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(Circulation Research. 2001;88:1267.)
© 2001 American Heart Association, Inc.

Cellular Biology

Mitochondrial ATP-Sensitive Potassium Channels Inhibit Apoptosis Induced by Oxidative Stress in Cardiac Cells

Masaharu Akao, Andreas Ohler, Brian O’Rourke, Eduardo Marbán

From the Institute of Molecular Cardiobiology, The Johns Hopkins University, Baltimore, Md. Current address of A.O. is Georg August Universität Göttingen, Abteilung Kardiologie und Pneumologie, Göttingen, Germany.

Correspondence to Eduardo Marbán, MD, PhD, Institute of Molecular Cardiobiology, The Johns Hopkins University, 720 Rutland Ave, 844 Ross Bldg, Baltimore, MD 21205. E-mail marban{at}jhmi.edu

Abstract

Abstract—Mitochondria can either enhance or suppress cell death. Cytochrome c release from mitochondria and depolarization of the mitochondrial membrane potential () are crucial events in triggering apoptosis. In contrast, activation of mitochondrial ATP-sensitive potassium (mitoK_ATP) channels prevents lethal ischemic injury in vivo, implicating these channels as key players in the process of ischemic preconditioning. We probed the relationship between mitoK_ATP channels and apoptosis in cultured neonatal rat cardiac ventricular myocytes. Incubation with 200 µmol/L hydrogen peroxide induced TUNEL positivity, cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and dissipation of . Pharmacological opening of mitoK_ATP channels by diazoxide (100 µmol/L) preserved mitochondrial integrity and suppressed all markers of apoptosis. Diazoxide prevented depolarization in a concentration-dependent manner (EC₅₀ 40 µmol/L, with saturation by 100 µmol/L), as shown by both flow cytometry and quantitative image analysis of cells stained with fluorescent indicators. These cytoprotective effects of diazoxide were reproduced by pinacidil, another mitoK_ATP agonist, and blocked by the mitoK_ATP channel antagonist 5-hydroxydecanoate (500 µmol/L). Our findings identify a novel mitochondrial pathway that is protective against apoptosis. The results also pinpoint mitoK_ATP channels as logical therapeutic targets in diseases of enhanced apoptosis and oxidative stress.

Key Words: apoptosis • ischemia • oxidative stress

Acute coronary syndromes remain the leading causes of death in developed countries. The classical notion that interruption of blood flow kills cells solely by necrosis (catastrophic cell rupture) has been challenged by evidence that apoptosis contributes to ischemic injury in the heart.^{1 2} Apoptosis is a genetically encoded, highly orchestrated mode of cell death in which mitochondria play a key role.^{3 4} Release of the electron transport protein cytochrome c into the cytosol activates caspases, culminating in DNA fragmentation and cytolysis.⁵ In contrast, another mitochondrial pathway promotes cell survival rather than cell death in "ischemic preconditioning."⁶ This endogenous process, well-documented to be operative in vivo in diverse species and tissues, refers to the paradoxical protection against lethal ischemia by brief episodes of prior "conditioning" ischemia. Similar cardioprotection can be recruited by drugs such as diazoxide that open mitochondrial ATP-sensitive potassium (mitoK_ATP) channels; conversely, mitoK_ATP channel blockers (5-hydroxydecanoate [5-HD] or glibenclamide) prevent both preconditioning and pharmacological cardioprotection.^{7 8 9} Thus, mitochondria are key determinants both of cell death and of cell survival.

These observations prompted us to investigate whether the cytoprotective effect of mitoK_ATP channel activation is related to inhibition of apoptosis. Oxidative stress induces cardiac myocyte apoptosis in vitro^{10 11} and contributes to tissue injury in ischemic syndromes^{12 13} and congestive heart failure.¹⁴ To test our hypothesis, we have investigated apoptosis induced by oxidative stress in cardiac myocytes. Our findings reveal that activation of mitoK_ATP channels suppresses programmed cell death.

Materials and Methods

Primary Culture of Neonatal Rat Cardiac Ventricular Myocytes
Cardiac ventricular myocytes were prepared from 1- to 2-day-old Sprague-Dawley rats and cultured as described.¹⁵ In brief, the hearts were removed, and the ventricles were minced in calcium- and bicarbonate-free Hanks’ buffer with HEPES. These tissue fragments were digested by stepwise trypsin dissociation. The dissociated cells were preplated for 1 hour to enrich the culture with myocytes. The nonadherent myocytes were then plated at a density of 1200 cells/mm² in plating medium consisting of DMEM (Mediatech) supplemented with 5% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), and 2 µg/mL vitamin B₁₂. The final myocyte cultures contained >90% cardiac myocytes at partial confluence, as detected by immunostaining with -sarcomeric actin antibody (Sigma). The cells were maintained at 37°C in the presence of 5% CO₂ in a humidified incubator. Bromodeoxyuridine (0.1 mmol/L) was in-cluded in the medium for the first 3 days after plating to inhibit fibroblast growth. Cultures were then placed in serum-free DMEM containing 3.8 g/L glucose, vitamin B₁₂, transferrin, and insulin 24 hours before the drug treatment.

