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Am J Physiol Heart Circ Physiol 294: H2088-H2097, 2008. First published February 22, 2008; doi:10.1152/ajpheart.01345.2007
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Effect of diazoxide on flavoprotein oxidation and reactive oxygen species generation during ischemia-reperfusion: a study on Langendorff-perfused rat hearts using optic fibers

Philippe Pasdois,1 Bertrand Beauvoit,1,2 Liliane Tariosse,1 Béatrice Vinassa,1 Simone Bonoron-Adèle,1 and Pierre Dos Santos1,2,3

1Institut National de la Santé et de la Recherche Médicale U828; 2Université Victor Segalen Bordeaux 2; and 3Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France

Submitted 16 November 2007 ; accepted in final form 12 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study analyzed the oxidant generation during ischemia-reperfusion protocols of Langendorff-perfused rat hearts, preconditioned with a mitochondrial ATP-sensitive potassium channel (mitoKATP) opener (i.e., diazoxide). The autofluorescence of mitochondrial flavoproteins, and that of the total NAD(P)H pool on the one hand and the fluorescence of dyes sensitive to H2O2 or O2bullet– [i.e., the dihydrodichlorofluoroscein (H2DCF) and dihydroethidine (DHE), respectively] on the other, were noninvasively measured at the surface of the left ventricular wall by means of optic fibers. Isolated perfused rat hearts were subjected to an ischemia-reperfusion protocol. Opening mitoKATP with diazoxide (100 µM) 1) improved the recovery of the rate-pressure product after reperfusion (72 ± 2 vs. 16.8 ± 2.5% of baseline value in control group, P < 0.01), and 2) attenuated the oxidant generation during both ischemic (–46 ± 5% H2DCF oxidation and –40 ± 3% DHE oxidation vs. control group, P < 0.01) and reperfusion (–26 ± 2% H2DCF oxidation and –23 ± 2% DHE oxidation vs. control group, P < 0.01) periods. All of these effects were abolished by coperfusion of 5-hydroxydecanoic acid (500 µM), a mitoKATP blocker. During the preconditioning phase, diazoxide induced a transient, reversible, and 5-hydroxydecanoic acid-sensitive flavoprotein and H2DCF (but not DHE) oxidation. In conclusion, the diazoxide-mediated cardioprotection is supported by a moderate H2O2 production during the preconditioning phase and a strong decrease in oxidant generation during the subsequent ischemic and reperfusion phases.

cardioprotection; 5-hydroxydecanoate

A MAJOR ROLE for reactive oxygen species (ROS) in the signaling of both ischemic and pharmacological preconditioning is now widely accepted. Exogenous administration of H2O2 can induce a preconditioned state in cardiomyocytes (28) and intact hearts (45), and a similar effect can be achieved by endogenous ROS formation, i.e., by the induction of the xanthine/xanthine oxidase reaction, or by the inhibition of the electron transport chain (21, 36). ROS have been described to act as second messengers on a number of targets implicated in preconditioning, including PKC and mitogen-activated kinases (23, 25, 37, 52). Although ROS may activate the mitochondrial ATP-sensitive potassium channel (mitoKATP) channel (53), there is also evidence that an activation of the mitoKATP may modulate the mitochondrial redox state and induce ROS production. Indeed, a well-known activator of the mitoKATP, i.e., diazoxide, when administrated to isolated mitochondria or cultured cells, induces flavoproteins (Fp) oxidation (13, 31, 43) and promotes ROS production. It has been shown that this ROS production is impaired in connexin 43 deficient mice (22). Two main theories have been proposed concerning the origin of ROS production induced by diazoxide: 1) a dependence on K+ influx into the matrix (3), and 2) an inhibition of the mitochondrial respiratory chain (33). Moreover, it has been reported that inhibition of the respiratory chain protects against ischemia-reperfusion damage (2, 29). Whatever the mechanism responsible for ROS generation, perfusion of ROS scavengers during the preconditioning phase blocks the diazoxide-induced cardioprotection (16, 38).

