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(Circulation. 2005;111:198-203.)
© 2005 American Heart Association, Inc.

Molecular Cardiology

Mitochondrial Ca²⁺-Activated K⁺ Channels in Cardiac Myocytes

A Mechanism of the Cardioprotective Effect and Modulation by Protein Kinase A

From the Department of Pharmacology, Chiba University Graduate School of Medicine, Chiba, Japan.

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

Received July 5, 2004; revision received August 30, 2004; accepted October 15, 2004.

	Abstract

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Background— The large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel in the cardiac inner mitochondrial membrane (mitoK_Ca channel) has been shown to protect the heart against ischemic injury. However, questions about the cardioprotective mechanism and the kinase-mediated regulation of mitoK_Ca channels remain to be answered.

Methods and Results— Flavoprotein fluorescence in guinea pig ventricular myocytes was measured to assay mitoK_Ca channel activity. The mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) and membrane potential (_m) were measured by loading cells with rhod-2 and JC-1, respectively. Cell death was assessed by trypan blue permeability. The BK_Ca channel opener NS1619 reversibly increased the flavoprotein oxidation in a concentration-dependent manner. NS1619 (30 µmol/L) attenuated the ouabain (1 mmol/L)-induced elevation of [Ca²⁺]_m with accompanying depolarization of _m. These effects of NS1619 were completely antagonized by the BK_Ca channel blocker paxilline (2 µmol/L) but not by the mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channel blocker 5-hydroxydecanoate (500 µmol/L). Paxilline, however, failed to block the oxidative effect of diazoxide (100 µmol/L), a mitoK_ATP channel opener. The combined application of submaximally effective concentrations of NS1619 (10 µmol/L) and diazoxide (30 µmol/L) produced additive effects. NS1619 (30 µmol/L) blunted the rate of cell death during exposure to ouabain; this cardioprotective effect was prevented by paxilline. Activation of cAMP-dependent protein kinase by 8-bromoadenosine 3'5'-cyclic monophosphate (0.5 mmol/L) and forskolin (10 µmol/L) potentiated the NS1619-induced flavoprotein oxidation.

Conclusions— Opening of mitoK_Ca channels, which is modulated by cAMP-dependent protein kinase, depolarizes the _m and attenuates the mitochondrial Ca²⁺ overload. Our study further indicates that mitoK_Ca channel activation confers cardioprotection in a manner similar to but independent of mitoK_ATP channel activation.

Key Words: potassium channels, calcium-activated • myocytes • mitochondria • cyclic AMP-dependent protein kinases

	Introduction

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A large amount of evidence has implicated mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels as a key element of cardioprotection.^1,2 A recent study by Xu et al³ has revealed that, besides the mitoK_ATP channels, large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are present on the mitochondrial inner membrane (mitoK_Ca channels) of guinea pig ventricular myocytes. They have further demonstrated that pharmacological preconditioning of rabbit heart with the BK_Ca channel opener NS1619 causes a significant reduction in infarct size, and such effect can be antagonized by the BK_Ca channel blocker paxilline.³ Despite these novel exciting observations, there are still many open questions about the nature of the mitoK_Ca channels. First, it remains to be established whether the primary target for BK_Ca channel-selective agents^4,5 in cardiac myocytes is the mitoK_Ca channels, although sarcolemmal BK_Ca channels have not been found in ventricular myocytes.⁶ Second, the mechanism by which opening of mitoK_Ca channels protects the heart against ischemic damage remains elusive. Third, although BK_Ca channel in smooth muscle cells can be activated by cAMP-dependent protein kinase (PKA),⁷ the regulatory mechanism of cardiac mitoK_Ca channels is unknown.

Therefore, the aim of the present study was to address these important questions. Flavoprotein oxidation has been used to determine the pharmacology and regulation of mitoK_ATP channels in intact cardiac myocytes.^2,8,9 It is to be expected that K⁺ entry through mitoK_Ca channels leads to net oxidation of the mitochondria in the same manner as mitoK_ATP channels. To determine whether NS1619 and paxilline can selectively act on mitoK_Ca channels, we examined the effects of these drugs on flavoprotein oxidation in guinea pig ventricular myocytes. It has been demonstrated that mitochondrial Ca²⁺ accumulation is attenuated by mitoK_ATP channel activation.^10–12 To explore the mechanism of cardioprotection, we determined whether opening of mitoK_Ca channels attenuates the mitochondrial Ca²⁺ overload. A previous study has shown that, with the use of the flavoprotein fluorescence method, mitoK_ATP channel activation is potentiated by protein kinase C (PKC).⁹ In the present study we investigated the effects of PKA and PKC activators on mitoK_Ca channel activity.

	Methods

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Cell Preparation
Adult guinea pig ventricular myocytes were isolated by collagenase digestion as previously described.¹³ Once isolated, the cells were suspended in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum at room temperature until use. All procedures complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, 1996 revision) and were approved by the Institutional Animal Care and Use Committee of Chiba University.