Experimental Protocol
Neonatal rat cardiac myocytes in primary culture were randomly assigned to one of four experimental groups, as follows: (1) control; (2) incubation with 200 µmol/L hydrogen peroxide (H₂O₂); (3) 100 µmol/L diazoxide, applied together with 200 µmol/L H₂O₂; and (4) 100 µmol/L diazoxide and 500 µmol/L 5-HD, applied together with 200 µmol/L H₂O₂. At the beginning of the experiment, culture media were replaced with fresh serum-free DMEM containing those drugs, and cells were exposed to those drugs during the entire experimental period.

Terminal Deoxynucleotidyl Transferase (TdT)–Mediated dUTP Nick End-Labeling (TUNEL) Staining
TUNEL staining was performed according to the manufacturer’s protocol (Roche). Fluorescein labels incorporated in nucleotide polymers were detected by laser scanning confocal microscopy at an excitation wavelength of 488 nm (argon laser). Cells were identified as apoptotic if they showed positive TUNEL staining in the nucleus.

Immunofluorescence Staining
For immunofluorescence, cells were plated on glass coverslips, treated with drugs as indicated, fixed in an ice-cold 1:1 mixture of methanol/acetone, blocked with 10% normal goat serum and 0.075% saponin in PBS, and incubated with primary antibody dissolved in blocking solution at a dilution of 1:100. For cytochrome c staining, mouse monoclonal anti–cytochrome c antibody (Pharmingen; 6H2.Ba4) was used; for caspase-3 staining, rabbit polyclonal antiserum raised against the activated form of caspase-3 (Pharmingen), which recognizes only the processed 20-kDa subunit of cleaved caspase-3, was used; and for poly(ADP-ribose) polymerase (PARP) staining, rabbit polyclonal antiserum raised against the activated form of PARP (New England Biolabs), which recognizes only the processed 89-kDa subunit of cleaved PARP, was used. After washing coverslips with PBS, cells were incubated with secondary antibody consisting of Alexa Fluor 546 goat anti-mouse IgG, F(ab')₂ (Molecular Probes, diluted 1:100) or Alexa Fluor 546 goat anti-rabbit IgG, F(ab')₂ (Molecular Probes, diluted 1:100), respectively. Finally, cells were counterstained with the DNA binding dye DAPI (5 µmol/L, Molecular Probes). Cells were examined by a laser scanning confocal microscope (Zeiss, LSM 410), using a 40x water-immersion lens and 2x optical zoom. Alexa 546 was excited and visualized by a helium/neon laser (543 nm), and DAPI by an UV laser (351 nm).

Immunoblot Analysis
For immunoblot detection of PARP, cells were harvested in ice-cold lysis buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, and 1% Triton X-100) with freshly added 1 mmol/L DTT and protease inhibitor mixture (Roche). Cells were triturated, and the resulting lysates were centrifuged at 12 000g for 10 minutes to remove cell debris. Protein content was determined using a Bradford assay kit (Bio-Rad). Equal amounts of protein (30 µg) were fractionated on 4% to 12% gradient NuPAGE gels (Invitrogen) and electroblotted to nitrocellulose membranes. Blots were stained with Ponceau red (Sigma) to ensure equal loading and transfer of proteins. Membranes were blocked for 2 hours at room temperature with 10% nonfat milk in Tris-buffered saline and were incubated overnight with specific mouse monoclonal anti–PARP antibody (Pharmingen; C2-10) at a 1:200 dilution in the same buffer. Unlike the polyclonal antibody that was used for immunofluorescence, this monoclonal antibody detects an epitope at the N-terminus of PARP holoenzyme. After washing, the blots were incubated for 1 hour with a 1:5000 dilution of horseradish peroxidase–conjugated anti-mouse IgG (Amersham) and were viewed using an enhanced chemiluminescence detection system (Amersham). Quantitative immunoreactivity was determined by densitometry of the developed film.

Assessment of Mitochondrial Membrane Potential ()
Loss of was assessed using either a laser scanning confocal microscope or flow cytometry analysis of cells stained with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbocyanide iodine (JC-1, Molecular Probes). Cells were incubated with 2 µg/mL JC-1 for 10 minutes at 37°C. After applying the dye, cells on dishes were scanned with a confocal microscope using a 10x objective lens. Fluorescence was excited by the 488-nm line of an argon laser and the 543-nm line of a helium/neon laser. The red emission of the dye is due to a potential-dependent aggregation in the mitochondria, reflecting . Green fluorescence reflects the monomeric form of JC-1, appearing in the cytosol after mitochondrial membrane depolarization. For flow cytometry, cells were harvested by trypsinization after loading of the dye and analyzed by FACScan (10 000 cells/sample). The excitation wavelength was 488 nm, and the emission fluorescence for JC-1 was monitored at 530 nm (FL-1) and 582 nm (FL-2). The flow cytometry data were analyzed using Cell Quest (Becton Dickinson). In this protocol, because green fluorescence intensity did not change significantly among groups, we simply plotted the changes in red fluorescence intensity.

