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(Circulation Research. 2001;88:802.)
© 2001 American Heart Association, Inc.

Cellular Biology

Diazoxide-Induced Cardioprotection Requires Signaling Through a Redox-Sensitive Mechanism

Robert A. Forbes, Charles Steenbergen, Elizabeth Murphy

From the Laboratory of Signal Transduction (R.A.F., E.M.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, and the Department of Pathology, Duke University Medical Center (C.S.), Durham, NC.

Correspondence to Elizabeth Murphy, PhD, NIEHS, National Institutes of Health, Laboratory of Signal Transduction, MD 2-03, 111 T.W. Alexander Dr, Research Triangle Park, NC 27709. E-mail murphy1{at}niehs.nih.gov

	Abstract

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Abstract—Diazoxide, a selective opener of the mitochondrial ATP-sensitive potassium channel, has been shown to elicit tolerance to ischemia in cardiac myocytes and in perfused heart. However, the mechanism of this cardioprotection is poorly understood. Because reactive oxygen species (ROS) are recognized as important intracellular signaling molecules and have been implicated in ischemic preconditioning, we examined diazoxide-induced ROS production in adult cardiomyocytes. Cells treated with 50 µmol/L diazoxide showed a 173% increase in ROS production relative to baseline. 5-Hydroxydecanoate was found to attenuate the diazoxide-induced increase in ROS generation. The diazoxide-induced increase in ROS also was abrogated by the addition of either the antioxidant N-acetylcysteine (NAC) or N-mercaptopropionylglycine. We also examined the ability of NAC to block the protective effects of diazoxide in the perfused rat heart. After 20 minutes of global ischemia and 20 minutes of reflow, hearts perfused with 100 µmol/L diazoxide before ischemia showed significantly improved postischemic contractile function relative to untreated hearts (84% versus 29% of initial left ventricular developed pressure, respectively). Hearts treated with diazoxide in the presence of 4 mmol/L NAC recovered 53% of initial left ventricular developed pressure, whereas hearts treated with NAC alone recovered 46% of preischemic function. Using ³¹P NMR spectroscopy, we found that, similar to preconditioning, diazoxide significantly attenuated ischemia-induced intracellular acidification and enhanced post- ischemic recovery of phosphocreatine levels, both of which were blocked by cotreatment with NAC. These data suggest that the cardioprotective actions of diazoxide are mediated by generation of a pro-oxidant environment.

Key Words: reactive oxygen species • cardioprotection • intracellular pH • dichlorofluorescin diacetate

	Introduction

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Ischemic preconditioning is a phenomenon whereby brief intermittent periods of ischemia are paradoxically protective against subsequent ischemic injury.¹ Although the molecular basis of this endogenous protective mechanism remains elusive, several key components have been identified. Among these, the ATP-sensitive potassium (K⁺_ATP) channel has been demonstrated to be an important component in perfused heart² and cellular³ models of ischemic preconditioning. Early studies suggested that the cardioprotective effects of K⁺_ATP channel agonists were mediated through the sarcolemmal channel.^{4 5 6} However, the following findings all support a mitochondrial site of action: (1) the evidence that the K⁺_ATP channel agonist diazoxide is 2000-fold more potent at the mitochondrial channel than at the surface channel⁷ ; (2) subsequent evidence demonstrating that diazoxide is cardioprotective in cellular models in which the concentration of diazoxide is sufficient to activate mitochondrial channels without activating sarcolemmal channels² ; and (3) the finding that this effect can be blocked with a potent mitochondrial K⁺_ATP channel antagonist, 5-hydroxydecanoate (5-HD). Nevertheless, a role for the sarcolemmal K⁺_ATP channel has not been totally dismissed.⁸

