

Measurement of lipofuscin-like pigments. Additional experiments were performed to verify that CIH is associated with increased oxidative stress and that NAC treatment prevents this effect. A separate group of rats was exposed to hypobaric hypoxia corresponding to the altitude of 5,500 m (Pb = 379 mmHg, 50.5 kPa; and PO2 = 79 mmHg, 10.5 kPa) for 8 h/day during 3 consecutive days. Hypoxic and normoxic animals were treated with NAC or saline as described in Animals and employed the next day following the third hypoxic exposure. The frozen LV myocardium was lyofilized, pulverized, and extracted for 1 h in a chloroform-methanol mixture (1:2, vol/vol). The samples were centrifuged at 2,000 g for 10 min, and the bottom chloroform layer was rinsed twice with water and used for the measurement of the concentration of lipofuscin-like pigments (LFP).

LFP are fluorescent end products of lipid peroxidation (9) that have been widely used as an indicator of tissue oxidative stress. Fluorescence emission and excitation spectra of chloroform extracts were measured on the Perkin-Elmer LS-5 fluorometer as previously described (46). The excitation spectra were measured in the range of 250–500 nm for emission adjusted between 300 and 500 nm with a step of 10 nm. The fluorometer was calibrated with the standard No. 2 of the instrument manufacturer, and the LFP concentration was expressed in relative fluorescence units per gram of dry tissue weight.

Data analysis. The results are expressed as means ± SE. Differences in the infarct size between the groups were compared by the Mann-Whitney U-test. One-way ANOVA and subsequent Student-Newman-Keuls test were used for comparison of differences in parametric variables between the groups. Differences were assumed as statistically significant when P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Weight parameters and hematocrit. In line with our previous studies, adaptation of rats to CIH led to a marked increase in hematocrit values and a significant retardation of body growth compared with age-matched normoxic animals. Treatment with NAC during the adaptation period slightly decreased the body weight in both normoxic and chronically hypoxic groups and did not affect the level of the hematocrit. The heart weight of chronically hypoxic rats increased due to hypertrophy of both ventricles. The right ventricular weight normalized to body weight increased to 177% and that of the LV to 128% of the respective normoxic values. NAC treatment had no effect on heart weight parameters (Table 1).


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  		Table 1. Body weight and heart weight parameters, hematocrit, and the relative size of the AR of infarction

 
Myocardial infarct size. The normalized AR (AR/LV) did not significantly differ between the groups (Table 1). CIH decreased the infarct size from 56.7 ± 4.5% of the AR in the normoxic control group to 27.7 ± 4.9%. NAC treatment decreased IA/AR in the normoxic animals to 42.0 ± 3.4%, but it abolished protection induced by CIH (to 41.1 ± 4.9%). The IA/AR did not differ between the NAC-treated groups (Fig. 2).


Figure 2
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  		Fig. 2. Myocardial infarct size expressed as a percentage of the area at risk (AR) [infarct area (IA)/AR] in control (Cont) and NAC-treated rats adapted to chronic hypoxia and in normoxic animals. Values are means ± SE from 7–10 hearts in each group. *P < 0.05 vs. corresponding normoxic group; {dagger}P < 0.05 vs. corresponding untreated group.

 
CIH increased mean arterial blood pressure (MAP) compared with normoxic groups, and the higher level of MAP persisted in the course of ischemia-reperfusion. In normoxic controls, MAP significantly decreased at the end of reperfusion compared with baseline values. This decrease was prevented by both NAC treatment and adaptation to hypoxia. Heart rate did not differ between the groups, and it significantly decreased at the end of reperfusion in all groups (Table 2).


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  		Table 2. Heart rate and mean arterial blood pressure

 
Myocardial glutathione, LFP, and antioxidant enzyme activities. Table 3 summarizes myocardial activities of total SOD, Mn SOD, catalase, and GPX. Neither CIH nor NAC treatment induced an appreciable effect on these enzymes.


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  		Table 3. Activities of myocardial antioxidant enzymes, the concentration of total glutathione, ratio of GSH to GSSG, and the concentration of LFP

 
CIH did not change the myocardial concentration of total glutathione, but it significantly increased the proportion of GSSG and decreased the GSH-to-GSSG ratio. NAC treatment prevented the effect of CIH on the GSH-to-GSSG ratio, and it increased the concentration of total glutathione without affecting the ratio in normoxic hearts (Table 3).

Myocardial concentration of LFP significantly increased already after three daily exposures to hypoxia of 5,500 m, and NAC treatment prevented this effect (Table 3).

Expression and distribution of PKC isoforms. Immunoreactivities of PKC-{delta} and PKC-{epsilon} were detected on Western blots as single bands that were confirmed by the respective blocking peptides, recombinant human PKC standards, and a positive control from rat brain homogenate (Fig. 3).