This knowledge of the implication of ROS production in ischemia-reperfusion injury and diazoxide-induced cardioprotection is mostly based on models of ischemia-reperfusion in isolated or cultured cells. Direct measurements of oxidant production and Fp oxidation have not yet been reported in perfused hearts. The aim of this study was to monitor the effects of diazoxide perfusion on Fp oxidation and ROS generation during both pharmacological treatment and ischemia-reperfusion protocols. The fluorescence of Fp, as well as that of oxidant-sensitive dyes, was monitored noninvasively via an optic fiber probe placed near the left ventricular wall of Langendorff-perfused rat hearts. We show, for the first time in intact hearts, that the protective effects of diazoxide-induced preconditioning involve a moderate H2O2 production during the preconditioning phase and a strong decrease in oxidant generation during the subsequent ischemic and reperfusion phases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Langendorff Perfusion

Male Sprague-Dawley rats weighing 350–375 g were used for the study. The procedure conforms to 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), and our protocol was submitted to and approved by the Ethical Independent Committee of University Bordeaux 2. The rats were anesthetized with 40 mg/kg of pentobarbital sodium injected intraperitoneally. The thorax was opened, and the hearts were rapidly excised and immediately perfused by an aortic canula delivering warm (37°C) buffer at a constant pressure of 100 mmHg. Hearts were perfused with a modified Krebs-Henseleit solution containing (in mM): 118 NaCl, 5.9 KCl, 1.75 CaCl2, 1.2 MgSO4, 0.5 EDTA, 25 NaHCO3, and 16.7 glucose. The perfusate was gassed with 95%O2-5%CO2, which resulted in a pO2 > 600 mmHg at the level of the aortic canula and a buffer pH of 7.4. A high glucose concentration was used to avoid limitations at the level of glucose entry in the absence of insulin. Diazoxide (100 µM), a selective mitoKATP activator, and/or 5-hydroxydecanoic acid (5HD; 500 µM), a selective mitoKATP blocker, were added to the buffer when indicated. Hearts were not paced, and left ventricular pressure was monitored from a water-filled latex balloon placed through the left atrial appendage and connected to a statham P23 Db pressure transducer. The volume of the intraventricular balloon was adjusted to produce an initial end-diastolic pressure between 5 and 8 and was kept constant throughout the experiments. Mechanical performance was evaluated as the product of heart rate and developed pressure (RPP).

Effects of Preconditioning by Diazoxide on Heart Function and Oxidant Generation

Experimental groups. Four groups of hearts were studied (Fig. 1). The ischemia-reperfusion control group (IR) was submitted to 30 min of zero-flow global ischemia followed by 20 min of reperfusion. The diazoxide-treated group (DIA), the diazoxide + 5-HD treated group (DIA + 5HD), and the 5-hydroxydecanoic acid treated group (5HD) were submitted to a 20-min perfusion with a buffer containing 100 µM of diazoxide, 100 µM diazoxide and 500 µM 5HD, or 500 µM 5HD, respectively, immediately before ischemia and reperfusion.


Figure 1
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  		Fig. 1. Perfusion protocols used in the study. This perfusion protocol was used to monitor the mechanical performances and the fluorescence of either flavoproteins (Fp) or fluorescent dyes. The ischemia-reperfusion group (IR) was stabilized for 40 min under aerobic conditions before a 30-min global zero-flow ischemia period followed by 20 min of reperfusion. The following 3 groups were stabilized for 20 min under aerobic conditions before 20 min of drug infusion followed by the ischemia-reperfusion sequence described above. DIA: 100 µM diazoxide; DIA + 5HD: 100 µM diazoxide + 500 µM 5-hydroxydecanoic acid; 5HD: 500 µM 5-hydroxydecanoic acid. In any case, reperfusion was carried out without the drug in the perfusion buffer.

 
Surface fluorescence measurements on perfused rat hearts. Experiments were conducted in a light-proofed black box to block incident light. The distal end of a bifurcated optic-fiber cable (5 mm diameter, 6.8 mm2 per bundle) was placed at ~1 cm of the left ventricular anterior wall. The two proximal ends of the optic-fiber cable were connected to a modified spectrofluoremeter (Xenius flx, SAFAS Monaco). The excitation light provided by a Xenon arc lamp at 150 W was focused onto the in-going fibers of the optic bundles. Emitted photons were collected by fibers of the second limb of the cable and measured at the appropriate wavelengths by a photomultiplier tube. High voltage value was set to 700–800 or 1,200 V for dyes, NAD(P)H or Fp fluorescence, respectively, and to 400–500 V for reflectances. The excitation/emission wavelength couples were alternatively set every 8 s by the multiwavelength software (SAFAS Monaco). At each wavelength couple, the averaged measurement time was 0.5 s. Bandwidth was equal to 10 nm for both excitation and emission light. Distance and motion artifacts were diminished by dividing the emitted photon intensity by the reflectance signal measured at the respective excitation and emission wavelengths (10). For plotting commodity, motion-corrected fluorescence signals were averaged over a sampling period of five measurements.


