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Cardiovascular Research 2002 55(3):567-575; doi:10.1016/S0008-6363(02)00456-X
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Copyright © 2002, European Society of Cardiology

Effects of mitochondrial KATP modulators on cardioprotection induced by chronic high altitude hypoxia in rats

Jan Neckára, Ondrej Szárszoia, Lukás Kotena, Frantisek Papouseka, Bohuslav Ost'ádala, Gary J Groverb and Frantisek Kolára,*

aInstitute of Physiology, Academy of Sciences of the Czech Republic and Centre for Experimental Cardiovascular Research, Vídenská 1083, 142 20 Prague 4, Czech Republic
bMetabolic and Cardiovascular Diseases Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Pennington, NJ, USA

* Corresponding author. Tel.: +420-2-4106-2559; fax: +420-2-4106-2125 kolar{at}biomed.cas.cz

Received 19 November 2001; accepted 25 April 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Adaptation of rats to intermittent high altitude hypoxia increases the tolerance of their hearts to acute ischemia/reperfusion injury. Our aim was to examine the role of mitochondrial ATP-sensitive potassium channels (KATP) in this form of protection. Methods: Adult male Wistar rats were exposed to hypoxia of 5000 m in a barochamber for 8 h/day, 5 days a week; the total number of exposures was 24–32. A control group was kept under normoxic conditions (200 m). Infarct size (tetrazolium staining) was measured in anesthetized open-chest animals subjected to 20-min regional ischemia (coronary artery occlusion) and 4-h reperfusion. Isolated perfused hearts were used to assess the recovery of contractile function following 20-min global ischemia and 40-min reperfusion. In the open-chest study, a selective mitochondrial KATP blocker, 5-hydroxydecanoate (5 mg/kg), or openers, diazoxide (10 mg/kg) or BMS-191095 (10 mg/kg), were administered into the jugular vein 5 and 10 min before occlusion, respectively. In the isolated heart study, 5-hydroxydecanoate (250 µmol/l) or diazoxide (50 µmol/l) were added to the perfusion medium 5 or 10 min before ischemia, respectively. Results: In the control normoxic group, infarct size occupied 62.2±2.0% of the area at risk as compared with 52.7±2.5% in the chronically hypoxic group (P<0.05). Post-ischemic recovery of contractile function (dP/dt) reached 60.0±3.9% of the pre-ischemic value and it was improved to 72.4±1.2% by adaptation to hypoxia (P<0.05). While 5-hydroxydecanoate completely abolished these protective effects of chronic hypoxia, it had no appreciable influence in normoxic groups. In contrast, diazoxide significantly increased the recovery of contractile function and reduced infarct size in normoxic groups only. The later effect was also observed following treatment with BMS-191095. Conclusion: The results suggest that opening of mitochondrial KATP channels is involved in the cardioprotective mechanism conferred by long-term adaptation to intermittent high altitude hypoxia.

KEYWORDS Contractile function; Hypoxia/anoxia; Infarction; Ischemia; K-ATP channel; Mitochondria; Reperfusion


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
An increasing body of evidence, based mostly on pharmacological experiments, indicates that mitochondrial ATP-sensitive potassium channels (mitoKATP) play a central role in preconditioning-induced protection of the heart against ischemia/reperfusion injury. Their involvement was demonstrated in various models and forms of preconditioning including acute [1–3] and delayed forms [4–6], pacing-induced protection [7], preconditioning on distance [8] and pharmacologically-induced protection [9–11]. Nevertheless, the mechanism by which mitoKATP opening leads to improved ischemic tolerance of the heart remains obscure.

Besides short-term preconditioning, long-term adaptation to hypoxic conditions can also increase cardiac ischemic tolerance. This phenomenon was demonstrated both in human populations living at high altitude [12] and in several animal studies which examined various manifestations of ischemia/reperfusion injury, such as infarct size [13–16], post-ischemic contractile dysfunction [17–19] or ischemia/reperfusion arrhythmias [20–23]. Although this form of protection is less robust, its duration is much longer than that of preconditioning which makes it interesting for potential therapeutic exploitation. However, the mechanism of protection by chronic hypoxia is much less understood as compared with classic ischemic preconditioning [24]. Studies dealing with both chronic hypoxia and preconditioning have shown that combination of these two phenomena did not provide better cardioprotective effect than preconditioning alone [16,22,25]. We hypothesized, therefore, that these long-term and short-term protective mechanisms might share the same signaling pathway or element.

