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(Circulation Research. 2001;89:856.)
© 2001 American Heart Association, Inc.

Reports

Opening of Mitochondrial K_ATP Channels Attenuates the Ouabain-Induced Calcium Overload in Mitochondria

Hideyuki Ishida, Yuki Hirota, Chokoh Genka, Hiroe Nakazawa, Haruaki Nakaya, Toshiaki Sato

From the Department of Physiology (H.I., Y.H., C.G., H. Nakazawa), Tokai University School of Medicine, Isehara, Japan; Department of Pharmacology (H. Nakaya, T.S.), Graduate School of Medicine, Chiba University, Chiba, Japan.

Correspondence to Toshiaki Sato, MD, PhD, Department of Pharmacology, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba, 260-8670 Japan. E-mail tsato{at}med.m.chiba-u.ac.jp

Abstract

We tested whether opening of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels depolarizes mitochondrial membrane potential (_m) and thereby prevents the mitochondrial Ca²⁺ overload. With the use of a Nipkow disk confocal system, the mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) and _m in rat ventricular myocytes were measured by loading cells with Rhod-2 and JC-1, respectively. Exposure to ouabain (1 mmol/L) for 30 minutes produced mitochondrial Ca²⁺ overload, and the intensity of Rhod-2 fluorescence significantly increased to 173±16% of baseline (P<0.001). Treatment of myocytes with the mitoK_ATP channel opener diazoxide (100 µmol/L) blunted the ouabain-induced mitochondrial Ca²⁺ overload (131±10% of baseline; P<0.001 versus ouabain). Moreover, diazoxide significantly depolarized the _m and reduced the intensity of JC-1 fluorescence during application of ouabain to 89±2% of baseline (P<0.05). These effects of diazoxide were blocked by the mitoK_ATP channel blocker 5-hydroxydecanoate (500 µmol/L). These results indicate that opening of mitoK_ATP channels prevents a mitochondrial Ca²⁺ overload in association with _m depolarization and thereby protects myocardium against ischemic damage.

Key Words: mitochondria • calcium • K_ATP channel • cardioprotection

Mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels are thought to play a key role in cardioprotection,¹ but the crucial question remains as to why the opening of mitoK_ATP channels can be so protective. Liu et al² originally hypothesized that the K⁺ entry through mitoK_ATP channels depolarizes the _m, which reduces the driving force for Ca²⁺ influx and, hence, results in the prevention of mitochondrial Ca²⁺ overload. Later on, Holmuhamedov et al³ demonstrated that diazoxide reduced the [Ca²⁺]_m in isolated cardiac mitochondria and neonatal cardiomyocytes. In contrast, Kowaltowski et al⁴ have shown that diazoxide has minimal effects on [Ca²⁺]_m and _m. Thus, investigations addressing the original hypothesis have yielded conflicting results. The present study therefore was aimed to carefully determine, using adult rat ventricular myocytes, whether opening of mitoK_ATP channels depolarizes _m and attenuates mitochondrial Ca²⁺ overload.

Materials and Methods

Cell Preparation
Adult rat ventricular myocytes were isolated by collagenase digestion, as previously described.⁵ Once isolated, the cells were resuspended in a culture medium composed of 5% fetal calf serum, 47.5% M199, and 47.5% modified Tyrode’s solution containing (mmol/L) NaCl 137, KCl 5.4, MgCl₂ 1, HEPES 5, dextrose 22, taurine 20, creatine 5, and sodium pyruvate 5 (pH 7.4) at room temperature until use.

Confocal Fluorescence Imaging of [Ca²⁺]_m and _m
The Ca²⁺ fluorophore Rhod-2 was used to measure changes of [Ca²⁺]_m. Myocytes were loaded with 10 µmol/L Rhod-2 acetoxymethyl ester (Molecular Probes) for 120 minutes at 4°C and then incubated for 30 minutes at 37°C in the culture medium. This two-step cold loading/warm incubation protocol achieves exclusive loading of Rhod-2 into the mitochondria.⁶ The _m was monitored with a fluorescent probe, JC-1 (Molecular Probes). Myocytes were incubated with 0.5 µmol/L JC-1 for 10 minutes at 37°C.⁷ We verified that the subcellular distribution of fluorescence arising from Rhod-2 and JC-1 was virtually identical to that observed by loading myocytes with the mitochondrial dye rhodamine-123.

Myocytes loaded with Rhod-2 and JC-1 were perfused with a HEPES-buffered physiological solution (37°C) containing (mmol/L) NaCl 123, KCl 5, CaCl₂ 2.7, MgCl₂ 1, HEPES 5, and glucose 5.5 (pH 7.4) and imaged with a Nipkow disk confocal system (CSU10, Yokogawa, Japan), as previously described.⁵ Rhod-2 was excited at 488 nm by an argon ion laser, with emission collected above 515 nm through a long-pass barrier filter. JC-1 was excited at 488 nm, and the red emission fluorescence was detected using a long-pass filter of 580 nm. The emission light was imaged through a relay lens to an intensified CCD camera. Images were recorded on a computer (Macintosh 8500/120) at video rate and analyzed with NIH Image 1.62f software.