The mechanism by which diazoxide induces cardioprotection is not clear. As preconditioning has been reported to be mediated via reactive oxygen species (ROS), we considered that diazoxide might alter ROS generation.^{9 10 11 12} It is difficult to predict what effect diazoxide might have on ROS production. A net transient increase in intramitochondrial K⁺ through K⁺_ATP channel activation has been shown to trigger mitochondrial swelling⁷ ; enhanced respiration; and augmented electron flux through the q cycle,¹³ which is suggested to be a major contributor to ROS generation.¹⁴ Boveris and Chance¹⁵ showed that a fully oxidized electron transport chain, as occurs with mild to moderate depolarization, was associated with reduced ROS generation. However, the effects of diazoxide are likely to be complex, and opening of a mitochondrial K⁺_ATP channel could differentially affect different sites of electron transport or could alter NADH levels and thereby alter nonmitochondrial generation of ROS.¹⁶ Pain et al¹⁷ recently reported that the diazoxide-induced reduction in infarct size is attenuated by addition of antioxidants. However, diazoxide-induced generation of ROS was not measured. These findings are in agreement with ours, as well as those of Obata and Yamanaka,¹⁸ who showed that K⁺_ATP channel agonists that activate both mitochondrial and sarcolemmal channels induce hydroxyl radical formation. One goal of our study is to determine whether agonists selective for the mitochondrial K⁺_ATP channel enhance ROS production in cardiac myocytes.

Although ROS have not traditionally been viewed as "signaling" molecules, alteration in cellular redox status, by slight imbalances in ROS generation and elimination, is now recognized as an important mediator of various cellular functions. Fluctuations in oxidant/antioxidant balance are known to influence the activation of transcription factors nuclear factor-B, activator protein-1, and hypoxia-inducible factor-1^{19 20} ; protein kinases and phosphatases^{21 22 23} ; Ca²⁺, K⁺, and Na⁺ channels^{24 25 26 27} ; and Na⁺-Ca²⁺ exchanger,²⁸ as well as GAPDH and pyruvate dehydrogenase.²⁹ ROS generated during preconditioning cycles of ischemia/reperfusion have been implicated in the triggering of the observed cardioprotective effects. Studies in the in situ heart, isolated perfused heart, and cardiomyocytes have demonstrated that brief exposure to H₂O₂ and/or superoxide (generated from hypoxanthine/xanthine oxidase) mimics the protective effects of preconditioning.^{9 10 11} Similarly, antioxidants have been shown to block the induction of preconditioning.^{11 12} This ability of ROS to trigger cardioprotection is proposed to be mediated by a slight to moderate increase in ROS and contrasts with the deleterious effects of a large bolus of ROS.

In the present study, we tested the hypothesis that diazoxide-induced cardioprotection is mediated through an increase in ROS production, by directly measuring ROS production in cardiac myocytes using a fluorescent indicator. We examined the ability of the K⁺_ATP channel agonists diazoxide and pinacidil to promote a pro-oxidant cellular environment in adult cardiomyocytes, as well as the K⁺_ATP channel antagonist 5-HD and antioxidants to block this effect. In addition, we have used the antioxidant N-acetylcysteine (NAC) to block the diazoxide-induced enhancement of postischemic recovery of function in the perfused rat heart. Also, to assess the effects of diazoxide treatment on cellular energetics, we used phosphorus NMR spectroscopy to examine high-energy phosphate levels and pH_i.

	Materials and Methods

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Isolated Rat Heart Preparation
Treatment of animals conformed with The Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1985). Male Sprague-Dawley rats weighing 200 to 250 g were anesthetized with pentobarbital. Animals were heparinized (200 U IV), and hearts were rapidly excised, cannulated, and perfused with Krebs-Henseleit buffer using a Langendorff perfusion model at a perfusion pressure of 100 cm H₂O. Krebs-Henseleit buffer contained (in mmol/L) NaCl 120, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 1.25, NaHCO₃ 25, and glucose 11. The buffer was continuously gassed with 95% O₂ and 5% CO₂ to a pH of 7.35 to 7.40 and maintained at a temperature of 37±0.5°C. A water-filled latex balloon, connected to a pressure transducer, was placed into the left ventricle through a left atriotomy and inflated to set the end-diastolic pressure to 5 cm H₂O. Left ventricular developed pressure (LVDP) was calculated as the difference between the peak-systolic and the end-diastolic pressure. Coronary flow was measured by timed collection of effluent. Hearts were allowed to equilibrate for 30 minutes before collection of baseline data.