ONLINE ASSESSMENT OF THE FP REDOX STATE. Tissue autofluorescence collected at {lambda}em 525 nm ({lambda}ex 460 nm) was used to measure the redox state of Fp (11, 26, 27, 41). To determine the percentage of Fp oxidation, signals were calibrated according to the procedure illustrated in Fig. 2A. The fully oxidized state (100%) was determined after perfusion of 500 µM dinitrophenol (DNP), whereas the fully reduced state (0%) was determined after perfusion of 1 mM cyanide (KCN).


Figure 2
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  		Fig. 2. Calibration of flavoprotein fluorescence and assessment of dichlorodihydrofluorescein (H2DCF) response to H2O2. A: calibration of the Fp fluorescence signal by perfusion of either 500 µM dinitrophenol [DNP; i.e., fully oxidized state (100%)] or 1 mM cyanide [KCN; i.e., fully reduced state (0%)]. B: effect of 100 µM H2O2 on DCF signal monitored in isolated heart. Data are means ± SE of 4 perfused hearts.

 

ONLINE ASSESSMENT OF THE NAD(P)H POOL REDOX STATE. Tissue autofluorescence collected at {lambda}em 460 nm ({lambda}ex 340 nm) was used to measure the redox state of NAD(P)H (40). Although fluorescence at 460 nm could arise from unknown intracellular constituents, the majority of the signal comes from the mitochondrial NADH and NAD(P)H (35).


ONLINE ASSESSMENT OF DICHLORODIHYDROFLUORESCEIN AND DIHYDROETHIDINE OXIDATION, AND LOADING OF FLUORESCENT DYES. ROS production was monitored using two fluorescent probes, namely 1) the dichlorodihydrofluorescein (H2DCF) oxidized to dichlorofluorescein (DCF), and 2) the dihydroethidine (DHE) oxidized to ethidium that intercalates with DNA to cause a red shift in fluorescence (9). Each of those fluorescent dyes has been described to be predominantly sensitive to H2O2 (51; for review, see Ref. 48) and O2bullet– (9, 51), respectively. The wavelength couples were {lambda}ex 480/{lambda}em 525 and {lambda}ex 518/{lambda}em 620 for DCF and ethidium-DNA, respectively. Background fluorescence was determined during a 10-min equilibration period at {lambda}em 525 or 620 nm.

The loading of dichlorodihydrofluorescein diacetate (H2DCFDA) or DHE in perfused hearts was carried out as described by Brandes et al. (10) with minor modifications. Briefly, hearts were perfused at 25°C by 85 ml of a recirculating modified Krebs-Henseleit solution containing: 10 µM H2DCFDA or 10 µM DHE (stock solution in dimethylsulfoxide, under N2 atmosphere), supplemented with 4% (wt/vol) Pluronic F127, 5% (vol/vol) FCS, and 0.1 mM probenecid (Sigma; stock solution in 1 M NaOH). Once the fluorescence signals were ~10-fold higher than the initial background signal (~25 min with DHE and ~45 min with H2DCFDA), the heart was then perfused at 37°C with a modified Krebs-Henseleit solution without dye and supplemented with 0.1 mM probenecid. The experimental protocol was started after the washout of the residual and interstitial probes for 20 min. Leakage of the loaded probes was taken into account in our plots by using the nonlinear regression of time-course recordings performed during aerobic perfusion.

Chemicals

H2DCFDA and DHE were purchased from Molecular Probes, and diazoxide, Pluronic F127, probenecid, and 5HD were purchased from Sigma-Aldrich. Diazoxide was dissolved in DMSO before being added to the Krebs-Henseleit solution. Therefore, control experiments were performed in the presence of 0.1% DMSO. 5HD, a mitoKATP blocker, was directly solubilized in the Krebs-Henseleit solution.

Statistical Analysis

Statistical analysis was carried out using a nonparametric Mann-Whitney test (NCSS97 software). A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of Diazoxide and 5HD on Cardiac Function

The effects of preconditioning by diazoxide on heart function are illustrated in Fig. 3. In the IR group, an ischemic contracture was observed after 10 min (Fig. 3A). After reperfusion, a discrete recovery of systolic function was observed (Fig. 3B). In the DIA group, the ischemic contracture was delayed and its amplitude was decreased compared with the IR group (Fig. 3A). Preconditioning by diazoxide also decreased left ventricular diastolic pressure and improved the recovery of RPP after reperfusion compared with the IR group (Fig. 3B). Perfusion of diazoxide induced a significant, 5HD-insensitive increase in coronary flow (Fig. 3C). Coperfusion of 5HD (DIA + 5HD group) significantly attenuated the beneficial effects of diazoxide on all of the parameters previously described. 5HD perfused alone had no significant effect on the cardiac function parameters compared with the IR control group.