Quantitative Image Analysis
Quantitative image analysis was performed for caspase-3 activation and JC-1 staining using image analysis software (ImageJ). Images were thresholded and masked to exclude background, and red- and/or green-field fluorescence was measured.

Time Course of Loss
For time-lapse analysis of , we used another fluorescent indicator, tetramethylrhodamine ethyl ester (TMRE, Molecular Probes). Cardiac myocytes plated on 35-mm dishes were loaded with 100 nmol/L TMRE for 15 minutes. Throughout the assay, cells were maintained at 37°C using a heater platform (Warner Instrument) installed on a microscope stage and were placed in phenol red–free and CO₂-independent Leibovitz’s L-15 medium (Life-Tech) supplemented with 50 nmol/L TMRE, to avoid pH change in a non–CO₂-equilibrated environment. After the desired temperature was reached, time-lapse confocal microscopy was started with a 5-minute interval using a 20x objective lens. TMRE was excited using a 543-nm line of a helium/neon laser. Fifty cells were randomly selected in each scan by drawing regions around individual cells, and red fluorescence intensity was sequentially monitored every 5 minutes.

Statistical Analysis
All quantitative data are presented as mean±SEM. Statistical analysis of apoptotic nuclei among four experimental groups, and of changes in protein levels for PARP holoenzyme among groups, were performed using 1-way ANOVA with Fisher’s least significant difference as the post hoc test. Differences in the mean values of caspase-3 activation between different groups as a function of time were tested using 2-way ANOVA. A level of P<0.05 was accepted as statistically significant.

Results

As an indicator of DNA fragmentation, Figures 1A through 1D demonstrate TUNEL staining in each experimental group. Control cells showed few TUNEL-positive nuclei (Figure 1A), but exposure to 200 µmol/L H₂O₂ for 16 hours increased the number of TUNEL-positive nuclei (Figure 1B). Diazoxide decreased the frequency of H₂O₂-induced TUNEL-positive nuclei (Figure 1C), indicating a protective effect of the mitoK_ATP channel agonist. This effect was blocked by the mitoK_ATP channel antagonist 5-HD (Figure 1D). Figure 1E shows a quantitative determination of apoptotic nuclei in each experimental group. Cells incubated for 16 hours were stained by DAPI, and the apoptotic cells were identified by the characteristic condensed, fragmented nuclei. An average of 300 to 500 nuclei from random fields were analyzed in each sample. The data indicate that diazoxide has a significant protective effect on the preservation of nuclear morphology, confirming the TUNEL results.

View larger version (15K): [in this window] [in a new window]   Figure 1. A through D, TUNEL staining in neonatal rat cardiac myocytes. A, Control cells show very sparse TUNEL-positive nuclei. B, Cells exposed to 200 µmol/L H2O2 for 16 hours become predominantly TUNEL positive. C, Diazoxide 100 µmol/L protects against H2O2-induced TUNEL positivity. D, Protective effect of diazoxide is blocked by 500 µmol/L mitoKATP channel blocker 5-HD. E, Quantitative determination of apoptotic nuclei (n=4). C indicates control; H, H2O2 200 µmol/L; DZ, H2O2+diazoxide 100 µmol/L; and 5HD, H2O2+diazoxide 100 µmol/L+5-HD 500 µmol/L. *P<0.05 vs C, P<0.05 vs H.

Figures 2A through 2D demonstrate cytochrome c immunofluorescence. In control, the distribution of cytochrome c is reticular and punctate (Figure 2A), indicative of a normal mitochondrial pattern. We have confirmed that the cytochrome c signals in control cells colocalize with the specific mitochondrial marker dye, MitoTracker (Molecular Probes) (data not shown). Incubation with 200 µmol/L H₂O₂ for 16 hours induced cytochrome c translocation to the cytoplasm, resulting in a homogeneous distribution (Figure 2B). Diazoxide inhibited the translocation of cytochrome c (Figure 2C), whereas the addition of 500 µmol/L 5-HD abrogated the effect of diazoxide (Figure 2D).

View larger version (111K): [in this window] [in a new window]   Figure 2. A through D, Cytochrome c translocation in neonatal rat cardiac myocytes. Blue fluorescent dye DAPI demonstrates nuclear morphology. A, Control cells show no translocation of cytochrome c to cytosol. B, Cells exposed to 200 µmol/L H2O2 for 16 hours exhibit extensive translocation of cytochrome c to cytosol, shown by homogeneous distribution. C, Diazoxide 100 µmol/L protects against H2O2-induced translocation of cytochrome c. D, Protective effect of diazoxide is blocked by 500 µmol/L 5-HD.