There is only limited evidence that opening of mitoKATP are also involved in improved ischemic tolerance of chronically hypoxic hearts and none of these studies examined the size of myocardial infarction as the end point of injury. We have shown that, in the open-chest adult rats, administration of the selective mitoKATP blocker, 5-hydroxydecanoate, completely abolished the protective antiarrhythmic effect of adaptation, whereas in control normoxic animals it had no effect [23]. In contrast, the selective mitoKATP opener, diazoxide, decreased the number of ischemic ventricular arrhythmias in isolated perfused hearts of normoxic rats to the same level as was achieved by adaptation but had no further effect in chronically hypoxic hearts [22]. Similarly, Eells et al. [26] have shown that mitoKATP play a role in enhanced post-ischemic recovery of the contractile function of isolated perfused hearts of chronically hypoxic neonatal rabbits. Another recent study of the same group has demonstrated that only a combination of 5-hydroxydecanoate and HMR 1098, a selective sarcolemmal KATP blocker, completely abolished improved ischemic tolerance of chronically hypoxic hearts in the same model [27]. It suggests that, unlike preconditioning, chronic hypoxia may exert its cardioprotective effect via both sarcolemmal KATP and mitoKATP. It is unclear to which extent the role of KATP in protecting the heart depends on the animal model of chronic hypoxia and on the end point of injury examined.

The objective of our study was, therefore, to examine the involvement of mitoKATP in the protection conferred by chronic high altitude hypoxia in adult rats using selective modulators of these channels, 5-hydroxydecanoate and diazoxide. We compared the effects of these agents on two end points of ischemia/reperfusion injury: infarct size in the in vivo open-chest model and contractile dysfunction in the isolated perfused heart. To further support our conclusion, additional experiments were performed examining the effect of a novel highly selective mitoKATP opener, BMS-191095 [28], on the infarct size.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animal model
Adult male Wistar rats (weighing 250–280 g) were exposed to intermittent high altitude (IHA) hypoxia of 5000 m in a hypobaric chamber for 8 h/day, 5 days a week. Barometric pressure (PB) was lowered stepwise, so that the level equivalent to an altitude of 5000 m (PB=405 mmHg, 54 kPa; PO2=85 mmHg, 11.3 kPa) was reached after nine exposures; the total number of exposures was 24–32. The animals were employed the next day after the last hypoxic exposure. 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; PO2=155 mmHg, 20.7 kPa). 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 US National Institutes of Health (NIH publication no. 85-23, revised 1996).

2.2 Drugs
5-Hydroxydecanoate and diazoxide were purchased from Sigma Chemical Co., USA and BMS-191095 was provided by Bristol-Myers Squibb, USA. Diazoxide was dissolved in a vehicle consisting of 1 M sodium hydroxide and 0.9% saline mixed in a 1:3 proportion. 5-Hydroxydecanoate was dissolved in either 0.9% saline or perfusion buffer. BMS-191095 was dissolved in ethanol, polyethylene glycol-400 and water mixed in a 2:3:5 proportion [29]. Control animals/hearts in each group were treated in the corresponding way with a solution containing the respective solvent. As no differences were observed between the control groups corresponding to 5-hydroxydecanoate- and diazoxide-treated rats/hearts in any parameter, their results were pooled. Controls to BMS-191095-treated animals are presented separately as these experiments were performed additionally.

2.3 Isolated perfused hearts
The rats were anesthetized with sodium pentobarbital (60 mg/kg i.p., Sanofi, France) and a sample of blood was taken from the tail to measure hematocrit by using the capillary micromethod. Hearts were rapidly excised and perfused according to Langendorff under constant pressure (100 cmH2O) with non-recirculating Krebs–Henseleit solution containing (mmol/l): NaCl 118.0, KCl 4.7, CaCl2 1.25, MgSO4 1.2, NaHCO3 25.0, KH2PO4 1.2 and glucose 7.0. The medium was saturated with 95% O2 and 5% CO2 (pH 7.4) and maintained at 37 °C. The left ventricle was vented at the apex and stimulated at 300 beats/min with platinum electrodes placed on the base of the right ventricle. The contractile function was measured with a non-elastic balloon inserted into the left ventricle via incision in the left atrium and connected to the pressure transducer (Hewlett-Packard 1280, USA). The balloon was gradually filled with water to give diastolic pressure of 7 to 10 mmHg. The amplified pressure signal was monitored and immediately analyzed on a computer using our software. The left ventricular systolic (LVSP), diastolic (LVDP), and developed pressure (LVDevP), and the peak rate of pressure developed [(+dP/dt)max] were expressed as the means of 10 cardiac cycles during a 2-s sampling period in selected time intervals. Coronary flow was measured by timed collection of coronary effluent and subsequently expressed per gram of heart weight.