Flavoprotein Fluorescence
The autofluorescence of mitochondrial flavoprotein was measured by a modification of method described by Sato et al.⁹ Briefly, the cells were superfused with glucose-free Tyrode’s solution containing the following (mmol/L): NaCl 140, KCl 5.4, CaCl₂ 1.8, NaH₂PO₄ 0.33, MgCl₂ 0.5, and HEPES 5 (pH 7.4) at room temperature (22°C). Flavoprotein fluorescence was excited at 480 nm (for 200 ms every 10 seconds) and emitted at 520 nm. Relative fluorescence was calibrated with signals recorded after application of the mitochondrial uncoupler 2,4-dinitrophenol (DNP) (100 µmol/L).

Mitochondrial Ca²⁺ Concentration and Membrane Potential Measurements
The mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) was measured by loading cells with the Ca²⁺ fluorophore rhod-2. Myocytes were loaded with 10 µmol/L rhod-2 acetoxymethyl ester for 120 minutes at 4°C and then incubated for 30 minutes at 37°C in the culture medium. This 2-step cold loading/warm incubation protocol achieves exclusive loading of rhod-2 into the mitochondria.¹⁴ The mitochondrial membrane potential (_m) was monitored with the fluorescent probe JC-1. Myocytes were incubated with 0.5 µmol/L JC-1 for 10 minutes at 37°C.¹⁵ Myocytes loaded with rhod-2 or JC-1 were perfused with normal Tyrode’s solution (37°C) containing the following (mmol/L): NaCl 140, KCl 5.4, CaCl₂ 2.7, NaH₂PO₄ 0.33, MgCl₂ 0.5, HEPES 5, and glucose 5.5 (pH 7.4). Rhod-2 fluorescence was excited at 540 nm, with emission monitored through a 605-nm (55-nm bandpass) barrier filter. JC-1 was excited at 488 nm, and the red emission fluorescence was detected with the use of a long-pass filter of 580 nm.

Cytosolic Ca²⁺ Concentration Measurement
The cytosolic Ca²⁺ concentration ([Ca²⁺]_c) was measured by loading cells with fluo-3. Myocytes were incubated in a 1-µmol/L fluo-3 acetoxymethyl ester-containing loading solution at 37°C in the dark for 30 minutes, as previously described.¹⁶ The loading solution was prepared by diluting a 10-µmol/L fluo-3 stock solution, which contained 0.45% Pluronic F127, 10% dimethyl sulfoxide, and 90% FBS. Myocytes loaded with fluo-3 were perfused with normal Tyrode’s solution (37°C), and fluo-3 fluorescence was excited at 480 nm and emitted at 520 nm.

Fluorescence Imaging
Emitted fluorescence was monitored with a cooled charge-coupled device digital camera (C4742-95, Hamamatsu Photonics). The imaging of flavoprotein, rhod-2, JC-1, and fluo-3 fluorescence was analyzed for average pixel intensities of regions of interest drawn to include whole cells, after correction for background, with the use of an Aquacosmos image-processing system (Hamamatsu Photonics).

Cell Viability
In another series of experiments, cells were exposed to ouabain without loading fluorescent dyes, and cell viability was determined by trypan blue exclusion assay. After 40-minute exposure to ouabain (1 mmol/L), 20 µL of 0.4% trypan blue was added into the recording chamber, and cells permeable to trypan blue were counted and expressed as a percentage of the total viable cells before application of drugs.

Chemicals
NS1619, paxilline, diazoxide, 5-hydroxydecanoate (5HD), ouabain, forskolin, 8-bromoadenosine 3'5'-cyclic monophosphate (8Br-cAMP), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. Rhod-2 acetoxymethyl ester, JC-1, fluo-3 acetoxymethyl ester, and Pluronic F127 were purchased from Molecular Probes. DNP was purchased from Wako Pure Chemicals. NS1619, paxilline, diazoxide, forskolin, 8Br-cAMP, and PMA were dissolved in dimethyl sulfoxide before they were added to the experimental solution, and final concentration of solvent was 0.1%. Ouabain, 5HD, and DNP were dissolved in the perfusate.

Statistical Analysis
Data are expressed as mean±SEM, and the number of cells or experiments is shown as n. Statistical comparisons were made with the use of Student t test or ANOVA combined with Fisher post hoc test, as appropriate. A value of P<0.05 was regarded as significant.