Measurement of Intracellular ROS Generation
Adult rat cardiac myocytes were isolated as previously described³⁰ with minor modifications. Briefly, cannulated hearts were perfused with Krebs-Henseleit buffer followed by calcium-free Krebs-Henseleit buffer for 5 minutes and finally calcium-free MEM (Joklik-modified), pH 7.4, with 0.1% collagenase (Sigma, type I), 0.03% hyaluronidase, 0.1% BSA, 20 mmol/L taurine, and 20 mmol/L creatine (buffer A) for 10 minutes. Ventricles were removed, minced, and shaken gently at 34°C in buffer A containing 50 µmol/L calcium and 2% BSA for 5 minutes followed by removal and dilution (1:3) of the cell suspension with MEM (Joklik-modified) containing 100 µmol/L calcium and 4% BSA. This digestion was repeated 4 times. Cells were then pelleted by centrifugation at 50g for 3 minutes at room temperature and resuspended in Joklik-modified MEM containing 1.25 mmol/L calcium. Freshly isolated cells were loaded with 2',7'-dichlorofluorescin diacetate (DCFH-DA, 10 µmol/L) for 1 hour at room temperature. Once inside the cell, the acetate group on DCFH-DA is cleaved by esterases yielding a polar, nonfluorescent product, DCFH, that accumulates intracellularly.³¹ Oxidation by ROS yields the fluorescent product dichlorofluorescein (DCF). After loading, cells were washed 3 times in Joklik-modified MEM with 1.25 mmol/L calcium by gentle resuspension and gravity sedimentation to remove extracellular DCFH-DA. Freshly isolated adult rat myocytes were placed in a medium-containing custom-built chamber that was placed on the stage of a Nikon inverted epifluorescence microscope coupled to a Photon Technology International photomultiplier tube and a Delta- scan dual-wavelength excitation spectrofluorometer. DCF fluorescence in single, rod-shaped cells was recorded at a rate of 1 point/second using Felix software and viewed at x200 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. Entrance and exit slit widths were set to 0.6 mm, corresponding to a 2.4-nm bandpass. Either vehicle (control) (n=18), diazoxide (n=18), pinacidil (n=7), diazoxide plus 5-HD (n=7), diazoxide plus NAC (n=7), or diazoxide plus N-mercaptopropionylglycine (MPG; n=7) was added to the recording chamber after 200 seconds (3.33 minutes) of baseline measurement. In an additional control group, cells were treated with NAC alone (n=7) to assess its effects on DCFH oxidation. Fluorescence was monitored in treated and untreated cells for 1000 seconds (16.67 minutes). Cells loaded with DCFH-DA exhibited fluorescence that was 3 to 4 times that of autofluorescence. Autofluorescence is subtracted from all DCFH-loaded cells. Data are expressed as the rate of increase in fluorescence after treatment minus the initial (pretreatment) rate of increase in fluorescence. Posttreatment measurement of the rate of fluorescence increase was made starting 1 minute after addition. Fluorescence changes were calculated as a percentage of initial fluorescence (time 0).

Perfused Heart Protocols
After hearts were excised and cannulated, they were allowed to stabilize for a period of 30 minutes before treatment. Four groups of hearts were used for ³¹P NMR and for LVDP measurements. The treatment period consisted of perfusion with either Krebs-Henseleit buffer alone (control) (n=7), 100 µmol/L diazoxide (n=9), 100 µmol/L diazoxide+4 mmol/L NAC (n=8), or 4 mmol/L NAC alone (n=7) for a period of 10 minutes. After the treatment period, hearts were subjected to 20 minutes of global ischemia followed by 20 minutes of reperfusion. There was no diazoxide or NAC in the buffer during reperfusion. Percentage recovery is expressed as LVDP after 20 minutes of reperfusion relative to pretreatment values. An additional 4 groups of perfused hearts were snap-frozen for analysis of glycogen and glucose-6-phosphate (G6P) content at the end of the treatment period and at the end of 20 minutes of ischemia. Glycogen and G6P were measured as described previously.³²

