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(Circulation. 1998;97:2463-2469.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Mitochondrial ATP-Dependent Potassium Channels

Novel Effectors of Cardioprotection?

Yongge Liu, PhD; Toshiaki Sato, MD, PhD; Brian O'Rourke, PhD; ; Eduardo Marban, MD, PhD

From the Section of Molecular and Cellular Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, Md. Yongge Liu is now at Maryland Research Laboratories, Otsuka America Pharmaceutical Inc, Rockville, Md.

Correspondence to Eduardo Marban, MD, PhD, Section of Molecular and Cellular Cardiology, Department of Medicine, Johns Hopkins University, Ross 844, 720 Rutland Ave, Baltimore, MD 21205. E-mail marban{at}welchlink.welch.jhu.edu

	Abstract

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Background—Brief interruptions of coronary blood flow paradoxically protect the heart from subsequent prolonged ischemia. The basis of such endogenous cardioprotection, known as "ischemic preconditioning," remains uncertain. Pharmacological evidence has implicated ATP-dependent potassium (K_ATP) channels in the mechanism of preconditioning; however, the effects of sarcolemmal K_ATP channels on excitability cannot account for the protection.

Methods and Results—We simultaneously measured flavoprotein fluorescence, an index of mitochondrial redox state, and sarcolemmal K_ATP currents in intact rabbit ventricular myocytes. Our results show that diazoxide, a K_ATP channel opener, selectively activates mitochondrial K_ATP channels. Diazoxide induced reversible oxidation of flavoproteins with an EC₅₀ of 27 µmol/L but did not activate sarcolemmal K_ATP channels. The subcellular site of diazoxide action is further localized to mitochondria by confocal imaging of fluorescence arising from flavoproteins and tetramethylrhodamine ethyl ester. In a cellular model of simulated ischemia, inclusion of diazoxide decreased the rate of cell death to about half of that in controls. Both the redox changes and protection are inhibited by the K_ATP channel blocker 5-hydroxydecanoic acid.

Conclusions—Our results demonstrate that diazoxide targets mitochondrial but not sarcolemmal K_ATP channels and imply that mitochondrial K_ATP channels may mediate the protection from K_ATP channel openers.

Key Words: ischemia • potassium channels • mitochondria

	Introduction

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Lethal injury to the heart can be dramatically blunted by brief conditioning periods of ischemia. Such ischemic preconditioning¹ exists in all species examined, including humans.² Despite intensive investigation, the mechanism of preconditioning remains poorly understood. K_ATP channel openers mimic and K_ATP channel inhibitors block ischemic preconditioning.³ Opening of sarcolemmal K_ATP channels shortens the action potential and thus depresses contractility⁴: this has been proposed as the mechanism for protection of ischemic myocardium.³ However, recent evidence contradicts this hypothesis. The degree of action potential shortening can be divorced from the extent of protection.^{5 6} Furthermore, K_ATP channel openers and ischemic preconditioning are protective even in unstimulated cardiac myocytes,^{7 8} in which action potential abbreviation cannot be a factor.

Cardiac myocytes have another type of K_ATP channel, in the inner mitochondrial membrane, which responds to many of the same openers and blockers as the sarcolemmal channels (albeit with different potencies).^{9 10 11 12} Although the physiological roles of mitochondrial K_ATP channels in cardiac myocytes remain unclear, opening of any potassium-selective ion channels in the inner mitochondrial membrane would tend to dissipate the membrane potential established by the proton pump.¹³ Such dissipation accelerates electron transfer by the respiratory chain and leads to net oxidation of the mitochondrial matrix. The fluorescence of FAD-linked enzymes can be used to index mitochondrial redox state.^{14 15} Low concentrations (1 to 100 µmol/L) of the K_ATP channel opener diazoxide have been reported to activate mitochondrial K_ATP channels,¹² whereas cardiac sarcolemmal K_ATP channels are quite resistant to this drug.^{12 16} To determine whether diazoxide can selectively open mitochondrial K_ATP channels in intact living cells, we simultaneously measured flavoprotein fluorescence and sarcolemmal K_ATP currents (I_K,ATP) in intact rabbit ventricular myocytes. Our results show that diazoxide selectively activates mitochondrial K_ATP channels. Diazoxide also protects myocytes against simulated ischemia. We propose that mitochondrial K_ATP channels may be the elusive effectors of preconditioning. Recognition of this role for mitochondrial K_ATP channels identifies a promising new target for the development of cardioprotective drugs and implicates the mitochondria in the process of lethal ischemic injury.