Figure 3
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  		Fig. 3. Assessment of heart function during perfusion protocols. Hearts were submitted to the perfusion protocols described in Fig. 1. A: end diastolic pressure (EDP) measured during the 30-min ischemic period followed by 20 min of reperfusion. B: recovery of left ventricular rate-pressure product (RPP) during the 20-min reperfusion period. C: coronary flow measured during the different periods of perfusion protocols described. Baseline corresponds to t = 20 min; preischemia corresponds to t = 40 min; and reperfusion corresponds to the minute 20 of reperfusion. *P < 0.01 vs. IR. Data are means ± SE of 7 perfused hearts.

 
Effect of Diazoxide and 5HD on Fp Oxidation During Ischemia-Reperfusion Protocol

Figure 4, A, B, and C, illustrates the time courses of Fp oxidation during the ischemia-reperfusion protocols obtained in the same four groups of hearts. Upon initiation of the zero-flow ischemia, and as expected by the lowered tissue oxygen tension, the percentage of Fp oxidation dropped within few seconds from ~50–25% (IR group, Fig. 4A). This decline was followed by a progressive and sustained increase in Fp oxidation, reaching a value of ~45% at the end of ischemia. Reperfusion did not induce any significant change in the Fp oxidation, the value of which remained below the baseline and close to 45%.


Figure 4
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  		Fig. 4. Time courses of flavoprotein oxidation state of isolated hearts during ischemia-reperfusion protocols. A: Fp oxidation state monitored in IR (black circles) and 5HD (grey circles) groups. B: fluorescence of flavoproteins monitored in IR (black circles) and DIA (white circles) groups. C: Fp oxidation monitored in DIA (white circles) and DIA + 5HD (grey circles) groups. D: effect of drug treatment on Fp oxidation state compared with IR group. The signal difference between IR group and DIA (white bar) or DIA + 5HD (grey bar) or 5HD (black bar) groups, respectively, was calculated and integrated over the total phase duration (i.e., PRECOND, preconditioning by diazoxide; ISCHEMIA, ischemia; REPERF, reperfusion) and then normalized to the value obtained in the IR group. Data are means ± SE of 4–8 perfused hearts. #P < 0.01 vs. DIA; £P < 0.01 vs. DIA.

 
When aerobically perfused, diazoxide induced an immediate increase in the oxidation steady state of Fp, which tended toward the baseline level during the intervening perfusion period (DIA group, Fig. 4B). Upon initiation of ischemia, Fp oxidation decreased to a value of 15% and then progressively increased to a value of 25% measured at the end of the ischemic period (Fig. 4B). Upon reperfusion, in contrast to what was observed in the IR group, Fp oxidation increased and stabilized close to a value of 60%.

The diazoxide effect on Fp oxidation during the preconditioning phase was abolished by coperfusion of 5HD (DIA + 5HD group, Fig. 4C). 5HD did not alter the effect of diazoxide on Fp oxidation during the ischemic period. Upon reperfusion, Fp oxidation increased to a value of 52% and stabilized below the value obtained in the DIA group.

When aerobically perfused, 5HD produced a progressive and sustained reduction of the Fp pool (Fig. 4A). Upon initiation of the zero-flow ischemia, the percentage of Fp oxidation dropped within a few seconds from ~50–15% and progressively increased to a value of ~30% below what was observed in the IR group. Upon reperfusion, Fp oxidation rapidly increased and reached a value of 52% (Fig. 4A).

The comparative effects of diazoxide and 5HD, alone or in combination, were further quantified during each phase of the protocol and compared with IR. Figure 4D shows that diazoxide induced a 5HD-sensitive Fp oxidation during normoxia and significantly prevented subsequent Fp oxidation during the index ischemia. Moreover, after reperfusion, the cardioprotective effect of diazoxide is characterized by a higher Fp oxidation state than in DIA + 5HD and 5HD groups (compare Figs. 3B and 4B). This is in agreement with the preservation of the RPP level and of the respiratory activity, consecutive to the preservation of the mitochondrial functionality (39).

Effect of Diazoxide and 5HD on NAD(P)H Fluorescence During the Ischemia-Reperfusion Protocol

Figure 5A shows changes in NAD(P)H autofluorescence before, during, and after ischemia. In the IR group, NAD(P)H fluorescence strongly increased at the beginning of ischemia and quickly reached a stable value throughout the ischemic period. Reperfusion induced a significant decrease in NAD(P)H fluorescence, reaching a value significantly below the baseline.