³¹P Nuclear Magnetic Resonance
Rat hearts were placed in a 20-mm NMR tube and continuously perfused in Langendorff mode with phosphate-free Krebs-Henseleit buffer. Temperature was maintained at 37±0.5°C. ³¹P NMR spectra were obtained at 161.92 MHz using a Varian (Unity Plus) wide-bore 400-MHz spectrometer. The proton signal from the heart was used for shimming, with a routine line width at one-half height of 0.2 ppm. Serial 5-minute spectra were acquired with a spectral width of ±3420 Hz and with 4000 data points/spectrum. The free-induction decay was multiplied by an exponential function corresponding to 20-Hz line broadening before Fourier transformation. pH_i was determined from the chemical shift difference between the inorganic phosphate and phosphocreatine (PCr) peaks.³³

Statistical Analysis
Values are expressed as mean±SEM. ANOVA, followed by Fisher’s post hoc test, was performed to identify significant differences. P<0.05 was considered significant.

Materials
DCFH-DA was purchased from Molecular Probes and prepared freshly in DMSO. Other chemicals were purchased from Sigma Chemical Co. Diazoxide and pinacidil were dissolved in DMSO before addition into experimental buffers. NAC, MPG, and 5-HD were dissolved in Krebs-Henseleit buffer.

	Results

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Diazoxide Stimulates an Increase in ROS Generation in Adult Cardiac Myocytes
To examine the effects of diazoxide on ROS generation by cardiac myocytes, we examined single-cell fluorescence of DCFH-loaded cells. Figure 1A shows the time-dependent changes in fluorescence in untreated and diazoxide-treated myocytes loaded with DCFH and in a representative trace of a diazoxide-treated myocyte without DCFH loading. Autofluorescence was also measured in the presence of NAC, MPG, or 5-HD, with no observed effect (data not shown). Figure 1B shows data compiled from multiple experiments showing treatment with either vehicle (control) (n=18), diazoxide (n=18), pinacidil (n=7), diazoxide+5-HD (n=7), diazoxide+NAC (n=7), diazoxide+MPG (n=7), or NAC alone (n=7). Diazoxide treatment (50 µmol/L) rapidly increased the rate of DCFH oxidation relative to baseline by 173%, indicative of an increase in ROS production. The basal rate was a 2.04% increase in fluorescence per minute, and with the addition of diazoxide, the rate increased to a 5.57% increase in fluorescence per minute. Thus, the difference in the rate was a 3.53% increase in fluorescence per minute, or 173% above baseline. Pinacidil (10 µmol/L) similarly increased the rate of DCFH oxidation. In control cells, DCFH oxidation increased by 17% over the same time period. The increase in DCFH oxidation with either diazoxide or pinacidil was significantly higher than the control increase. In the presence of 100 µmol/L 5-HD, addition of diazoxide resulted in a DCFH fluorescence increase of 56% above the basal rate, a value that was not significantly different from the control value but was significantly less than that for diazoxide alone. Furthermore, we show in Figure 1B that ROS generation subsequent to diazoxide exposure is attenuated by cotreatment with 1 mmol/L NAC or 300 µmol/L MPG, which reduces ROS production to less than baseline. Likewise, with addition of NAC alone, the rate of DCFH oxidation was less than in control cells. The baseline rate of DCFH oxidation in control cells is likely due to constitutive production of ROS. There were no significant differences among control, NAC alone, diazoxide+NAC, diazoxide+MPG, and diazoxide+5-HD.