Statistical Analysis
Data are expressed as mean±SEM. Intergroup comparisons are made by Student’s t test for two groups and by ANOVA followed by Tukey’s test for multiple groups. A value of P<0.05 was regarded as significant.

Results

Confocal images of rat ventricular myocytes loaded with Rhod-2, obtained before and after application of diazoxide, are shown in Figure 1A. The resting [Ca²⁺]_m in intact cells was low as revealed by the dim baseline signal of Rhod-2 fluorescence (Figure 1A, a). Subsequent exposure to diazoxide (100 µmol/L) did not affect the [Ca²⁺]_m, and the Rhod-2 fluorescence remained unchanged (Figure 1A, b). However, we occasionally observed the cells with high intensity of Rhod-2 fluorescence (Figure 1A, c; shown in pink), which might have damage caused by the isolation procedure. In such cells with mitochondrial Ca²⁺ overload, exposure to diazoxide dramatically decreased the intensity of Rhod-2 fluorescence (Figure 1A, d). As summarized in Figure 1B, diazoxide significantly decreased the [Ca²⁺]_m to 41±9% of baseline (P<0.05) only when the resting [Ca²⁺]_m was high. Here, the intensity of Rhod-2 fluorescence of baseline in Ca²⁺-overloaded cells was 3-fold greater than that in intact cells (Figure 1B, inset). The effect of diazoxide was antagonized by 5-hydroxydecanoate (500 µmol/L).

View larger version (75K): [in this window] [in a new window]   Figure 1. A, Two-dimensional confocal images of Rhod-2 fluorescence before (Baseline) and after treatment with diazoxide (DIAZ) in intact (a and b) and Ca2+-overloaded (c and d) cells. B, Summarized data for the effects of diazoxide and 5-hydroxydecanoate (5HD) on Rhod-2 fluorescence in intact and Ca2+-overloaded cells. *P<0.001 vs baseline; #P<0.05 vs DIAZ in Ca2+-overloaded cells. Inset shows summarized data for the arbitrary units of Rhod-2 fluorescence of baseline in intact and Ca2+-overloaded cells. C, Two-dimensional confocal images of Rhod-2 fluorescence before and after treatment with diazoxide and/or ouabain. D, Summarized data for the time courses of changes in Rhod-2 fluorescence. *P<0.001 vs baseline; #P<0.001 vs OUAB; and P<0.001 vs OUAB+DIAZ.

In the next series of experiments, cells were exposed to the Na⁺/K⁺-ATPase inhibitor ouabain in an attempt to produce mitochondrial Ca²⁺ overload. Treatment of myocytes with 1 mmol/L ouabain increased the intensity of Rhod-2 fluorescence, suggesting that Ca²⁺ overload in mitochondria was evoked (Figure 1C, a and b). Treatment with diazoxide (100 µmol/L) attenuated the mitochondrial Ca²⁺ overload during exposure to ouabain (Figure 1C, c and d). As summarized in Figure 1D, Rhod-2 fluorescence after 15 minutes and 30 minutes of exposure to ouabain significantly increased to 130±3% and 173±16% of baseline (P<0.001), respectively. Diazoxide significantly attenuated the ouabain-induced mitochondrial Ca²⁺ overload (116±7% at 15 minutes, 131±10% at 30 minutes; P<0.001), and the effect was antagonized by 5-hydroxydecanoate (500 µmol/L). 5-Hydroxydecanoate alone slightly increased the Rhod-2 fluorescence (123±3% at 30 minutes, n=4; P=NS versus control), so that the Rhod-2 fluorescence in the ouabain+diazoxide+5-hydroxydecanoate group was insignificantly higher than that in the ouabain group. Like diazoxide, nicorandil (100 µmol/L), a potent mitoK_ATP channel opener,⁸ significantly reduced the ouabain-induced Ca²⁺ overload (see Figure in the online data supplement, available at http://www.circresaha.org). These results indicate that opening of mitoK_ATP channels attenuates the ouabain-induced Ca²⁺ overload in mitochondria.

We then examined whether the protective effect of diazoxide on the ouabain-induced Ca²⁺ overload is associated with the depolarization of _m. Diazoxide (100 µmol/L) alone reduced the intensity of JC-1 fluorescence to 93±2% of baseline (n=9), but this change was not statistically significant. In the experiments shown in Figure 2A, although ouabain alone did not affect the JC-1 fluorescence (Figure 2A, a and b), diazoxide apparently reduced the intensity of JC-1 fluorescence during application of ouabain (Figure 2A, c and d). As summarized in Figure 2B, diazoxide significantly reduced the intensity of JC-1 fluorescence to 89±2% of baseline (P<0.05), and the effect was antagonized by 5-hydroxydecanoate (500 µmol/L). These results suggest that diazoxide attenuates the mitochondrial Ca²⁺ overload in association with the depolarization of _m.