Figure 3
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  		Fig. 3. Western blot analysis of PKC isoforms in myocardial homogenate (Hom) and in cytosolic (Cyt) and particulate (Part) fractions. A and B: representative Western blots of PKC-{delta} and PKC-{epsilon}, respectively, in fractions from the control normoxic heart in the absence (left) and presence (right) of the respective blocking peptides. C and D: representative Western blots comparing the expression of PKC-{delta} and PKC-{epsilon}, respectively, in fractions from normoxic (N), normoxic NAC-treated (NN), chronically hypoxic (H), and chronically hypoxic NAC-treated (HN) rats. Total amounts of protein loaded were as follows: PKC-{delta}: cytosol, 16 µg; particles, 5 µg; homogenate, 10 µg; and PKC-{epsilon}: cytosol, 18 µg; particles, 8 µg; homogenate, 14 µg. For details, see MATERIALS AND METHODS. Numbers on the right indicate the positions of prestained molecular mass standards (in kDa). R, recombinant human PKC-{delta} or PKC-{epsilon} standards; B, extract from rat brain homogenate.

 
CIH significantly increased the relative protein content of PKC-{delta} in the homogenate from the myocardium (Fig. 4A). This effect was predominantly due to increased abundance of the isoform in the particulate fraction (Fig. 4C). NAC treatment did not affect PKC-{delta} expression and distribution in normoxic hearts, but it prevented its upregulation by CIH in both homogenate and the particulate fraction.


Figure 4
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  		Fig. 4. The expression of PKC-{delta} (A and C) and PKC-{epsilon} (B and D) in homogenate (A and B) and their distributions between cytosolic and particulate fractions (C and D) from the myocardium of control and NAC-treated rats adapted to chronic hypoxia and of normoxic animals. Values are expressed as arbitrary units: the sum of densitometric volumes (related to 1 µg of protein) measured in all groups on the same blot equals 100. Values are means ± SE from 5 (PKC-{delta}) or 6 (PKC-{epsilon}) hearts in each group. *P < 0.05 vs. corresponding normoxic group; {dagger}P < 0.05 vs. corresponding untreated group.

 
CIH tended to decrease the relative protein content of PKC-{epsilon} in the homogenate, but the difference reached statistical significance only in a comparison of NAC-treated normoxic with hypoxic groups (Fig. 4B). The abundance of the isoform in the particulate fraction of chronically hypoxic hearts was also significantly lower than in normoxic hearts (Fig. 4D). NAC treatment had no appreciable effect on PKC-{epsilon} expression.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
In this study, we demonstrated for the first time that the antioxidant NAC completely prevented the development of cardioprotection in chronically hypoxic rats. Beneficial reduction of myocardial infarct size induced by CIH did not appear in animals treated with NAC every day before the hypoxic exposure during the whole 5-wk adaptation period. The decrease in myocardial GSH-to-GSSG ratio, which reflects tissue oxidative stress in chronically hypoxic animals, was eliminated by NAC treatment. Myocardial concentration of LFP, another marker of oxidative stress, was already elevated after three daily hypoxic exposures, and this effect was absent in NAC-treated hypoxic animals. It is in agreement with our previous observation that the infarct size-limiting effect of chronic hypoxia is attenuated by increased concentration of CO2 in the air during the period of adaptation to hypoxia (30); the increased CO2 level is considered to act by a decrease of oxidative stress as well (27). Besides protection by CIH, a growing body of evidence indicates that ROS are also involved in intracellular signaling cascades of various forms of early and delayed preconditioning (e.g., 2, 6, 10–12, 20, 45). It appears, therefore, that the generation of the ROS signal before ischemia-reperfusion insult plays an important role in the induction of both short- and long-term protective programs.

In the past, ROS were considered solely injurious, but now it is generally accepted that they may exert both deleterious and beneficial actions (5). Our observation of opposite effects of NAC treatment on ischemic tolerance of normoxic and chronically hypoxic hearts, i.e., decreased and increased infarct size, respectively, is in line with this concept. Infarct size was about the same in the two NAC-treated groups, which means that hearts of treated hypoxic rats were still moderately protected compared with those of untreated normoxic rats. It suggests that the ischemic tolerance of NAC-treated chronically hypoxic hearts resulted from an interplay between the protective action of the antioxidant and the abrogation of the protective adaptive response induced by CIH. Generally, this dual effect might, at least partially, explain why clinical trials with antioxidants failed to confirm promising data obtained in a number of animal studies. It is obvious that beneficial consequences of antioxidant supplementation in normal healthy myocardium cannot be used to predict an outcome in adapted or diseased hearts.

Cardioprotective properties of NAC have been demonstrated in several experimental studies using various in vivo or in vitro models (15, 23, 40). NAC is a sulfhydryl-containing compound that exerts its complex antioxidant effect both through direct interaction with ROS and as a precursor of L-cysteine and glutathione. In this study, we assessed cardiac ischemic tolerance on the next day following the last administration of NAC. It seems unlikely that direct scavenging activity of NAC itself was responsible for its protective effect at this time since the elimination half-life of total plasma NAC is about 2 h in rats (18). NAC is rapidly converted to L-cysteine, which is also cardioprotective as a ROS scavenger (39) or can enter in the synthesis of glutathione, a central component of the cellular antioxidant defense system. The potential role of glutathione in the infarct size-limiting effect of NAC treatment in normoxic animals cannot be excluded since its total myocardial concentration was increased although the GSH-to-GSSG ratio remained unchanged in our study. In addition, NAC increases nitric oxide availability by scavenging ROS and stimulating endothelial nitric oxide synthase activity and protein expression in the heart (36) that might contribute to the protective effect of a prolonged treatment.