	Methods

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The investigation conforms with The Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85–23, revised 1985).

Chemicals
Collagenase (type II) was purchased from Worthington. Diazoxide was obtained from Sigma Chemical Co. Pinacidil and 5-HD were purchased from Research Biochemical Int. TMRE was obtained from Molecular Probes. Diazoxide, pinacidil, and TMRE were dissolved in DMSO before being added into experimental solutions. The final concentration of DMSO was <0.1%.

Electrophysiology and Flavoprotein Fluorescence Measurement
Ventricular myocytes were isolated from adult rabbit hearts by conventional enzymatic dissociation,¹⁷ then washed several times with calcium-free solution. Calcium concentration was gradually brought back to 1 mmol/L. Cells were then cultured on laminin-coated coverslips in M199 culture medium with 5% fetal bovine serum at 37°C. Experiments were performed over the next 2 days. For whole-cell patch recordings, the internal pipette solution contained (in mmol/L) potassium glutamate 120, KCl 25, MgCl₂ 0.5, K-EGTA 10, HEPES 10, and MgATP 1 (pH 7.2 with KOH). The external solution included (in mmol/L) NaCl 140, KCl 5, CaCl₂ 1, MgCl₂ 1, and HEPES 10 (pH 7.4 with NaOH). Whole-cell currents were elicited every 6 seconds from a holding potential of -80 mV by two consecutive steps to -40 mV (for 100 ms) and 0 mV (for 380 ms). Currents at 0 mV were measured 200 ms into the pulse. Endogenous flavoprotein fluorescence was excited with a xenon arc lamp with a band-pass filter centered at 480 nm, but only during the 100-ms step to -40 mV to minimize photobleaching. Emitted fluorescence was recorded at 530 nm by a photomultiplier tube and digitized (Digidata 1200, Axon Instruments).¹⁸ Relative fluorescence was averaged during the excitation window and calibrated with the values after DNP and CN exposure. In some cells, contracture occurred before the fully reduced level (after CN exposure) could be determined. In these cells, data were expressed as a percentage of the DNP-induced fluorescence, because the basal redox state was nearly fully reduced (average 5% oxidation, n=5).

Flavoprotein Fluorescence and Mitochondrial Imaging
Confocal images were obtained with a Diaphot 300 inverted fluorescence microscope with a PCM-2000 confocal scanning attachment (Nikon, Inc). Fluorescence was excited by the 488-nm line of an argon laser, and the emission at 505 to 535 nm was recorded. A time series of images was collected at intervals of 10 seconds, and baseline, diazoxide, DNP, and CN images were enhanced by averaging 8 to 10 sequential images having stable mean fluorescence intensities during the exposure to each agent. To localize mitochondria, cells were loaded with 100 nmol/L TMRE, which distributes into negatively charged cellular compartments,¹⁹ for 10 minutes. TMRE fluorescence was excited with the 535-nm line of a helium neon laser and recorded at >605 nm. A pseudocolor palette was applied to visualize the relative increase in mitochondrial flavoprotein oxidation state. Images were analyzed on a personal computer with the software program ImageTool (University of Texas Health Sciences Center in San Antonio). All the recordings were performed at room temperature (21°C to 22°C).