Figures 3A through 3D demonstrate the immunofluorescent staining for caspase-3, visible as red, using polyclonal antiserum that recognizes only the active form of the enzyme. In control, cells showed little cytosolic fluorescence (Figure 3A), but 200 µmol/L H₂O₂ markedly increased the red signals (Figure 3B). Incubation with 100 µmol/L diazoxide inhibited the H₂O₂-induced activation of caspase-3 (Figure 3C), whereas addition of 500 µmol/L 5-HD negated the effect of diazoxide (Figure 3D). From images such as these, mean red fluorescence intensity was measured as an index of caspase-3 activity (Figure 3E). Two-way ANOVA revealed that the treatment group and the exposure duration were both independent and significant factors. Additional analysis with the Bonferroni method indicates that the activities in the H₂O₂ and 5-HD groups (category 1) are significantly higher than those in control and diazoxide groups (category 2), but there are no significant differences between groups within each category. The activity at 8 hours is significantly higher than that at 4 hours and does not decrease at 16 hours. Furthermore, diazoxide delayed the onset of caspase-3 activation and also blunted the overall response. Similar results were obtained for activated caspase-7 (data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 3. A through D, Caspase-3 activation in neonatal rat cardiac myocytes. Red fluorescence represents 20-kDa active form of caspase-3. A, Control cells show no caspase-3 activation. B, Cells exposed to 200 µmol/L H2O2 for 16 hours become predominantly caspase-3 positive. C, Diazoxide 100 µmol/L protects against H2O2-induced activation of caspase-3. D, Protective effect of diazoxide is blocked by 500 µmol/L 5-HD. E, Quantitative determination of caspase-3 activation in neonatal rat cardiac myocytes. Each data point is a relative ratio of red fluorescence intensity against control value of the same time points (n=4 for each data point). Control value in each time point is defined as 100%. There are no actual data for 0 hours. C indicates control (); H, H2O2 200 µmol/L (); DZ, H2O2+diazoxide 100 µmol/L (); and 5HD, H2O2+diazoxide 100 µmol/L+5-HD 500 µmol/L (). *P<0.05 vs C or DZ.

Next, we examined PARP, one of the main targets of caspase-3 or -7 in vivo,¹⁶ using a polyclonal antibody that detects only the cleaved form (Figures 4A through 4D). In parallel with the activation of caspase-3 or -7, PARP cleavage was augmented in H₂O₂-treated cells (Figure 4B). Diazoxide inhibited the H₂O₂–induced PARP cleavage (Figure 4C), and addition of 500 µmol/L 5-HD prevented the diazoxide effect (Figure 4D). To confirm these findings using complementary methods, we examined PARP protein levels by immunoblot analysis. As shown in Figure 4E, the PARP holoenzyme was detected as a 117-kDa band. H₂O₂ exposure enhanced the cleavage of PARP, resulting in a loss of the holoenzyme band in a time-dependent manner. Diazoxide attenuated the loss of PARP holoenzyme, and 5-HD abolished the protective effect of diazoxide. Densitometry was performed on PARP holoenzyme immunoreactivity (Figure 4F). Although the effect of diazoxide on preservation of PARP holoenzyme did not reach statistical significance at 8 hours (26.5±3.4% in the H₂O₂ group, 43.2±9.5% in the diazoxide group, and 27.2±5.3% in the 5-HD group; P=0.068 between the H₂O₂ and diazoxide groups and P=0.078 between the 5-HD and diazoxide groups; n=3), it became statistically significant at 16 hours (6.9±1.4% in the H₂O₂ group, 18.7±5.1% in the diazoxide group, and 9.7±2.0% in the 5-HD group; P=0.017 between the H₂O₂ and diazoxide groups and P=0.050 between the 5-HD and diazoxide groups; n=3). All of these features indicate that oxidative stress induces apoptosis, which is inhibited by mitoK_ATP channel activation.

View larger version (52K): [in this window] [in a new window]   Figure 4. A through D, PARP cleavage in neonatal rat cardiac myocytes. Red fluorescence represents 89-kDa cleaved form of PARP. A, Control cells show no PARP cleavage. B, Cells exposed to 200 µmol/L H2O2 for 16 hours become predominantly cleaved PARP positive. C, Diazoxide 100 µmol/L protects against H2O2-induced cleavage of PARP. D, Protective effect of diazoxide is blocked by 500 µmol/L 5-HD. E, Immunoblot showing time-dependent changes in protein levels of 117-kDa PARP holoenzyme in neonatal rat cardiac myocytes. Cell lysates from each experimental group were subjected to immunoblot analysis (30 µg protein/lane) with monoclonal antibody against PARP holoenzyme. Cells were treated for either 8 or 16 hours. F, Densitometric analysis of protein levels for PARP holoenzyme (n=3). Results are expressed as relative ratio of band intensity against control. *P<0.05 vs H2O2 200 µmol/L. E and F, Abbreviations as in Figure 1E.