View larger version (22K): [in this window] [in a new window]   Figure 1. A, Time-dependent fluorescence changes in untreated and diazoxide-treated DCFH-DA–loaded cells and in diazoxide (DZX)–treated cells not loaded with DCFH-DA (autofluorescence). B, Rate of increase in fluorescence in DCFH-DA–loaded cardiac myocytes after addition of vehicle (control), diazoxide, pinacidil, diazoxide+NAC, diazoxide+MPG, diazoxide+5-HD, and NAC. Fluorescence is measured as percentage of initial fluorescence, and rates of increase are expressed as percentage change per minute. Baseline rates of fluorescence increase were determined for 2 minutes for each individual cell, after which the addition was made. After 1 minute, the rate of fluorescence increase was determined during the next 2 minutes, and the initial baseline rate was subtracted from the rate measured after addition, to determine the rate of fluorescence increase attributable to the treatment. Values are mean±SEM. *Significantly different from control (P<0.05); #Significantly different from diazoxide (P<0.05).

Preischemic and Postischemic Hemodynamics
To investigate the effects of diazoxide and NAC on LVDP and energetics, we also studied Langendorff-perfused hearts. As indicated in the Table, there were no significant differences in LVDP, coronary flow, or heart rate after treatment. Furthermore, postischemic heart rate and coronary flow rate were similar in each of the 4 treatment groups.

View this table: [in this window] [in a new window]   Table 1. Pre- and Postischemic Hemodynamic Properties

Diazoxide-Induced Improvement in Postischemic Contractile Function Is Blocked by NAC
Figure 2 shows that a 10-minute treatment of perfused rat heart with 100 µmol/L diazoxide before 20 minutes of sustained ischemia significantly improved postischemic contractile function. Diazoxide-treated hearts recovered 84±2% (n=9) of their preischemic contractile function (LVDP) compared with control hearts, which recovered 29±6% (P<0.05) (n=7). If diazoxide-induced cardioprotection is mediated through an increased production of ROS, then the antioxidant NAC should inhibit the diazoxide-induced improvement in postischemic contractile function. Postischemic recovery of LVDP in hearts treated with 100 µmol/L diazoxide and 4 mmol/L NAC (53±5%, n=8) was significantly lower than in hearts treated with diazoxide alone (P<0.05) and was virtually identical to that in hearts treated with NAC alone (48±3%, n=7).

View larger version (25K): [in this window] [in a new window]   Figure 2. Recovery of LVDP after 20 minutes of sustained ischemia and 20 minutes of reperfusion, as percentage of LVDP in the pretreatment baseline period. Values are mean±SEM. *P<0.05 in comparison with untreated hearts; #P<0.05 in comparison with diazoxide (DZX)–treated hearts.

Diazoxide-Triggered PCr Overshoot Is Blocked by NAC
The effects of K⁺_ATP channel agonists on mitochondrial membrane potential are debated.^{34 35} Therefore, it was of interest to determine whether diazoxide treatment had any effect on ATP or PCr levels in intact myocardium. Using ³¹P NMR spectroscopy, we examined the effects of 100 µmol/L diazoxide treatment on ATP and PCr levels in the perfused heart. As shown in Figures 3 and 4, addition of diazoxide for 10 minutes during the treatment period before ischemia had no effect on steady-state ATP or PCr content. Furthermore, NAC treatment alone did not significantly affect ATP or PCr levels. There were no significant differences among any of the groups in the decline of ATP or PCr during the 20-minute period of ischemia. After 20 minutes of reperfusion, ATP content was similar among treatment groups. Elevated postischemic PCr levels have been observed in the preconditioned heart.³⁶ Preischemic treatment with diazoxide resulted in a significant enhancement of postischemic PCr levels (Figure 4). Furthermore, this "PCr overshoot" was abolished by NAC cotreatment (Figure 4).

View larger version (20K): [in this window] [in a new window]   Figure 3. Time course of changes in ATP content (percentage of initial pretreatment value) during pretreatment and treatment periods, 20 minutes of ischemia, and 20 minutes of reperfusion. Values are mean±SEM. DZX indicates diazoxide.

View larger version (20K): [in this window] [in a new window]   Figure 4. Time course of PCr levels (percentage of initial pretreatment value) during treatment period, 20 minutes of ischemia, and 20 minutes of reperfusion. ANOVA indicated significant differences. Values are mean±SEM. *P<0.05 in comparison with untreated hearts. DZX indicates diazoxide.