Simulated Ischemia and Cellular Injury
The procedure to determine cell injury was modified from Vander Heide et al.²⁰ After cell isolation, cells were washed with incubation buffer (in mmol/L): NaCl 119, NaHCO₃ 25, KH₂PO₄ 1.2, KCl 4.8, MgSO₄ 1.2, HEPES 10, and glucose 11 and supplemented with creatine, taurine, and amino acids (pH 7.4). Calcium was added into the buffer stepwise (0.25 mmol/L every 5 minutes) to a final concentration of 1 mmol/L. An aliquot of each cell suspension (0.5 mL) was placed into a 0.5-mL microcentrifuge tube and centrifuged for 20 seconds into a pellet. Each pellet occupied a volume of 0.2 mL. Approximately 0.25 mL of excess supernatant was removed to leave a thin fluid layer above the pellet, and 0.2 mL of mineral oil was layered on the top of the pellet to exclude gaseous diffusion. After 60 minutes and 120 minutes of pelleting, 5 µL of cell pellet was sampled through the oil layer and mixed with 75 µL of 85 mosm hypotonic staining solution (in mmol/L): NaHCO₃ 11.9, KH₂PO₄ 0.4, KCl 2.7, MgSO₄ 0.8, and CaCl₂ 1 and 0.5% glutaraldehyde, 0.5% trypan blue. Microscopic examination was performed 2 to 5 minutes after mixing to determine the permeability of the cells to trypan blue. Cells permeable to trypan blue were counted as dead and expressed as a percentage of the total cells counted (>200 for each sample). The killing of cells by ischemia was quantified as percentage of the vital cells at the beginning of each experiment (78% to 90% of total, mean=82±1%, n=24). The small percentage of cells (18%) that were nonviable at the beginning of the experiment were mostly rounded and had been damaged as a known consequence of the enzymatic isolation process.²¹ Individual experiments in each group were performed on cells isolated from different hearts. Four groups of experiments were performed. In the control group (Cont), cells were pelleted and sampled at 60 minutes and 120 minutes. For the diazoxide-treated group (DIAZO), 50 µmol/L of diazoxide was added to the solution 15 minutes before the pelleting. In the third group (5-HD), 100 µmol/L of 5-HD was added to the cell suspension 20 minutes before pelleting. Cells in the DIAZO+5-HD group were treated the same as in the third group, except that 50 µmol/L of diazoxide was added to the cell suspension 15 minutes before pelleting. Once applied, drugs were not washed out and thus were present throughout the period of simulated ischemia.

Statistical Analysis
Data are presented as mean±SEM, and the number of cells or experiments is shown as n. ANOVA combined with Tukey's honestly significant difference post hoc test was used to test for differences among groups for electrophysiological and fluorescence data. Cell pelleting data were analyzed by two-way ANOVA combined with Tukey's highly significant difference post hoc test. P<.05 was considered significant.

	Results

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Figure 1 shows results from simultaneous measurements of flavoprotein fluorescence and membrane I_K,ATP in cells exposed to diazoxide. The periods of drug treatment are marked with horizontal bars. Diazoxide (100 µmol/L) induced reversible oxidation of the flavoproteins (Figure 1A) but did not activate I_K,ATP (Figure 1B). The redox signal was calibrated by exposing the cells to DNP followed by CN at the end of the experiments. DNP, a protonophore that uncouples respiration from ATP synthesis and collapses the mitochondrial potential, induced maximal oxidation, whereas CN, which inhibits the cytochrome oxidase and thus stops electron transfer, caused complete reduction of the flavoproteins (Figure 1A and 1C). Although membrane currents were unchanged by diazoxide, I_K,ATP eventually turned on after prolonged exposure to DNP (Figure 1B and 1D), indicating that these channels are operable under our experimental conditions despite the inability of diazoxide to open them. Diazoxide 100 µmol/L [DIAZO(1)] reversibly increased mitochondrial oxidation to 48±3% of the DNP value (Figure 1E). This oxidation was reproducible, because after washout of the response, a second exposure to diazoxide [DIAZO(2)] in the same cells increased flavoprotein oxidation to 43±5%. 5-HD 100 µmol/L attenuated the oxidative effect of diazoxide by about half [DIAZO+5-HD(100)], whereas 500 µmol/L 5-HD further reduced oxidation to 8±3% [DIAZO+5-HD(500); P<.01 versus DIAZO(1), DIAZO(2), and DIAZO+5-HD(100) groups]. Treatment with diazoxide and 5-HD did not activate I_K,ATP, whereas prolonged exposure (>6 minutes) to DNP did turn on I_K,ATP (Figure 1F). The EC₅₀ for diazoxide to induce mitochondrial oxidation is 27 µmol/L, as shown in Figure 1G.

View larger version (28K): [in this window] [in a new window]   Figure 1. Diazoxide effect on flavoprotein fluorescence and IK,ATP. A, Diazoxide (DIAZO) 100 µmol/L induced a reversible increase of mitochondrial oxidation. B, Diazoxide did not activate IK,ATP. C, 5-HD 500 µmol/L completely blocked oxidative effect of diazoxide. D, Diazoxide and 5-HD did not activate IK,ATP. E and F, Pooled data for fluorescence and IK,ATP. DIAZO(1) indicates first exposure to diazoxide; DIAZO+5-HD(100), diazoxide in presence of 100 µmol/L 5-HD; DIAZO+5-HD(500), diazoxide in presence of 500 µmol/L 5-HD; DIAZO(2), second exposure to diazoxide; and DNP, exposure to DNP. Bar indicates periods when cells were exposed to drug. G, Dose-response curve for diazoxide. Each point constitutes measurements from 5 to 6 cells. *P<.01 vs DIAZO(1), DIAZO(2), and DNP groups.