View larger version (35K): [in this window] [in a new window]   Figure 2. A, Two-dimensional confocal images of JC-1 fluorescence before (Baseline) and after treatment with ouabain (OUAB) and/or diazoxide (DIAZ). B, Summarized data for the relative changes of JC-1 fluorescence measured 30 minutes after treatment with drugs. *P<0.05 vs control (CONT); #P<0.05 vs OUAB+DIAZ.

Discussion

Among putative mechanisms of cardioprotection,¹ the hypothesis of mitochondrial Ca²⁺ handling seems to be plausible. Indeed, Holmuhamedov et al³ showed that diazoxide inhibited the Ca²⁺ uptake and depolarized _m in isolated cardiac mitochondria. Their results, however, raise methodological criticism that mitoK_ATP channels would be already open because of the ATP-free assay condition.⁹ Conflicting observations in isolated cardiac mitochondria have been reported, showing that diazoxide has little effect on Ca²⁺ uptake and _m.⁴ Thus, the results so far obtained from isolated mitochondria remain inconclusive.

The present results demonstrate that diazoxide does not affect the basal [Ca²⁺]_m in intact rat ventricular myocytes, which is inconsistent with previous findings observed in neonatal rat cardiomyocytes.³ Although such disparity may stem from the difference in cell preparations used, our results suggest that the mitoK_ATP channel does not play a significant role in regulating [Ca²⁺]_m under normal conditions. However, it should be noted that diazoxide could decrease [Ca²⁺]_m when the resting mitochondrial Ca²⁺ level was raised. In this study, we evoked mitochondrial Ca²⁺ overload experimentally by exposure to ouabain. Elevation in cytosolic Ca²⁺ concentration occurs during ischemia, and it eventually results in mitochondrial Ca²⁺ accumulation.¹⁰ More recently, functional interaction between mitoK_ATP channels and Na⁺/K⁺-ATPase in preconditioned rat hearts has been reported.¹¹ Accordingly, our results obtained in the presence of ouabain are of interest and notable. The salient finding is that diazoxide could prevent the ouabain-induced Ca²⁺ overload in mitochondria. Also, nicorandil produced similar effects to diazoxide. Moreover, 5-hydroxydecanoate abolished these effects of diazoxide and nicorandil. Although the pathways for Ca²⁺ uptake and efflux need to be defined, these results indicate that opening of mitoK_ATP channels attenuates mitochondrial Ca²⁺ overload in adult rat ventricular myocytes.

It is debatable whether K⁺ influx through mitoK_ATP channels can depolarize _m. Holmuhamedov et al³ showed that diazoxide depolarized _m by 15 mV, whereas Kowaltowski et al⁴ observed only a 1- to 2-mV depolarization of _m in isolated mitochondria. The present study has demonstrated that diazoxide can depolarize _m, and notably the degree of depolarization is augmented in the presence of ouabain. These results suggest that the effect of diazoxide on _m seems to depend on Ca²⁺ concentration. Such Ca²⁺-dependent effect of diazoxide has been demonstrated for protection against Ca²⁺ paradox injury in rat myocardium.¹² Although the Ca²⁺-dependent mechanism has not been defined, Ca²⁺-mediated signaling cascades including protein kinase C might augment the opening of mitoK_ATP channels by diazoxide. We further found that the reduction of _m by 10% resulted in >50% reduction of [Ca²⁺]_m after 30 minutes of exposure to ouabain. Because membrane-impermeable Rhod-2 free acids are not excluded from the depolarized mitochondria,⁶ the reduction of Rhod-2 fluorescence cannot be ascribed to exclusion of dye from mitochondria, but reflects the reduction of [Ca²⁺]_m. Therefore, our present results suggest that even small changes in the electrochemical driving force could potentially lead to large differences in total mitochondrial Ca²⁺ accumulation over long periods of time.

In conclusion, we have shown convincing evidence that opening of mitoK_ATP channels attenuates the Ca²⁺ overload in mitochondria, a phenomenon associated with the depolarization of _m. These results support the original hypothesis and provide insight into the mechanism by which the opening of mitoK_ATP channels confers cardioprotection.

Acknowledgments

This study was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (Nos. 13670743, 13670080, and 17670044) and by the Mitsui Life Social Welfare Foundation.

Received July 27, 2001; revision received September 28, 2001; accepted October 8, 2001.

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This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 89/10/856    most recent hh2201.100341v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ishida, H. Articles by Sato, T. Search for Related Content PubMed PubMed Citation Articles by Ishida, H. Articles by Sato, T. Related Collections Ischemic biology - basic studies Ion channels/membrane transport