Redistribution of cytochrome c has been linked to the loss of mitochondrial membrane potential () and the opening of the mitochondrial permeability transition pore.¹⁷ We assessed in H₂O₂-stimulated myocytes using a fluorescent probe, JC-1. Control cells exhibit punctate red staining (Figure 5A), indicative of normal mitochondrial uptake driven by maintained . Cells exposed to 200 µmol/L H₂O₂ for 2 hours lose punctate red staining (Figure 5B), in favor of a diffuse green cytosolic signal indicative of the loss of . Incubation with 100 µmol/L diazoxide protects against H₂O₂-induced loss of mitochondrial integrity (Figure 5C). The protective effect of diazoxide is blocked by 500 µmol/L 5-HD (Figure 5D). These observations were rendered quantitative by image analysis. Figure 5E shows the representative data for red fluorescence, an indicator of . Exposure to 200 µmol/L H₂O₂ for 2 hours resulted in mitochondrial depolarization, whereas diazoxide prevented loss of in a concentration-dependent manner; the EC₅₀ of 40 µmol/L is close to the value of 27 µmol/L for mitoK_ATP channel activation in intact heart cells.⁸ Addition of 500 µmol/L 5-HD antagonized the salutary effect of diazoxide.

View larger version (56K): [in this window] [in a new window]   Figure 5. Mitochondrial membrane potential () in neonatal rat cardiac myocytes. A, Control cells show predominantly punctate red staining with a fluorescent dye, JC-1, indicative of normal mitochondrial uptake driven by a maintained . B, Cells exposed to 200 µmol/L H2O2 for 2 hours lose punctate red staining. C, Diazoxide 100 µmol/L protects against H2O2-induced loss of , as shown by the preservation of red signals. D, Protective effect of diazoxide is blocked by 500 µmol/L 5-HD. E, Representative data of quantitative analysis from low-power confocal images. Bars, from left to right, show data from the following: bar 1, H2O2; bar 2, H2O2+diazoxide 20 µmol/L; bar 3, H2O2+diazoxide 50 µmol/L; bar 4, H2O2+diazoxide 100 µmol/L; bar 5, H2O2+diazoxide 200 µmol/L; bar 6, H2O2+diazoxide 100 µmol/L+5-HD 500 µmol/L; bar 7, H2O2+diazoxide 100 µmol/L+glibenclamide 10 µmol/L; and bar 8, H2O2+pinacidil 100 µmol/L. Concentration of H2O2 was 200 µmol/L for all groups. Results are expressed as arbitrary units of red fluorescence intensity. Value of control group was 95.06. F through J, Results of flow cytometry analysis. Histograms of FL-2 channel (red fluorescence) are shown. In all histograms, those from control and 200 µmol/L H2O2-treated groups are overlaid as references. F, H2O2+diazoxide 20 µmol/L; G, H2O2+diazoxide 50 µmol/L; H, H2O2+diazoxide 100 µmol/L; I, H2O2+diazoxide 100 µmol/L+5-HD 500 µmol/L; J, H2O2+5-HD 500 µmol/L. Concentration of H2O2 was 200 µmol/L for all groups. Results of flow cytometry analysis shown here are representative data of 3 independent experiments.

To further confirm the central role of mitoK_ATP channels, we examined another mitoK_ATP channel opener, pinacidil.¹⁸ As shown in Figure 5E, incubation with 100 µmol/L pinacidil was equi-effective to diazoxide in preventing the loss of induced by 200 µmol/L H₂O₂. Moreover, the classical K_ATP blocker glibenclamide (10 µmol/L), which blocks both the surface K_ATP and the mitoK_ATP channel, reversed the salutary effect of diazoxide. To rule out the possibility that diazoxide cancels the effect of H₂O₂ by a direct chemical interaction, we measured the activity of H₂O₂ in diazoxide-containing medium using a commercially available fluorometric assay kit (Amplex Red Hydrogen Peroxide Assay Kit; Molecular Probes). The content of H₂O₂ was not affected by the presence of diazoxide (data not shown). Furthermore, addition of diazoxide did not affect the transient p38 MAPK activation induced by H₂O₂ (authors’ unpublished observation, 2000). Therefore, the protective effect of diazoxide against H₂O₂-induced cell death reflects a genuine and specific biological response, not a radical-scavenging effect of diazoxide.

We further quantified the effects of mitoK_ATP channel activation by flow cytometry (Figure 5F through 5J). Incubation with 200 µmol/L H₂O₂ for 2 hours decreased the red fluorescence and shifted the distribution curve leftward. In agreement with the confocal image analysis, diazoxide prevented the H₂O₂-induced dissipation of in a concentration-dependent manner, as shown by the progressive rightward shift of the distribution curve with increasing concentrations of diazoxide (Figures 5F through 5H; in µmol/L, F 20, G 50, and H 100). 5-HD (500 µmol/L) abolished the effect of diazoxide and reverted the distribution to that of the H₂O₂ group (Figure 5I). Importantly, 5-HD alone had no effect on in the absence of diazoxide (Figure 5J). 5-HD alone did not promote any of the apoptotic markers examined here compared with the control group (data not shown), and its incubation together with H₂O₂ did not further aggravate those markers compared with the H₂O₂-treated drug-free group.