We also examined another K_ATP channel opener, pinacidil, which opens sarcolemmal K_ATP channels and is known to induce pharmacological preconditioning.²² As shown in Figure 2, pinacidil 100 µmol/L induced 35±8% mitochondrial oxidation, comparable to the effect of diazoxide exposure in the same cell (41±5%). Unlike diazoxide, pinacidil activated sarcolemmal I_K,ATP (0.74±0.54 nA measured at 0 mV) in addition to inducing flavoprotein oxidation, suggesting that pinacidil activates both mitochondrial and sarcolemmal K_ATP channels.

View larger version (24K): [in this window] [in a new window]   Figure 2. Effect of pinacidil on flavoprotein fluorescence and IK,ATP. A, Flavoprotein fluorescence changes induced by 100 µmol/L diazoxide or pinacidil in representative cell. B, Pinacidil also activates IK,ATP, whereas diazoxide has no effect. C and D, Summarized data for percentage of flavoprotein oxidation and IK,ATP, respectively.

We further localized the subcellular site of diazoxide action by imaging flavoprotein fluorescence (Figure 3A). Fluorescence is low under control conditions, but exposure to diazoxide increased fluorescence in strips parallel to the myofibril orientation. Subsequent exposure to DNP increased fluorescence even further, in a pattern similar to that revealed by diazoxide. CN reduced the fluorescence to the basal level. The distribution of fluorescence induced by diazoxide and DNP is as expected for mitochondria, which occupy 35% of cardiomyocyte volume and are clustered longitudinally between myofibrils.²³ This correspondence was further confirmed by use of TMRE (Figure 3B), which distributes into negatively charged cellular compartments, to localize mitochondria.¹⁹ The pattern of TMRE fluorescence was virtually identical to that of the flavoprotein fluorescence induced by diazoxide.

View larger version (102K): [in this window] [in a new window]   Figure 3. A, Confocal images of a cell at baseline (Control), after 3 minutes' exposure to diazoxide (Diazo.), and after exposure to DNP and CN. B, Confocal image after TMRE loading of same cell.

Figure 4 plots the fraction of cells killed by 60 or 120 minutes of ischemia as a percentage of the total number of viable cells before ischemia. Pelleting for 60 minutes and 120 minutes killed 35±2% and 46±4% of cells, respectively, in the controls. However, inclusion of 50 µmol/L diazoxide significantly decreased cell death during simulated ischemia to about half of that in the controls (18±3% after 60 minutes and 23±4% after 120 minutes, P<.01 versus control). The protection by diazoxide was completely blocked by 100 µmol/L 5-HD (31±2% after 60 minutes and 41±2% after 120 minutes). 5-HD alone did not significantly alter the percentage of cells killed by simulated ischemia: 31±2% after 60 minutes and 47±2% after 120 minutes. Glibenclamide 1 µmol/L also blocked the protection from diazoxide (data not shown). Diazoxide at 100 µmol/L had a similar protective effect (data not shown). For each experiment, there was always an isochronal nonischemic group in which cells were not pelleted. In these groups, <5% of trypan blue–resistant cells became permeable to trypan blue during the 2-hour experiments.

View larger version (52K): [in this window] [in a new window]   Figure 4. Pooled data show that diazoxide protects rabbit ventricular myocytes from ischemia. Cells killed (%) was calculated as number of cells killed by ischemia as a percentage of total viable cells before ischemia. Cont indicates control; Diazo, 50 µmol/L diazoxide; 5-HD, 100 µmol/L 5-HD; and Diazo+5-HD, diazoxide in presence of 5-HD. *P<.01 vs other three groups.