Figure 5
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  		Fig. 5. Time courses of NAD(P)H fluorescence of isolated hearts during ischemia-reperfusion protocols. A: NAD(P)H fluorescence monitored in IR (black circles), DIA (white circles), and DIA + 5HD (grey circles) groups. B: effect of drug treatment on NAD(P)H fluorescence compared with IR group. Signal difference between IR group and DIA (white bar) or DIA + 5HD (grey bar) groups, respectively, was calculated and integrated over the total phase duration and then normalized to the value obtained in the IR group. Data are means ± SE of 4–8 perfused hearts. #P < 0.01 vs. DIA; £P < 0.05 vs. DIA.

 
When aerobically perfused, diazoxide induced an immediate decrease in NAD(P)H fluorescence (DIA group, Fig. 5A). Upon initiation of ischemia, NAD(P)H fluorescence increased about the same as the value that was obtained in the IR control group. Upon reperfusion, in contrast to what was observed in the IR group, NAD(P)H fluorescence strongly decreased and then increased to reach a value close to the baseline. The recovery of the NAD(P)H pool at reperfusion is a well-described end point in cardioprotection studies (40). Indeed, the preservation of NAD(P)H fluorescence at the end of the reperfusion period in the DIA group further demonstrates the beneficial effect of diazoxide on cell damage.

The diazoxide-induced decreased in NAD(P)H during the preconditioning phase was attenuated by coperfusion of 5HD (DIA + 5HD group, Fig. 5A). Interestingly, during ischemia, the NAD(P)H fluorescence was significantly higher in the DIA + 5HD group than in the DIA or IR groups. Upon reperfusion, the fluorescence signal decreased and then increased to a value corresponding to the one obtained at the end of the preconditioning period.

The comparative effects of diazoxide alone or in combination with 5HD were further quantified during each phase of the protocol and compared with IR. Figure 5B shows that diazoxide induced a 5HD-sensitive NAD(P)H oxidation during normoxia. During ischemia, diazoxide had no significant effect on NADP(H) fluorescence. In contrast, in presence of 5HD, NAD(P)H strongly accumulated in accordance to its possible metabolism by the intramitochondrial β-oxidation enzymes. After reperfusion, the cardioprotective effect of diazoxide was characterized by a recovery of NAD(P)H fluorescence compared with the IR control group. Nevertheless, in the DIA + 5HD group, the NAD(P)H pool remained highly reduced, probably as a consequence of the 5HD metabolism, whereas hearts were not protected at all (see Fig. 3 and Ref. 39).

To further analyze the effect of diazoxide and 5HD perfused alone on the mitochondrial redox state during the preconditioning period, the Fp fluorescence was plotted against the time-corresponding NAD(P)H fluorescence. Figure 6 shows that diazoxide and 5HD have antagonist effects on the mitochondrial redox poise, i.e., diazoxide oxidized both the Fp and NAD(P) pools, whereas 5HD reduced both of them.


Figure 6
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  		Fig. 6. Fp fluorescence as a function of NAD(P)H fluorescence of isolated hearts during DIA or 5HD treatment. Fp and NAD(P)H fluorescences were monitored as described in MATERIALS AND METHODS and plotted one against the other. Fluorescence values were obtained during the preischemia period in DIA and 5HD groups. Dashed lines indicate the baseline NAD(P)H and Fp fluorescence values. Data are means ± SE of 4 perfused hearts.

 
Effect of Diazoxide and 5HD on DCF Fluorescence During the Ischemia-Reperfusion Protocol

Figure 7, A–C, illustrates the time courses of the H2DCF oxidation obtained in the four groups of hearts during the ischemia-reperfusion protocols.


Figure 7
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  		Fig. 7. Time courses of DCF fluorescence of isolated hearts during ischemia-reperfusion protocols. After a 45-min loading period of H2DCFDA and as described in MATERIALS AND METHODS, hearts were perfused with dye-free Krebs solution for 20 min to allow for the dye deesterification by esterases and then were submitted to the ischemia-reperfusion protocols described in Fig. 1A. A: fluorescence of DCF monitored in the IR group (black circles) and 5HD (grey circles) groups. B: fluorescence of DCF monitored in the IR (black circles) and DIA (white circles) groups. C: fluorescence of DCF monitored in the DIA (white circles) and DIA + 5HD (grey circles) groups. D: effect of drug treatment on DCF fluorescence compared with IR group. The signal difference between IR group and DIA (white bar) or DIA + 5HD (grey bar) or 5HD (black bar) group, respectively, was calculated, integrated over the total phase duration, and then normalized to the value obtained in the IR group. Data are means ± SE of 4–6 perfused hearts. #P < 0.01 vs. DIA.