After 25 min, during which the contractile parameters were allowed to stabilize, hearts were submitted to 20-min global no-flow ischemia followed by 40 min of reperfusion. Global ischemia was induced by clamping the perfusate inflow line while the hearts were placed in a bath of Krebs-Henseleit solution saturated with 95% N2 and 5% CO2 (pH 7.4) and maintained at 35 °C. After a restoration of flow, the functional parameters were recorded in 5-min intervals and their recovery was expressed as percentage of initial pre-ischemic values. The hearts were treated with either 5-hydroxydecanoate (250 µmol/l) or diazoxide (50 µmol/l) starting 5 min or 10 min before ischemia, respectively. 5-Hydroxydecanoate was delivered into the infusion port directly above the aortic cannula at 0.2 ml/min by infusion pump (Hugo Sachs Electronic, Germany); solution of diazoxide was diluted directly in the perfusion medium (0.2 ml/l); its pH remained at 7.4. At the end of the experiment, the hearts were dissected into left and right ventricles and the septum, and all parts were weighed.

2.4 Open-chest rats
Animals were anesthetized as above. Heparinized cannulas were placed in the left carotid artery for blood pressure monitoring with a pressure transducer (Gould P23Gb, USA) and in the right jugular vein for drug administration. Tracheotomy was performed, the rats were intubated with a cannula connected to a rodent ventilator (Ugo Basile, Italy) and ventilated with room air at 65–70 strokes/min (tidal volume of 1.2 ml/100 g body weight). Blood pressure signal was stored in a computer and subsequently analyzed by our computer program. The heart rate was derived from the blood pressure curve. The rectal temperature was maintained between 36.5 and 37.5 °C by a heated table throughout the experiment.

Left thoracotomy was performed and the pericardium was removed to reveal the location of the left anterior descending (LAD) coronary artery. Then a polyester ligature 6/0 (Ethibond-Ethicon, Scotland) was placed around the LAD coronary artery about 1–2 mm distal to the origin and an occlusive snare was placed around it. The ends of the suture were threaded through a polyethylene tube. After the surgical preparation, the rats were allowed to stabilize for 10 min before the ischemic intervention. Regional myocardial ischemia was induced by the tightening of the ligature placed around the coronary artery. Transient decrease in blood pressure (by about 40%) verified the coronary artery occlusion. After the occlusion, the snare was released; reperfusion of previously ischemic tissue was indicated by transient decrease of blood pressure and appearance of reperfusion arrhythmias.

Groups of chronically hypoxic and normoxic rats were divided into four subgroups: (1) 5-hydroxydecanoate-treated (5 mg/kg), (2) diazoxide-treated (10 mg/kg) and (3, 4) corresponding controls treated with a solvent. In addition, other groups of hypoxic and normoxic animals were treated with BMS-191095 (10 mg/kg) or its solvent. The drugs or a corresponding solvent were administered into the jugular vein as a single bolus (1 ml/kg) 15 min (5-hydroxydecanoate) and 10 min (diazoxide, BMS-191095) before ischemia. All animals were subjected to a test 20-min coronary artery occlusion followed by 4-h reperfusion.

After reperfusion, the hearts were arrested in diastole with 0.25 mg verapamil (Isoptin, Knoll, Germany) injected into the jugular vein. The hearts were excised and washed with 20 ml saline through the cannulated aorta. The area at risk and the infarct size were determined as described earlier [16] by staining with 5% potassium permanganate dissolved in water and by 1% 2,3,5-triphenyltetrazolium chloride (Sigma, St. Louis, MO, USA) dissolved in 0.1 mol/l phosphate buffer (pH 7.4). The hearts were cut perpendicularly to the long axis of the ventricle into slices 1 mm in thickness and stored overnight in 10% neutral formaldehyde solution. The day after the infarct size staining, the right ventricular free wall was separated and both sides of the slices were photographed. The infarct size (IS), the size of the area at risk (AR) and the size of the left ventricle (LV) were determined by a planimetric method. The IS was normalized to the AR and LV (IS/AR and IS/LV, respectively); the size of AR was normalized to the LV (AR/LV).