To examine the time-dependent changes of on a single-cell basis, we used time-lapse confocal analysis of cardiac myocytes loaded with another indicator dye, TMRE, at 5-minute intervals. Throughout the period of observation, TMRE fluorescence intensities of control cells remained unchanged (Figure 6A). In contrast, cells treated with 200 µmol/L H₂O₂ progressively lost red fluorescence intensity, indicating irreversible dissipation of (Figure 6B). Diazoxide remarkably inhibited this catastrophic loss of (Figure 6C), and this protective effect was abrogated by 5-HD (Figure 6D). Fifty cells were randomly and prospectively selected in each group, and the mean values of red fluorescence intensity from each cell are plotted in Figures 6F through 6I. Diazoxide prevented the loss of induced by 200 µmol/L H₂O₂ in the majority of cells, and 5-HD inhibited the protective effect of diazoxide. The duration of loss of each cell was abrupt (5 to 10 minutes) and complete, regardless of the time elapsed since the oxidative stress was applied. Thus, although the averages of the individual cell data appear as a progressive decline in signals (Figure 6E), a more rigorous analysis of the cumulative first latency to loss for individual cells reveals that the majority of cells lost within 1 hour (Figure 6J). Diazoxide not only decreased the number of cells undergoing dissipation of , but also delayed the onset of loss. However, it did not change the duration required for loss in those cells that did lose their inner membrane potential. Movie files reconstituted from time-lapse confocal images are available in the online data supplement (http://circres.ahajournals.org/).

View larger version (75K): [in this window] [in a new window]   Figure 6. Time-lapse analysis of loss of mitochondrial membrane potential () in neonatal rat cardiac myocytes. A, Control cells maintain constant level of a indicator dye, TMRE, over the time course. B, Cells exposed to 200 µmol/L H2O2 show progressive loss of TMRE fluorescence. C, Diazoxide 100 µmol/L protects against H2O2-induced loss of , as shown by the preservation of red signals and intact morphology. D, 5-HD 500 µmol/L abrogates the protective effect of diazoxide. E, Mean fluorescence intensity from 50 cells randomly and prospectively selected in each group. Concentration of H2O2 was 200 µmol/L for all groups. Results are normalized red fluorescence intensity. Similar results were obtained in 3 independent experiments. F through I, Time course of red fluorescence in each individual cell. F, Control cells; G, H2O2 200 µmol/L; H, H2O2 200 µmol/L+diazoxide 100 µmol/L; I, H2O2 200 µmol/L+diazoxide 100 µmol/L+5-HD 500 µmol/L. All of 50 cells are shown. J, Cumulative first latency plot of number of cells that have undergone dissipation. Results are mean±SEM from 3 independent experiments. E and J, Abbreviations and symbols as in Figure 3E.

Discussion

Here we have identified a mechanistic link between mitoK_ATP channels and the mitochondrial apoptotic pathway. The principal findings are as follows. (1) In isolated cardiac myocytes, the mitoK_ATP channel opener diazoxide inhibits activation of the mitochondrial apoptotic pathway induced by oxidative stress in a concentration-dependent manner. (2) The channel blocker 5-HD (as well as glibenclamide) abolishes the antiapoptotic effect of diazoxide. These observations support the hypothesis that activation of mitoK_ATP channels inhibits apoptosis, thereby contributing, at least in part, to the infarct size-limiting effect of this agent.

Besides the activation of mitoK_ATP channels, diazoxide has been argued to have other pharmacological actions, such as succinate dehydrogenase (SDH) inhibition.¹⁹ However, we believe that the antiapoptotic properties of diazoxide are exclusively attributable to mitoK_ATP activation and not to SDH inhibition, for the following reasons. (1) The concentration needed to achieve SDH inhibition is much higher (400 µmol/L).^{19 20} (2) SDH inhibition is insensitive to K_ATP channel blockers.²¹ (3) Pinacidil, which is equi-effective in cardioprotection, is not known to inhibit SDH. The concentration range of diazoxide for cardioprotection is 10 to 100 µmol/L,^{8 9} which is quite consistent with the antiapoptotic range observed in this study. Although this range is higher than that for mitoK_ATP channel opening in reconstituted liposomes or isolated mitochondria,²² the accessibility of diazoxide to mitochondria may differ in isolated mitochondria and in intact myocytes, given the net negative charge of this agent at physiological pH.¹⁹ Finally and most importantly, the present results show that diazoxide protects cells against loss and cell death induced by oxidative stress, a property that cannot be written off as a toxic side effect of the drug.²³ Furthermore, diazoxide treatment alone had no ill effects on cell viability compared with controls.