 
At initiation of the zero-flow ischemia, the DCF signal decreased within 1 min (IR group, Fig. 7A). This drop was followed by a progressive and sustained increase throughout the ischemic period. Upon reperfusion, DCF oxidation strongly increased above the baseline level and remained markedly elevated throughout the reperfusion period (Fig. 7A).

Diazoxide perfusion induced an immediate increase in the DCF signal (DIA group, Fig. 7B) followed by progressive return to the baseline level after 10 min of treatment. Initiation of ischemia resulted in an abrupt decrease of the DCF signal followed by a progressive increase toward a value below that observed in the IR group. Upon reperfusion, the DCF signal rapidly increased but, in contrast to what was observed in the IR group, returned abruptly to the baseline level (Fig. 7B).

The diazoxide-induced increase in H2DCF oxidation during the preconditioning phase was decreased by >50% in the presence of 5HD (Fig. 7C). During both the ischemic and reperfusion periods, there were strong differences between the DIA and DIA + 5HD groups, corresponding to a higher H2DCF oxidation when 5HD was coperfused to diazoxide. In contrast to what was observed in Fp oxidation, 5HD, when aerobically perfused, did not produce any decrease in DCF fluorescence (Fig. 7A). At initiation of the zero-flow ischemia, the DCF signal rapidly decreased to a level similar to what was observed in the IR group and then progressively increased toward a value below what was observed in the IR group. Upon reperfusion, the DCF signal rapidly increased to a level similar to that observed in the IR group.

To take into account the abrupt, and probably artifactual (see Ref. 7, in isolated mitochondria), variations in the DCF signal at the beginning of both ischemia and reperfusion phases, the effects of diazoxide and 5HD, alone or in combination, were further quantified during each protocol phase and compared with the IR group. Figure 7D shows that diazoxide treatment induced H2DCF oxidation during normoxia and significantly prevented subsequent dye oxidation during both the index ischemia and reperfusion phases. Moreover, 5HD coperfusion significantly decreased both of these effects. Appropriate control experiments showed that the drug vehicle (i.e., DMSO) or the 5HD alone did not directly affect the DCF fluorescence.

Effect of Diazoxide on Ethidium-DNA Fluorescence During the Ischemia-Reperfusion Protocol

Figure 8 illustrates the time courses of DHE oxidation during ischemia-reperfusion protocols. Diazoxide perfusion did not affect the ethidium fluorescence pattern, compared with the time control experiment (i.e., performed in the presence of the drug vehicle DMSO). Ischemia resulted in an abrupt increase in DHE oxidation, which was significantly attenuated by diazoxide treatment (Fig. 8A). DHE oxidation decreased upon reperfusion in both groups but persisted at a higher level in IR than in DIA group. Overall, as illustrated in Fig. 8B, diazoxide preconditioning did not evoke any O2bullet– accumulation during normoxia but significantly prevented O2bullet– production during both the index ischemia and the subsequent reperfusion period.


Figure 8
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  		Fig. 8. Time courses of ethidium-DNA fluorescence in isolated hearts during ischemia-reperfusion protocols. After a 25-min loading period of dihydroethidine (DHE) and as described in MATERIALS AND METHODS, hearts were perfused with a dye-free Krebs solution for 20 min before initiation of the ischemia-reperfusion protocols described in Fig. 1A. A: fluorescence of ethidium-DNA monitored in the IR (white circles) and DIA (black circles) groups. B: effect of diazoxide treatment on ethidium-DNA fluorescence compared with IR group. The signal difference between IR and DIA groups was calculated and integrated over the total phase duration and then normalized to the value obtained in the IR group. Data are means ± SE of 3 perfused hearts.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Fp and NAD(P)H Oxidation During Pharmacological Preconditioning

An important experimental feature of our work is that diazoxide induces a transient, reversible, and 5HD-sensitive Fp oxidation. These original data obtained in intact hearts confirm and extend previous results obtained in isolated mitochondria (44) and in cultured cardiomyocytes (13, 31, 43). A widely used assay of mitoKATP activity within cells is the ability of a substituted short chain fatty acid (i.e., 5HD) to inhibit the drug-mediated Fp oxidation. However, several authors (13) pointed out the difficulty to reproduce this assay using diazoxide when applied on unstarved cultured cardiomyocytes, thus questioning the specificity of 5HD. Moreover, other authors (13, 33) have proposed that diazoxide can act like an uncoupler because this drug can dissipate mitochondrial membrane potential, which is associated with an increase in Fp oxidation, even in absence of potassium, thus questioning the existence of the mitoKATP.