2.5 Statistics
The results are expressed as means±S.E.M. Differences in the percentage recovery of (+dP/dt)max and in the infarct size between the groups were compared by the Mann–Whitney U-test. One-way ANOVA or ANOVA for repeated measures and subsequent Student–Newman–Keuls test were used for comparison of differences in all other parametric variables between the groups. Differences were assumed as statistically significant when P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Weight parameters and hematocrit
Adaptation of rats to IHA hypoxia significantly decreased body weight (BW) and increased relative weight of the right ventricle (RV/BW) as compared with age-matched normoxic controls. Relative weight of the left ventricle (LV/BW) and septum (S/BW) were not influenced by adaptation. Hypoxic animals exhibited significantly increased hematocrit level by 32% as compared with controls (Table 1).


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  		Table 1 Hematocrit, body weight and relative weight of the left and right ventricles and the septum of rats adapted to chronic hypoxia and of normoxic animals

 
3.2 Isolated perfused hearts
Baseline pre-ischemic values of contractile parameters and coronary flow did not differ between the normoxic and hypoxic groups. Neither diazoxide nor 5-hydroxydecanoate affected the cardiac contractile function in any group. Diazoxide significantly increased the coronary flow in both hypoxic and normoxic groups (Table 2). The time course of post-ischemic recovery of contractile function is shown in Fig. 1 and maximum values of recovery are summarized in Fig. 2. Diazoxide facilitated the recovery of (+dP/dt)max in both normoxic and hypoxic groups. Maximum recovery reached 60.0±3.9% of pre-ischemic value in the control normoxic group and it was improved to 72.4±1.2% by adaptation to IHA hypoxia. 5-Hydroxydecanoate completely abolished this protective effect of chronic hypoxia, whereas in the normoxic group its effect was not significant. On the other hand, diazoxide increased the recovery of the contractile function in the normoxic group to a similar level as was achieved by adaptation to IHA hypoxia. This mitoKATP opener had no additive protective effect in the hypoxic group.


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  		Table 2 Effects of 5-hydroxydecanoate (5-HD) and diazoxide (DX) on baseline contractile parameters and coronary flow of the isolated perfused hearts of rats adapted to IHA hypoxia and of normoxic animals

 

Figure 1
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  		Fig. 1 Recovery of peak rate of pressure development [(+dP/dt)max] during reperfusion after 20 min of global ischemia, expressed as percentage of pre-ischemic values, in the control, 5-hydroxydecanoate-treated (5-HD; upper graph) and diazoxide-treated (DX; lower graph) hearts of rats adapted to IHA hypoxia and of normoxic animals. Values are means±S.E.M.

 

Figure 2
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  		Fig. 2 Maximum recovery of peak rate of pressure development [(+dP/dt)max] during reperfusion after 20 min of global ischemia, expressed as percentage of pre-ischemic values, in the control (Cont), 5-hydroxydecanoate-treated (5-HD) and diazoxide-treated (DX) hearts of rats adapted to IHA hypoxia and of normoxic animals. Values are means±S.E.M. *P<0.05 vs. corresponding controls; {dagger}P<0.05 vs. corresponding normoxic group.

 
3.3 Open-chest rats
Tables 3 and 4Go summarize values of heart rate (HR) and mean arterial blood pressure (MAP) in all groups, determined at baseline (before ischemia and drug treatment), after treatment (pre-ischemic), at the end of test ischemia and at the end of 4-h reperfusion. No significant differences were found in the baseline values of these variables between the groups. Ischemia did not influence HR and MAP in normoxic and hypoxic controls, whereas both parameters decreased (or tended to decrease) at the end of reperfusion, except for the hypoxic group, which maintained MAP relatively unchanged. Neither drug affected hemodynamics during the pre-ischemic period as compared with baseline values. However, HR was significantly lower in diazoxide-treated hypoxic animals as compared with untreated ones and this effect persisted throughout ischemia and reperfusion. On the other hand, pre-ischemic MAP values were lower in both normoxic and hypoxic groups treated with diazoxide and higher in the hypoxic group treated with 5-hydroxydecanoate as compared with corresponding untreated groups. Unlike these two drugs, BMS-191095 abolished the difference in MAP values between normoxic and hypoxic animals at the end of reperfusion.