The mechanisms by which mitoK_ATP channel opening protects against apoptosis are still unknown. However, as evidenced in this study, the opening of mitoK_ATP channels may act quite early in the apoptotic cascade by inhibiting cytochrome c release and depolarization; these are the earliest alterations in the cascade, and the two events are closely associated.²⁴ In good agreement with a previous report,¹¹ we observed the dissipation of within 1 hour in the majority of cells that were exposed to H₂O₂ (Figure 6); once initiated, dissipation is rapid, complete, and irreversible. Diazoxide not only decreased the number of cells undergoing dissipation of , but also delayed the onset of loss, whereas it did not change the duration of loss in each cell. These observations suggest that diazoxide could modulate the initiation process of loss but not the dissipating process itself. In other words, diazoxide appears to delay or block the entry into the point of no return. Such inhibition could result from a decrease in mitochondrial calcium overload, which is a reported consequence of diazoxide.^{25 26} However, further investigation is needed to elucidate the precise mechanism.

Along with significant advances in our understanding of the biochemical and molecular basis of apoptosis, the development of novel therapeutic strategies against apoptosis-related diseases has drawn considerable attention.²⁷ Caspases were among the first obvious therapeutic targets for modulating apoptosis. In various models of ischemia-reperfusion injury, including heart and brain, caspase inhibition has shown remarkable efficacy, demonstrated by the reduction of infarct size or preservation of organ function.^{28 29 30} However, it has also been reported that caspase inhibitors do not inhibit cytochrome c release or dissipation,^{31 32} giving reason to wonder whether such drugs effectively preserve mitochondrial function. In this context, diazoxide has the potential advantage that it blocks the apoptotic pathway upstream of cytochrome c release or dissipation, thus preserving mitochondrial integrity and minimizing functional loss. Paradoxically, isolated mitochondria have been reported to release cytochrome c in response to diazoxide³³ ; however, this effect was observed in highly calcium-loaded isolated mitochondria and was not blocked by mitoK_ATP channel inhibitors, suggesting that this permeability transition may not have been due to specific mitoK_ATP opening.

Our findings have various conceptual and therapeutic implications. First, the fact that mitoK_ATP channel activation inhibits apoptosis provides further evidence for the central role of these organelles in programmed cell death. Second, the potent cytoprotection by mitoK_ATP channel recruitment suggests that ischemic preconditioning reflects inhibition of apoptosis in addition to necrosis.³⁴ Finally, and most importantly, mitoK_ATP channel opening represents a novel therapeutic strategy against apoptosis induced by oxidative stress. A lead compound, diazoxide, already exists and has been used safely for decades to treat hypertension. The spectrum of diseases potentially susceptible to such pharmacological intervention is staggering,^{35 36} given the implication of accelerated apoptosis in pathologies ranging from heart failure to autoimmunity or neurodegeneration.

Acknowledgments

This study was supported by NIH Grant R37 HL36957, a Banyu Fellowship in Cardiovascular Medicine (M.A.), and the Deutsche Forschungsgemeinschaft (A.O.). E.M. holds the Michel Mirowski, MD Professorship of Cardiology. We thank D.C. Johns, R. Li, N. Sasaki, M. Murata, and J. Seharaseyon for technical advice and helpful discussions. We also thank H. Tachimoto for assistance with the analysis of flow cytometry data, H. Kusuoka for statistical analysis, and A. Rosen for critical reading of the manuscript.

Footnotes

Original received January 16, 2001; resubmission received March 20, 2001; revised resubmission received April 23, 2001; accepted April 26, 2001.

This manuscript was sent to Richard A. Walsh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

References

1. Anversa P, Cheng W, Liu Y, Leri A, Redaelli G, Kajstura J. Apoptosis and myocardial infarction. Basic Res Cardiol. 1998;93:8–12.

2. Haunstetter A, Izumo S. Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ Res. 1998;82:1111–1129.

3. Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998;281:1309–1312.

4. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 1997;275:1132–1136.

5. Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996;86:147–157.

6. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124–1136.

7. Gross GJ. ATP-sensitive potassium channels and myocardial preconditioning. Basic Res Cardiol. 1995;90:85–88.

8. Liu Y, Sato T, O’Rourke B, Marbán E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection? Circulation. 1998;97:2463–2469.

9. Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D’Alonzo AJ, Lodge NJ, Smith MA, Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K⁺ channels: possible mechanism of cardioprotection. Circ Res. 1997;81:1072–1082.

10. von Harsdorf R, Li PF, Dietz R. Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation. 1999;99:2934–2941.

11. Cook SA, Sugden PH, Clerk A. Regulation of Bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential. Circ Res. 1999;85:940–949.

12. Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest. 1994;94:1621–1628.

13. Vanden Hoek TL, Li C, Shao Z, Schumacker PT, Becker LB. Significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion. J Mol Cell Cardiol. 1997;29:2571–2583.