Diazoxide-Attenuated Ischemic Acidification Is Blocked by NAC
Ischemia is known to promote intracellular acidification, and preconditioning has been shown by several groups to attenuate this fall in pH_i.^{36 37} As shown in Figure 5, in the untreated (control) heart, the pH_i fell to 6.20±0.07 during the course of 20 minutes of ischemia. Interestingly, similar to what has been observed in preconditioned hearts, diazoxide treatment resulted in a significant attenuation in acidification during ischemia, with a decline in pH_i to 6.42 ± 0.05 (P<0.05 versus control). NAC inhibited the diazoxide-induced decrease in acid production; during ischemia, pH_i fell to 6.23 ± 0.06. Treatment of hearts with NAC alone resulted in a fall in pH_i that was not significantly different from that of control (6.33±0.09). Lactate measurements correlated with these findings (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 5. Time course of changes in pHi during pretreatment and treatment period, 20 minutes of ischemia, and 20 minutes of reperfusion. ANOVA indicated significant differences. Values are mean±SEM. *P<0.05 in comparison with untreated hearts. DZX indicates diazoxide.

Diazoxide Treatment Significantly Lowers Preischemic Glycogen Content
The reduction in intracellular acidification during ischemia in diazoxide-treated hearts could be due to a diazoxide-induced decrease in glycogen content or inhibition of glycolysis. To investigate this possibility, hearts were snap-frozen after treatment (before ischemia) and after 20 minutes of ischemia. The glycogen measurements (Figure 6A) show that diazoxide caused a significant decrease in preischemic glycogen levels relative to control hearts. Diazoxide treatment for 10 minutes significantly decreased glycogen content from a control value of 81 to 64 µmol/g dry weight. This diazoxide-induced decline in glycogen was blocked by addition of NAC. Addition of NAC alone had no effect on glycogen levels. After 20 minutes of ischemia, glycogen fell to similar levels in all groups. If diazoxide-generated ROS were resulting in an inhibition of glycolysis, one might expect an increase in G6P in diazoxide versus control hearts; however, there was no significant difference (see Figure 6B). Furthermore, at the end of ischemia, diazoxide-treated hearts had a significantly lower G6P content compared with control (0.89 versus 2.45 µmol/g, respectively), which is consistent with preischemic glycogen depletion in diazoxide-treated hearts. Thus, the differences in intracellular acidification during ischemia can be attributed to differences in glycogen levels before ischemia and are not indicative of glycolytic inhibition.

View larger version (33K): [in this window] [in a new window]   Figure 6. Glycogen content (A) and G6P content (B) in hearts snap-frozen after treatment with Krebs-Henseleit buffer alone (control), diazoxide (DZX), diazoxide+NAC, or NAC alone, or treatment followed by ischemia (+Isch). n=5 per group; *P<0.05 in comparison with untreated hearts (A); #P<0.05 in comparison with control ischemic hearts (B).

	Discussion

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Diazoxide-Mediated Protection Requires a Pro-Oxidant Environment
Change in redox state is proposed as a potent and selective cellular signal, influencing cell death as well as survival.^{38 39} Indeed, ROS have been implicated in preconditioning.^{9 10 11 12} Supporting a role for ROS in triggering protection, we found that diazoxide and pinacidil significantly enhance the production of ROS in adult cardiac myocytes and that this production is markedly attenuated by 5-HD, as well as by antioxidants. The data in our study are consistent with recent findings by Pain et al,¹⁷ who showed that antioxidants block the diazoxide-induced reduction in infarct size when administered during diazoxide treatment. Our study is also consistent with that of Obata and Yamanaka,¹⁸ who showed that K⁺_ATP channel agonists increase hydroxyl radical production. Furthermore, our study is consistent with previous work^{9 10 12} suggesting that a pro-oxidant environment before ischemia is important for cardioprotection such as occurs with ischemic preconditioning.