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  		Table 3 Heart rate and mean arterial blood pressure in control, 5-hydroxydecanoate-treated (5-HD) and diazoxide-treated (DX) rats adapted to IHA hypoxia and in normoxic animals

 

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  		Table 4 Heart rate and mean arterial blood pressure in control and BMS-191095-treated (BMS) rats adapted to IHA hypoxia and in normoxic animals

 
The normalized area at risk (AR/LV) did not differ among the groups (Table 5). The infarct size reached about 60% of the AR in normoxic controls and adaptation of rats to hypoxia significantly decreased IS/AR by ~10%. Administration of 5-hydroxydecanoate did not change IS/AR in normoxic hearts but it abolished the cardioprotective effect of adaptation to chronic hypoxia. Diazoxide induced an opposite effect as it did not change IS/AR in hypoxic animals but significantly reduced the infarct size in normoxic animals (Fig. 3). Similarly as diazoxide, BMS-191095 exhibited protective effect on myocardial infarction in the normoxic group only (Fig. 4). When the infarct size was normalized to the size of the left ventricle (IS/LV), the protective effect of hypoxia was also evident but effects of the drugs did not reach statistical significance (Table 5).


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  		Table 5 The relative size of the area at risk (AR/LV) and the relative infarct size (IS/LV) in control, 5-hydroxydecanoate-treated (5-HD), diazoxide-treated (DX) and BMS-191095-treated (BMS) rats adapted to IHA hypoxia and in normoxic animals

 

Figure 3
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  		Fig. 3 Myocardial infarct size expressed as percentage of the area at risk (IS/AR) in the control (Cont), 5-hydroxydecanoate-treated (5-HD) and diazoxide-treated (DX) rats adapted to IHA hypoxia and in normoxic animals. Circles indicate individual experiments. Values are means±S.E.M. *P<0.05 vs. corresponding controls; {dagger}P<0.05 vs. corresponding normoxic group.

 

Figure 4
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  		Fig. 4 Myocardial infarct size expressed as percentage of the area at risk (IS/AR) in the control (Cont) and BMS-191095-treated (BMS) rats adapted to IHA hypoxia and in normoxic animals. Circles indicate individual experiments. Values are means±S.E.M. *P<0.05 vs. corresponding controls; {dagger}P<0.05 vs. corresponding normoxic group.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We confirmed earlier reports showing that adaptation of rats to IHA hypoxia increased the tolerance of their hearts to ischemia/reperfusion injury, determined as improved recovery of post-ischemic contractile function and reduction of infarct size. These protective effects were modest but statistically significant and well reproducible. The extent of infarct size reduction depends on the degree of chronic hypoxia, as the IS/AR decreased by only 10% in the present study as compared with 16% in our recent study which used a more severe model of IHA hypoxia corresponding to 7000 m [16]. Here, we reduced the severity of hypoxia because the majority of 5-hydroxydecanoate-treated hearts of animals adapted to 7000 m exhibited no reflow of the ischemic area after release of an occluder. The cause of this effect, which was not observed following adaptation to 5000 m, is unclear.

Using the modulators, which are believed to be selective for mitoKATP, we have shown that these channels play a crucial role in the cardioprotective mechanism of chronic hypoxia. Whereas the blocker, 5-hydroxydecanoate, completely abolished both the improvement of post-ischemic recovery of contractility and the reduction of infarct size in animals adapted to IHA hypoxia it had no appreciable influence in the normoxic group. In contrast, the opener, diazoxide significantly reduced contractile dysfunction and infarct size in normoxic hearts to the same extent as achieved by chronic hypoxia, but it had no additive protective effect in the IHA-adapted group. Furthermore, the novel highly selective mitoKATP opener, BMS-191095, exhibited similar protective effect on myocardial infarction in the normoxic group (and the absence of any effect in the hypoxic group) as diazoxide. These results are in accordance with our previous studies, which have shown that mitoKATP are involved in the mechanism of the antiarrhythmic effect of adaptation to chronic hypoxia. Likewise, pretreatment with 5-hydroxydecanoate prevented the decrease in the number of premature ventricular complexes during ischemia in animals adapted to IHA hypoxia and diazoxide was antiarrhythmic in the normoxic controls but not in the adapted group [22,23]. These effects cannot be attributed to hemodynamic actions of the drugs as they were demonstrated in both open-chest animals and isolated hearts perfused under either constant pressure or constant flow. Moreover, unlike diazoxide, BMS-191095 is devoid of peripheral vasodilator activity [28].