Adaptation to CIH led to a marked upregulation of PKC-{delta} that was significant in both myocardial homogenate and particulate fraction. This observation confirms the results of our recent study that demonstrated that CIH-induced increase in the relative protein content of PKC-{delta} was most prominent in mitochondrial and nuclear fractions (28). We also showed that the PKC-{delta} isoform-selective inhibitor rottlerin, administered before the acute ischemia-reperfusion insult, attenuated the infarct size-limiting effect of CIH, suggesting that this isoform is involved in the cardioprotective mechanism. The novel finding of the present study is that the preventive treatment of chronically hypoxic rats with NAC eliminated the upregulation of PKC-{delta}. This observation suggests that the induction of this isoform during the adaptation period is dependent on oxidative stress. The absence of both protection and PKC-{delta} upregulation in NAC-treated hypoxic animals further supports our previous conclusion regarding the role of this isoform in increased ischemic tolerance of chronically hypoxic hearts (28).

PKC-{delta} may be both protective and detrimental as reported by a growing number of studies. It appears that protective effects of PKC-{delta} are manifested when the enzyme is activated well before ischemia-reperfusion insult (19). This condition was satisfied in our experiments. Consequences of PKC-{delta} activation also depend on its localization to various subcellular compartments that is controlled by phosphorylation at multiple sites (42). For example, phosphorylation of PKC-{delta} at serine-643 is associated with its translocation to mitochondria and activation of mitoKATP channels following a protective stimulus (44). Opening of mitoKATP channels is considered to play a crucial role in various forms of myocardial protection, including that afforded by CIH (3, 31, 48). The sequence of signaling events linking ROS, PKC-{delta}, and mitoKATP channels in the protective mechanism of CIH remains to be elucidated. Nevertheless, our data are compatible with the view that ROS generation precedes PKC-{delta} activation and mitoKATP opening as recently demonstrated in cardioprotection induced by the volatile anesthetic sevoflurane (6). Obviously, the infarct size-limiting pathways induced by CIH may also involve other redox-sensitive steps (24) that were not addressed in the present study.

Unlike PKC-{delta}, the abundance of PKC-{epsilon}, the key enzyme isoform involved in the mechanism of preconditioning, was rather decreased in the myocardium of chronically hypoxic rats, and NAC treatment did not exert any appreciable effect. More detailed analysis performed in our recent study did not reveal any significant change in PKC-{epsilon} abundance and subcellular distribution due to CIH (28). Taken together, these data suggest that this isoform does not seem to play a major role in the increased cardiac ischemic tolerance in our model of severe CIH. In contrast, cardioprotection afforded by permanent chronic hypoxia in neonatal rabbits appears to involve PKC-{epsilon} activation and translocation (38), suggesting that the role of PKC isoforms differs in species- and/or age-dependent manner. However, we cannot exclude that, apart from PKC-{delta}, other PKC isoform(s) contribute to protection in our experimental model. It should be noted that a moderately increased expression of PKC isoforms-{alpha}, -{delta}, and -{epsilon} was observed in the myocardial particulate fraction isolated from rats adapted to much less severe hypoxia (13).

It has been well documented that ROS can induce myocardial antioxidant enzymes. In particular, the expression and activity of Mn SOD, a key enzyme that converts superoxide to hydrogen peroxide in mitochondria, increase under various conditions associated with oxidative stress. It has been demonstrated to play a role in delayed preconditioning elicited by ischemia, heat stress, inflammatory cytokines, or exercise training (e.g., 17, 20, 21); a close correlation exists between the increase in Mn SOD activity and the reduction of infarct size under these conditions (21). Increased activities of Mn SOD and catalase were also observed in hearts of rats exposed to CIH just after birth for 60 days (49). However, the present study failed to detect any effect of long-term adaptation of adult rats to CIH and/or NAC treatment on total myocardial activities of Mn SOD and other major antioxidant enzymes. We cannot exclude that CIH had a stimulatory effect during the first exposures, which disappeared later on when the animals became fully adapted. Nevertheless, the increased ischemic tolerance of adult chronically hypoxic hearts seems unlikely to be mediated by the increased capacity of enzymic antioxidant defense.

In conclusion, oxidative stress, acting during adaptation of rats to CIH, plays an important role in the induction of endogenous cardioprotective mechanism, which involves the upregulation of PKC-{delta} but not PKC-{epsilon}. Moreover, our data point to a potentially adverse effect of antioxidant supplementation under conditions, which alone evoke ROS-dependent adaptive responses. This might be considered as one of the reasons why clinical data are rather weak and do not justify the use of antioxidants for the prevention and treatment of cardiovascular diseases.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Grant Agency of the Czech Republic Grant 305/04/0465, Grant Agency of the Charles University Grant 153/2005/B-Bio/PrF, and the institutional research project AVOZ 50110509.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Kolar, Inst. of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic (e-mail: kolar{at}biomed.cas.cz)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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