Although it is well established that high levels of ROS are detrimental,⁴⁰ moderate levels of H₂O₂ and ·O₂^- have been shown to elicit a cardioprotective effect similar to that observed with ischemic preconditioning.^{9 10} Cellular antioxidant capacity is observed to fall during preconditioning, as total tissue glutathione levels drop to below 70% of initial preischemic values,¹² suggesting thiol oxidation in the course of ischemic preconditioning. Furthermore, promotion of a reducing environment, as exemplified by the administration of NAC, a glutathione precursor that buffers the cellular antioxidant capacity and maintains glutathione levels, is effective at inhibiting ischemic preconditioning¹² ; additionally, as demonstrated in this study, NAC also blocked the protection afforded by diazoxide.

There is some controversy regarding the relationship between opening of K⁺_ATP channels and generation of ROS. Our data show that addition of diazoxide or pinacidil results in an increase in ROS, which is blocked by coadministration of 5-HD or antioxidants. These data are in agreement with those of Obata and Yamanaka,¹⁸ who reported that addition of the K⁺_ATP channel openers cromakalin and nicorandil to a perfused rat heart resulted in an increase in ROS that was blocked by 5-HD. Our diazoxide data indicate that opening of mitochondrial K⁺_ATP channels can increase ROS generation; additionally, the data of Obata and Yamanaka¹⁸ are consistent with this finding, although they used nonselective K⁺_ATP channel openers that activate both sarcolemmal and mitochondrial channels. Obata and Yamanaka¹⁸ also examined the rate of ROS generation after ischemia and found that K⁺_ATP channel blockers attenuated ROS generation during reperfusion, suggesting that opening of the K⁺_ATP channel is a major mechanism responsible for ROS generation.¹⁸ This conclusion contrasts with the data of Vanden Hoek et al,¹⁶ in neonatal chick myocytes, which showed that pinacidil, given at the onset of reperfusion, caused a reduction in ROS generation on reperfusion, which was blocked by 5-HD. The differences might be explained by a difference in experimental model, as Vanden Hoek et al¹⁶ used neonatal chick myocytes and Obata and Yamanaka¹⁸ used adult rat heart, as well as a difference in timing of treatment. Thus, the data are conflicting as to whether K⁺_ATP channel opening during reperfusion after ischemia enhances or diminishes ROS generation, but the direct measurements of ROS generation (this study and that of Obata and Yamanaka¹⁸ ), as well as the findings of Pain et al,¹⁷ suggest that mitochondrial K⁺_ATP channel openers lead to ROS generation in the absence of ischemia and that this is an important component of the protective effect elicited by these agents.

A recent study by Pain et al¹⁷ found that diazoxide is protective even if it is only present before, rather than during, the sustained period of ischemia, suggesting that diazoxide acts as a trigger of preconditioning. 5-HD added 5 minutes before sustained ischemia and after washout of diazoxide did not block the protective effects of diazoxide, although 5-HD or the antioxidant MPG added concurrently with diazoxide did block the protection. These data suggest that diazoxide acts as a trigger to mediate preconditioning and that opening of the K⁺_ATP channel during the sustained period of ischemia is not required for the protective effect. However, there are data to the contrary,⁴¹ and it is possible that opening of a K⁺_ATP channel may be involved as both a trigger and a mediator (see Fryer et al⁴¹ ). ROS have been previously suggested to act as a trigger of preconditioning, given that the ROS can be washed away before sustained ischemia without attenuation of the cardioprotection.^{9 10} Thus, the diazoxide-induced generation of ROS is likely to be a trigger for preconditioning, but this does not preclude an additional role for the mitochondrial K⁺_ATP channel as a mediator of preconditioning.