14. Ide T, Tsutsui H, Kinugawa S, Suematsu N, Hayashidani S, Ichikawa K, Utsumi H, Machida Y, Egashira K, Takeshita A. Direct evidence for increased hydroxyl radicals originating from superoxide in the failing myocardium. Circ Res. 2000;86:152–157.

15. Johns DC, Nuss HB, Marbán E. Suppression of neuronal and cardiac transient outward currents by viral gene transfer of dominant-negative Kv4.2 constructs. J Biol Chem. 1997;272:31598–31603.

16. Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, Munday NA, Raju SM, Smulson ME, Yamin TT, Yu VL, Miller DK. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature. 1995;376:37–43.

17. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT, Thompson CB. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell. 1997;91:627–637.

18. Sato T, O’Rourke B, Marbán E. Modulation of mitochondrial ATP-dependent K⁺ channels by protein kinase C. Circ Res. 1998;83:110–114.

19. Schafer G, Wegener C, Portenhauser R, Bojanovski D. Diazoxide, an inhibitor of succinate oxidation. Biochem Pharmacol. 1969;18:2678–2681.

20. Grimmsmann T, Rustenbeck I. Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria. Br J Pharmacol. 1998;123:781–788.

21. Lenzen S, Panten U. Characterization of succinate dehydrogenase and -glycerophosphate dehydrogenase in pancreatic islets. Biochem Med. 1983;30:349–356.

22. Garlid KD, Paucek P, Yarov-Yarovoy V, Sun X, Schindler PA. The mitochondrial K_ATP channel as a receptor for potassium channel openers. J Biol Chem. 1996;271:8796–8799.

23. Kowaltowski AJ, Seetharaman S, Paucek P, Garlid KD. Bioenergetic consequences of opening the ATP-sensitive K⁺ channel of heart mitochondria. Am J Physiol Heart Circ Physiol. 2001;280:H649–H657.

24. Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol. 2000;2:156–162.

25. Jaburek M, Yarov-Yarovoy V, Paucek P, Garlid KD. State-dependent inhibition of the mitochondrial K_ATP channel by glyburide and 5-hydroxydecanoate. J Biol Chem. 1998;273:13578–13582.

26. Holmuhamedov EL, Wang L, Terzic A. ATP-sensitive K⁺ channel openers prevent Ca²⁺ overload in rat cardiac mitochondria. J Physiol (Lond). 1999;519:347–360.

27. Nicholson DW. From bench to clinic with apoptosis-based therapeutic agents. Nature. 2000;407:810–816.

28. Yaoita H, Ogawa K, Maehara K, Maruyama Y. Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation. 1998;97:276–281.

29. Mocanu MM, Baxter GF, Yellon DM. Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury. Br J Pharmacol. 2000;130:197–200.

30. Endres M, Namura S, Shimizu-Sasamata M, Waeber C, Zhang L, Gomez-Isla T, Hyman BT, Moskowitz MA. Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family. J Cereb Blood Flow Metab. 1998;18:238–247.

31. Finucane DM, Bossy-Wetzel E, Waterhouse NJ, Cotter TG, Green DR. Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J Biol Chem. 1999;274:2225–2233.

32. Bialik S, Cryns VL, Drincic A, Miyata S, Wollowick AL, Srinivasan A, Kitsis RN. The mitochondrial apoptotic pathway is activated by serum and glucose deprivation in cardiac myocytes. Circ Res. 1999;85:403–414.

33. Holmuhamedov EL, Jovanovic S, Dzeja PP, Jovanovic A, Terzic A. Mitochondrial ATP-sensitive K⁺ channels modulate cardiac mitochondrial function. Am J Physiol. 1998;275:H1567–H1576.

34. Takashi E, Wang Y, Ashraf M. Activation of mitochondrial K_ATP channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway. Circ Res. 1999;85:1146–1153.

35. Haunstetter A, Izumo S. Toward antiapoptosis as a new treatment modality. Circ Res. 2000;86:371–376.

36. Yuan J, Yankner BA. Apoptosis in the nervous system. Nature. 2000;407:802–809.

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				J. M. Hare Oxidative Stress and Apoptosis in Heart Failure Progression Circ. Res., August 3, 2001; 89(3): 198 - 200. [Full Text] [PDF]

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				Z. Yang, R. J. Cerniway, A. M. Byford, S. S. Berr, B. A. French, and G. P. Matherne Cardiac overexpression of A1-adenosine receptor protects intact mice against myocardial infarction Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H949 - H955. [Abstract] [Full Text] [PDF]

				M. Murata, M. Akao, B. O'Rourke, and E. Marban Mitochondrial ATP-Sensitive Potassium Channels Attenuate Matrix Ca2+ Overload During Simulated Ischemia and Reperfusion: Possible Mechanism of Cardioprotection Circ. Res., November 9, 2001; 89(10): 891 - 898. [Abstract] [Full Text] [PDF]

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