	Discussion

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Although much evidence demonstrates the cardioprotective effects of K_ATP channels and their involvement in ischemic preconditioning, the underlying mechanisms for such protection are poorly understood. One of the early hypotheses proposed that opening of sarcolemmal K_ATP channels shortens the action potential duration. By a cardioplegic effect, energy consumption and calcium overload would be attenuated during ischemia. Although preconditioning has been shown to accelerate action potential shortening slightly during lethal ischemia,²⁴ several recent studies indicate that abbreviation of action potential duration may not be necessary for the protection from preconditioning and K_ATP channel openers. Grover et al⁶ showed that dofetilide, a class III antiarrhythmic agent, abolished the action potential shortening during ischemia but did not abolish ischemic preconditioning in dogs. Yao and Gross²⁵ found that bimakalim, a K_ATP channel opener, had minimal effect on action potential duration but still reduced infarction. Such dissociation has also been shown in several other studies.^{5 26} Furthermore, K_ATP channel openers and ischemic preconditioning are protective even in models using unstimulated cardiac myocytes.^{7 8} Because adult ventricular myocytes are electrically quiescent in these models, action potential duration shortening should not be a factor. These experimental results challenge the idea that the protective effect of K_ATP channels is targeted to sarcolemmal K_ATP channels.

Cardiac myocytes and other cells have another type of K_ATP channel. Inoue et al⁹ were the first to demonstrate the existence of K_ATP channels in the inner mitochondrial membrane by patch clamping mitoplasts prepared from rat liver mitochondria. Later on, a fraction containing mitochondrial K_ATP channel activity was purified from the inner membranes of rat liver and beef heart mitochondria.¹⁰ Using reconstituted mitochondrial vesicles or isolated mitochondria and measuring potassium flux, Garlid et al¹² demonstrated that heart and liver mitochondrial K_ATP channels share some pharmacological properties with the channels found in sarcolemma while possessing a distinct profile. The outstanding pharmacological signature of mitochondrial channels is their high sensitivity to opening by diazoxide, which exceeds the sensitivity of sarcolemmal channels 2000-fold.¹² To study the selectivity of diazoxide in intact cells, we simultaneously measured endogenous flavoprotein fluorescence and sarcolemmal K_ATP currents by whole-cell patch clamp. Although the physiological and pathophysiological roles of the mitochondrial K_ATP channel are not yet very clear, opening of mitochondrial K_ATP channels dissipates the inner mitochondrial membrane potential established by the proton pump. This dissipation accelerates electron transfer by the respiratory chain and if uncompensated by increased production of electron donors (such as NADH), leads to net oxidation of the mitochondria. Mitochondrial redox state can be monitored by recording the fluorescence of FAD-linked enzymes in the mitochondria.^{14 15} Our data show that diazoxide reversibly oxidizes the mitochondrial matrix, as would be expected if it opens the mitochondrial K_ATP channel. Diazoxide had no effect on sarcolemmal K_ATP channels. This insensitivity is consistent with the phenotype of the cardiac sarcolemmal isoform of K_ATP channels.¹⁶ Considering the diffusion barriers between extracellularly applied diazoxide and the mitochondria and other differences in the experimental conditions, our value of 27 µmol/L for the EC₅₀ of diazoxide induction of mitochondrial oxidation is not inconsistent with the EC₅₀ of 3 µmol/L for enhanced potassium flux in isolated mitochondria.¹² We found that pinacidil, another K_ATP channel opener, is also capable of causing mitochondrial oxidation. This indicates that the ability of K_ATP channel openers to induce mitochondrial oxidation may be a general property of such drugs. Nevertheless, pinacidil in addition activates K_ATP channels in the surface membrane, whereas diazoxide can specifically open K_ATP channels in mitochondria without turning on sarcolemmal I_K,ATP. This finding is consistent with the known pharmacology of various K_ATP channel agonists, which indicates that diazoxide is unique in its selectivity for mitochondrial K_ATP channels.¹²

The specificity of diazoxide for mitochondrial K_ATP channels is further supported by the fact that 5-HD, which has been shown to inhibit K_ATP channels in sarcolemma²⁷ and isolated mitochondria,²⁸ reversibly blocked the flavoprotein oxidation induced by diazoxide (Figure 1C). Although Notsu et al²⁷ showed that 5-HD blocked action potential shortening and K_ATP channel openings induced by metabolic inhibition in guinea pig ventricular myocytes, McCullough et al²⁹ did not resolve any effect of 5-HD on cromakalim-activated sarcolemmal I_K,ATP. 5-HD is widely used to block ischemic preconditioning and cardioprotection induced by K_ATP channel openers. Results from this study as well as others²⁸ show that 5-HD is an effective blocker of mitochondrial K_ATP channels. The possibility that 5-HD is selective for mitochondrial K_ATP channels merits further investigation. We also tested another K_ATP channel inhibitor, glibenclamide. We did not observe consistent blockade of mitochondrial oxidation, probably because glibenclamide alone caused oxidation of the flavoproteins especially at concentrations >1 µmol/L (data not shown). This is consistent with the finding that glibenclamide uncouples mitochondria with a K_d of 4 µmol/L.³⁰ Similarly, high concentrations of glibenclamide have been shown to affect the function of isolated mitochondria nonspecifically.²⁸ Therefore, we caution against the interpretation at face value of studies using glibenclamide to test the involvement of K_ATP channels in cardioprotection.