Effects of Diazoxide on ATP and PCr
Studies by Halestrap¹³ demonstrate that transient inward mitochondrial K⁺ flux is sufficient to trigger an increase in intramitochondrial volume, which is proposed to play a role in the regulation of mitochondrial metabolism, including activation of pyruvate decarboxylation, respiration, and ATP production. Opening the mitochondrial K⁺_ATP channel has been reported to slightly uncouple mitochondria, resulting in a decreased maximum rate of ATP synthesis in isolated mitochondria.³⁴ However, Fryer et al⁴¹ demonstrated that 5-HD, a selective mitochondrial K⁺_ATP channel antagonist, attenuated the ischemic preconditioning–induced preservation of mitochondrial ATP synthesis capability. Thus, it is unclear what effect diazoxide might have on ATP levels in intact myocardium. We found that ATP levels in diazoxide-treated hearts are similar to those observed in control hearts and that ATP levels remain similar throughout the course of global ischemia and through reperfusion. Our findings are consistent with those of Fryer et al,⁴¹ who found that the rate of ATP synthesis in isolated mitochondria from diazoxide-treated hearts and ischemic/reperfused hearts are similar.

Interestingly, in terms of cellular energetics, we have found that in diazoxide-treated hearts, postischemic PCr levels rebounded to levels significantly higher than in untreated (control) hearts. This finding is consistent with a protective effect of diazoxide. The fact that NAC cotreatment attenuates this "PCr overshoot" is consistent with the hypothesis that a pro-oxidant environment can promote the preservation of cellular energetics, as is suggested in the preconditioned heart.

Attenuation of Ischemic Acidification by Diazoxide
A reduction in intracellular acidification during sustained ischemia has been consistently demonstrated after ischemic preconditioning.^{36 37 42 43} Diazoxide is also shown to significantly reduce intracellular acidification during ischemia. Attenuation of the decline in pH_i during ischemia has been attributed to (1) inhibition of H⁺ production secondary to a reduction in glycolysis or glycogenolysis⁴³ or, alternatively, to preischemic glycogen depletion, and (2) increased H⁺ extrusion to the extracellular space, lowering pHo but attenuating the intracellular acidification. However, arguing against the hypothesis that preconditioning enhances H⁺ extrusion, Gabel et al⁴² found that preconditioning attenuated the fall in both pHi and pHo. It is possible that ROS generation with diazoxide may inhibit glycolysis through inhibition of GAPDH, which has a thiol in its active site,²⁹ thereby reducing acid production. If diazoxide resulted in inhibition of glycolysis via inhibition of GAPDH, one would expect an elevation of G6P, a glycolytic intermediate proximal to GAPDH. However, the increase in G6P during ischemia is less in diazoxide-treated hearts than in untreated ischemic hearts. In diazoxide-pretreated hearts, preischemic glycogen levels are shown to be significantly lower than control, suggesting that diazoxide stimulates glycogen utilization or decreases glycogen synthesis. The decreased G6P levels in the diazoxide-treated ischemic hearts are consistent with glycogen depletion at the onset of ischemia, which limits glycogenolysis during ischemia. Thus, the data suggest that a diazoxide-induced decrease in glycogen is responsible for the decrease in acid production during ischemia.

In summary, we find that diazoxide promotes the generation of ROS in cardiac myocytes, and this is blocked by 5-HD or antioxidants. We have shown that diazoxide does not significantly interfere with energy metabolism before ischemia, and, consistent with the protective effect of diazoxide, we find improved recovery of PCr on reperfusion in diazoxide-treated hearts. We find also that NAC is effective in blocking the diazoxide-induced improvement in postischemic function and energetics. Thus, we conclude that a pro-oxidant environment created by treatment with diazoxide is required for diazoxide-mediated cardioprotection. Specific signaling targets for redox-mediated changes involved in this protective effect have not been elucidated but may include protein kinase C,²² p38 mitogen-activated protein kinase,²¹ and K⁺_ATP channels.⁴⁴

	Acknowledgments

 
R.A.F. and E.M. were supported by the National Institute of Environmental Health Sciences intramural program. C.S. was supported by NIH Grant RO1-HL-39752.

	Footnotes

 
Original received September 12, 2000; resubmission received January 3, 2001; revised resubmission received February 8, 2001; accepted February 27, 2001.

	References

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