In our study, the reducing capability of 5HD in both the mitochondrial Fp and NAD(P)H pools was clearly demonstrated in situ (see Figs. 4, 5, and 6). This is in line with the ability of 5HD to be metabolized in vitro (18, 19), thereby generating NADH via the β-oxidation pathway and ensuring electron supply to the respiratory chain via the electron transfer protein. Therefore, we can reasonably hypothesize that the mechanism by which 5HD attenuated the diazoxide-induced Fp oxidation is a metabolic (i.e., redox) effect rather than a pharmacological effect per se.

ROS Generation During Pharmacological Preconditioning

We have shown, for the first time in intact heart, that diazoxide perfusion during normoxia was associated with an oxidation of H2DCF (mainly sensitive to H2O2) in a 5HD-sensitive manner. Meanwhile, no significant DHE oxidation (mainly sensitive to O2bullet–) could be detected. Thus, a predominant ROS pathway during diazoxide preconditioning appears to involve H2O2 rather than O2bullet– accumulation. However, the specificity of these fluorescent probes for ROS detection in vivo has already been discussed (see for review Ref. 49), so the precise delineation of each ROS may be questionable. Nevertheless, the suggested role of H2O2 as a trigger of preconditioning is supported by the fact that exogenous H2O2 in normoxia can induce preconditioning-like protection in the intact heart (6). Moreover, SOD inhibition, which increases O2bullet– generation (i.e., DHE oxidation) relative to H2O2 formation (i.e., H2DCF oxidation), abolishes the protective effect of hypoxic preconditioning in cardiomyocytes (50). We believe that the loaded H2DCF is probably reporting the generation of H2O2, formed at both sides of the mitochondrial inner membrane by the spontaneous and SOD-linked dismutation of the released O2bullet–. Therefore, H2O2 has presumably a sufficient half life in vivo to play a signaling role during heart preconditioning.

From our results, it seems to be clear that ROS, most likely H2O2, are produced during diazoxide treatment, associated with a transient change in the mitochondrial redox poise, i.e., an oxidation of both the NAD(P) and Fp pools. These findings suggest an interaction between the drug and mitochondrial electron carrier(s) in accordance with the concept that a mild and regulated impairment of the respiratory chain can lead to cardioprotection (for review see Ref. 12). However, the causal links between diazoxide, electron carrier-dependent ROS production and mitoKATP-dependent K+ movement are not entirely elucidated to date. For instance, several in vitro studies (13, 15) pointed out the existence of an H2O2-independent, but complex III-dependent oxidation of H2DCF in isolated heart mitochondria treated with diazoxide, whereas others (3) showed a H2O2- and complex I-dependent oxidation of H2DCF in the presence of either diazoxide or low concentrations of the K+ ionophore valinomycin. Marban et al. (5) proposed an interaction between the complex II and KATP channels in heart mitochondria, whereas other authors (20) postulated that complex II is the primary target of diazoxide during cardioprotection. In this context, we have previously demonstrated in vivo that diazoxide treatment induces a transient and reversible inhibition of the latter complex (39). Unfortunately, the use of optic fibers to study Fp fluorescence does not allow us to discriminate between the different mitochondrial Fp pools (27). Therefore, we were unable to determine which flavin enzyme(s) were specifically oxidized during diazoxide treatment. Nevertheless, in isolated rat hearts, the complex I inhibition, mediated by a cardioprotective drug (i.e., the amobarbital), has been shown to induce an O2bullet– production, probed by DHE, without any change in the Fp oxidation state (1). In contrast, the complex II inhibition, mediated by another cardioprotective drug (i.e., the 3-nitropropionate; see Ref. 36), induces both a Fp oxidation and an H2DCF oxidation (Pasdois P, et al., unpublished data). Taking together the literature data and ours, it is reasonable to hypothesize that the primary effect of diazoxide treatment is a partial inhibition of the respiratory chain (as elicited by the diazoxide modifications of the mitochondrial redox poise), thus inducing oxidant generation and, in turn, stimulating the mitoKATP-signaling pathway.