To test the idea that mitochondrial K_ATP channels may play a role in cardioprotection, we examined the effect of diazoxide in a cellular ischemia model. Cells were centrifuged into a pellet to simulate the restricted extracellular space and reduced oxygen supply during ischemia, sampled at designated time points and stained with a hypotonic (85 mosm) trypan blue solution to test the osmotic fragility of the membrane.²⁰ Previous studies have shown that simulated ischemia preconditions myocytes in this model^{7 31} and that the underlying mechanisms for the protection are similar to those in intact hearts.³¹ Our results demonstrated that diazoxide treatment protects rabbit ventricular myocytes to the same extent as preconditioning in our previously published results. Interestingly, a cardioprotective EC₂₅ of 11 µmol/L diazoxide has been reported in intact hearts.³² This concentration corresponds closely to that which we observed to induce flavoprotein oxidation (Figure 1G). Using the same cellular model as in the present study, Armstrong et al³³ showed that pinacidil afforded protection. Notably, if pinacidil was only added into the cell pellet without preincubation, there was no protection. In the present study, diazoxide was added before pelleting and was present during the simulated ischemia. Further studies are required to dissect the time course of the cardioprotective effect of diazoxide.

We have previously shown that adenosine and protein kinase C can synergistically activate sarcolemmal I_K,ATP.¹⁷ It will be very interesting to investigate whether adenosine and protein kinase C have similar effects on mitochondrial K_ATP channels. Our preliminary unpublished results suggest that protein kinase C activation can indeed augment diazoxide-induced flavoprotein fluorescence. A detailed study in this area is currently in progress in the laboratory.

Because of the lack of a single-cell model of ischemic preconditioning, we were unable to investigate the involvement of mitochondrial K_ATP channels in ischemic preconditioning in the present experimental system. Important differences are known to exist between ischemic preconditioning and the cardioprotective effects of K_ATP channel openers in terms of efficacy and memory.³⁴ Nevertheless, the protection from ischemic preconditioning and K_ATP channel openers is blocked by glibenclamide and 5-HD.³ Thus, although it is reasonable to propose that mitochondrial K_ATP is the target for both, further studies are warranted to bolster the links between pharmacological and genuine ischemic preconditioning.

Our results demonstrate that diazoxide targets only mitochondrial K_ATP channels but not sarcolemmal K_ATP channels and suggest that mitochondrial K_ATP channels may serve as effectors of cardioprotection by K_ATP channel openers. The question remains as to how opening of mitochondrial K_ATP channels might protect myocytes against ischemic damage. One possibility is that dissipation of mitochondrial membrane potential decreases the driving force for calcium influx through the calcium uniporter. Inhibition of the mitochondrial calcium uniporter by ruthenium red protects hearts against ischemia and reperfusion injury,^{35 36 37} consistent with this hypothesis. Another possibility is that opening of mitochondrial K_ATP channels, by decreasing the membrane potential, could promote the binding of the endogenous mitochondrial ATPase inhibitor IF₁³⁸ and thus conserve ATP during ischemia. Finally, a change of mitochondrial membrane potential could alter glycolytic pathways during ischemia in favor of myocyte survival. Further studies on mitochondrial K_ATP channels will help us not only to dissect the mechanism of cardioprotection from K_ATP channels and ischemic preconditioning but also to understand the pathogenesis of ischemic and reperfusion injury.

	Selected Abbreviations and Acronyms

 

CN = sodium cyanide DNP = dinitrophenol 5-HD = 5-hydroxydecanoic acid sodium KATP = ATP-dependent potassium TMRE = tetramethylrhodamine ethyl ester

	Acknowledgments

 
This study was supported by the National Institutes of Health (R01-HL-44065 to Dr Marban) and by a Banyu Fellowship in Lipid Metabolism and Atherosclerosis (to Dr Sato). We thank Dr Dmitry Romashko for demonstrating the utility of diazoxide as a selective activator of mitochondrial oxidation.

Received November 24, 1997; revision received December 22, 1997; accepted January 9, 1998.

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