The role of KATP channels in the mechanism of cardioprotection by adaptation to hypoxia was also studied in neonatal rabbits subjected to permanent normobaric hypoxia. This model is known to induce similar cardiopulmonary and metabolic changes as our model of intermittent hypobaric (high altitude) hypoxia of a comparable degree. It has been shown that a non-selective KATP blocker, glibenclamide, completely abolished the protective effect of chronic hypoxia on post-ischemic contractile dysfunction, whereas an opener, bimakalim, induced cardioprotection in normoxic hearts only [19,26]. According to a recent study by the same group [27], both mitoKATP and sarcolemmal KATP contribute to the cardioprotection in their model of chronic hypoxia. This protection was completely abolished by co-administration of selective mitoKATP and sarcolemmal KATP blockers, whereas 5-hydroxydecanoate alone exhibited only a partial inhibitory effect. Thus, our results obtained on adult rats do not fully confirm the above conclusion, as mitoKATP blockade led to a complete inhibition of protection. Both species (rat vs. rabbit) and ontogenetic (adult vs. neonatal) differences may account for these results. Nevertheless, both our studies and those of Baker's group provide clear evidence that mitoKATP are involved in the cardioprotective mechanism of chronic hypoxia, regardless of their quantitative importance.

MitoKATP have also been implicated in the protection of the heart induced by various forms of preconditioning in several experimental models, as demonstrated in a number of reports. It appears, therefore, that these channels may represent the common central component of both short-term and long-term cardioprotective mechanisms. The assumption that various protective phenomena may share the same molecular pathway or its element is further supported by the observation that protective effects of chronic hypoxia and classic ischemic preconditioning are not additive [16,25]. Moreover, the newborn rat heart, which is ‘adapted’ to hypoxic conditions in utero, is also more tolerant to ischemic injury and cannot be preconditioned [30].

It has been proposed that chronic hypoxia leads to sustained, tonic activation of cardiac mitoKATP channels [22,26] that may explain the insensitivity of these hearts to diazoxide or BMS-191095. However, little is known about factor(s) which maintain these channels in the active state under conditions of chronic hypoxia and also following a restoration of normoxic conditions. Chronic hypoxia induces a large variety of adaptive changes in the myocardium, which may be protective [24] but their potential link to mitoKATP activation was not yet clearly established. Myocardial mitoKATP can be activated, for instance, by protein kinase C (PKC) which appears to be a key player in signal transduction of various cardioprotective phenomena [5,31–33]. Limited information is available suggesting that PKC is up-regulated and permanently activated under conditions of chronic hypoxia [34]. Our preliminary data suggest that IHA hypoxia leads to up-regulation of the PKC-{delta} isoform in the mitochondrial fraction [35]. Chronic hypoxia also increases myocardial concentration of phosphatidylinositol, the substrate of PKC-activating signaling cascade [36]. Another potential candidate for endogenous mitoKATP activator in chronic hypoxia is nitric oxide (NO). Rouet-Benzineb et al. [34] detected abundance and higher enzyme activity of inducible NO synthase in chronically hypoxic adult rat hearts due to hypoxia-inducible factor 1. Baker et al. [37] showed that adaptation of neonatal rabbits to hypoxia increased NO production by up-regulation of constitutive NO synthase. Inhibition of NO synthase activity by L-NAME led to a complete abolition of protection by chronic hypoxia whereas NO donor, GSNO, was protective in normoxic but not in chronically hypoxic hearts. Increased NO production in these hearts may activate KATP by both cGMP-dependent [38] and cGMP-independent [39] pathways.

In conclusion, we have shown that long-term adaptation of adult rats to IHA hypoxia increases the tolerance of their hearts to ischemia/reperfusion injury. Both the infarct size reduction and improvement of post-ischemic contractile function of these hearts were completely inhibited by mitoKATP blockade and a similar degree of protection, as induced by chronic hypoxia, was achieved in normoxic hearts by mitoKATP openers. Activation of these channels therefore appears to play an important role in the cardioprotective mechanism of adaptation to IHA hypoxia.

Time for primary review 28 days.


   Acknowledgements
 
This study was supported by the Grant Agency of the Czech Republic (grants no. 306/98/0470 and 305/01/0279 to FK). The authors thank Mrs. A. Dedicová for her assistance in statistical evaluation.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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