Regarding the mechanism of 5HD inhibition, a direct effect of this compound on the fluorescent dye is likely to be excluded under our experimental conditions. However, taking into account the reducing power of 5HD discussed above, we can hypothesize that 5HD abrogates the diazoxide-induced H2DCF oxidation through its ability to reduce the flavin-containing electron carrier(s), which are oxidized during diazoxide treatment.

Cardioprotection and ROS Generation During Ischemia and Reperfusion

It was originally proposed that a "burst" of ROS generation occurs when molecular O2 is returned to ischemic cells. However, in vitro studies (8, 14) carried out on cultured cardiomyocytes revealed that both O2bullet– and H2O2 production, probed by DHE and H2DCFDA, respectively, increase during the simulated ischemia, despite the lowered in situ O2 levels. The present study, which used the same fluorescent dyes, confirms and extends this concept to the intact beating heart. Indeed, DCF and ethidium-DNA signals strongly suggest that H2O2 production occurs during the index ischemia and upon reperfusion (see Fig. 7), whereas O2bullet– accumulates mostly during the ischemic period (Fig. 8; see Fig. 4 in Ref. 24). These data strengthen the idea that, in the isolated heart, ischemia is accompanied by a progressive tissue hypoxia, creating an opportunity for significant ROS generation. Moreover, we demonstrated that diazoxide preconditioning significantly attenuated the oxidant generation during both the ischemic and reperfusion periods (Figs. 7D and 8B). These results were in accordance with the cardioprotective effect obtained after diazoxide preconditioning (Figs. 3 and 4).

Previous studies (14) have identified the ubisemiquinone sites of the respiratory chain complexes I and III as the primary sources of ROS in isolated heart mitochondria (46) and in cardiomyocytes. Furthermore, in isolated mitochondria, ROS generation can be supported by the flavin mononucleotide group of complex I (17, 30) and by the flavin adenine dinucleotide group of {alpha}-lipoamide dehydrogenase (47). Moreover, the purified succinate dehydrogenase is also capable of flavin-dependent superoxide production in the absence of electron acceptor (54). All these enzymes may participate in the overall oxidation of the flavin groups observed during ischemia and, in turn, to the oxidation of the H2DCF and DHE dyes. The mechanisms by which diazoxide preconditioning may prevent this phenomenon during ischemia may rely on the fact that some enzyme complexes (e.g., complex I, pyruvate dehydrogenase, and {alpha}-ketoglutarate dehydrogenase complexes) are strongly regulated by calcium- and redox-dependent covalent modifications (4, 12, 32, 34, 42).

Limitations of the Study

Noninvasive fluorescence measurements of Fp and DCF fluorescence at the surface of the left ventricular wall by means of optic fibers are likely limited to events occurring in the epicardium. It is well known that the epicardium is a very different environment than the endocardium and mid-myocardium during ischemia and that the outermost surface is usually spared from necrosis in isolated-perfused rat hearts, even after 30 min of zero-flow global ischemia. It is therefore likely that the alterations in Fp, NAD(P)H, ethidium-DNA, and DCF fluorescence observed during the ischemic phase were underestimated compared with what is actually occurring in the endocardium. Moreover, our method of continuous fluorescence measurements in the intact heart permits the study ROS formation. Nevertheless, the dye specificity and the quantitative aspect of the measurements may be somehow questioned in the light of the in vitro data. For instance, whereas dihydroethidine oxidation is relatively specific to anion superoxide (Refs. 9, 51; see for review Ref. 49), H2O2 at very high dose (i.e., 100 mM) has been shown to quench the ethidium-DNA fluorescence (51). Although H2DCF has been described to be relatively specific to H2O2, it can be oxidized by heme-containing proteins (for review, see Ref. 49), thus raising the possibility of a direct oxidation by respiratory chain components (13).

In conclusion, this study shows, for the first time in intact hearts, an induction of ROS production and Fp oxidation in a 5HD-sensitive manner by diazoxide. Moreover, it shows a transient and moderate ROS generation during the preconditioning phase, likely consecutive to mitoKATP activation, followed by a decrease in ROS production during both the ischemic and reperfusion phases, thus eliciting the final cardioprotection. These in vivo data strengthen the concept that ROS generation is an essential component of the signaling pathway activated during diazoxide preconditioning.


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During this work, P. Pasdois was supported by a grant from Inserm and the Conseil Régional d'Aquitaine. This work was also supported by a grant from Fondation pour la Recherche Médicale to P. Dos Santos.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Dos Santos, Inserm U828, Ave. du Haut Lévêque, 33600 Pessac, France (e-mail: pierre.dossantos{at}wanadoo.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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