	Results

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Figure 1A through 1C shows representative time course of changes in flavoprotein fluorescence in a cell exposed to NS1619 and/or paxilline. NS1619 (30 µmol/L) reversibly oxidized flavoproteins, and subsequent exposure to DNP (100 µmol/L) led to full oxidation of flavoproteins (Figure 1A). In the presence of paxilline (2 µmol/L), NS1619 failed to oxidize flavoproteins (Figure 1B). Furthermore, the NS1619-induced flavoprotein oxidation was suppressed by subsequent application of paxilline (Figure 1C). Figure 1D shows that NS1619 oxidized flavoproteins even in the presence of the mitoK_ATP channel blocker 5HD (500 µmol/L). On the other hand, as shown in Figure 1E, paxilline did not inhibit the flavoprotein oxidation induced by the mitoK_ATP channel opener diazoxide (100 µmol/L). Figure 1F further shows that oxidative effects of NS1619 and diazoxide were additive: diazoxide (30 µmol/L) augmented the flavoprotein oxidation when applied after the effect of NS1619 (10 µmol/L) had reached steady state. As summarized in Figure 1G, NS1619 (3 to 100 µmol/L) oxidized flavoproteins in a concentration-dependent manner. Paxilline completely inhibited the oxidative effects of NS1619 (30 µmol/L) from 25±3% to 4±1% of the DNP value (P<0.0001). 5HD (500 µmol/L) did not affect the oxidative effect of 30 µmol/L NS1619 (27±3%; P=NS versus NS1619 alone). In addition, diazoxide (100 µmol/L) increased flavoprotein oxidation (32±4%) even in the presence of paxilline, and this degree of oxidation was comparable to that obtained by applying diazoxide alone (33±3%). The combined application of NS1619 (10 µmol/L) and diazoxide (30 µmol/L) had an additive effect and significantly increased flavoprotein oxidation to 41±7% (P<0.05) compared with NS1619 (16±2%) or diazoxide (21±3%) alone. These results suggest that NS1619 activates whereas paxilline inhibits the mitoK_Ca channels, independently of the mitoK_ATP channels.

View larger version (39K): [in this window] [in a new window]   Figure 1. Effects of NS1619 and paxilline on flavoprotein (FP) fluorescence. A, NS1619 (NS) (30 µmol/L) induced a reversible flavoprotein oxidation. B and C, Paxilline (PX) (2 µmol/L) completely inhibited the oxidative effect of NS1619. D, 5HD (500 µmol/L) did not affect the oxidative effect of NS1619. E, Paxilline did not block the oxidative effect of diazoxide (DZ) (100 µmol/L). F, Additive effects of NS1619 (10 µmol/L) and diazoxide (30 µmol/L) on flavoprotein oxidation. Bars indicate periods when the cells were exposed to drug. Flavoprotein fluorescence was calibrated by exposing the cells to DNP (100 µmol/L) at the end of experiments. G, Summarized pooled data for percentage of flavoprotein oxidation. NS(3, 10, 30, 100) indicates NS1619 (3, 10, 30, 100 µmol/L, respectively); DZ(30, 100), diazoxide (30, 100 µmol/L, respectively).

We then examined whether opening of mitoK_Ca channels by NS1619 attenuates the mitochondrial Ca²⁺ overload. As summarized in Figure 2A, exposing cells to ouabain (1 mmol/L) for 30 minutes evoked the elevation of [Ca²⁺]_m, and the intensity of rhod-2 fluorescence increased to 233±11% of baseline (P<0.0001 versus control). Coadministration of 10 and 30 µmol/L NS1619 significantly and dose-dependently attenuated the ouabain-induced increase in rhod-2 fluorescence to 184±19% (P<0.05 versus ouabain) and 138±6% (P<0.01 versus ouabain) of baseline, respectively. The effect of 30 µmol/L NS1619 was antagonized by 2 µmol/L paxilline (216±19% of baseline; P<0.05 versus ouabain plus NS1619). Contrarily, even in the presence of 5HD (500 µmol/L), NS1619 (30 µmol/L) attenuated the elevation of [Ca²⁺]_m during exposure to ouabain (147±7% of baseline; P<0.01 versus ouabain). Paxilline did not prevent the protective effect of diazoxide (100 µmol/L), and the intensity of rhod-2 fluorescence was attenuated to 151±9% of baseline (P<0.01 versus ouabain), a value comparable to that achieved with diazoxide (100 µmol/L) alone. The combined application of submaximally effective concentrations of NS1619 (10 µmol/L) and of diazoxide (30 µmol/L) produced an additive effect (145±9% of baseline; P<0.01 versus ouabain).

View larger version (65K): [in this window] [in a new window]   Figure 2. Summarized data for relative changes in rhod-2 (A), JC-1 (B), and fluo-3 (C) fluorescence measured after 30-minute exposure to drugs. CONT indicates control; OUAB, ouabain (1 mmol/L); NS(10, 30), NS1619 (10, 30 µmol/L, respectively); PX, paxilline (2 µmol/L); 5HD, 5-hydroxydecanoate (500 µmol/L); and DZ(30, 100), diazoxide (30, 100 µmol/L, respectively). *P<0.05, **P<0.01 vs OUAB; ¶P<0.05 vs OUAB+NS(30).

Figure 2B shows the summarized data for the relative changes of JC-1 fluorescence measured 30 minutes after treatment of drugs. NS1619 (30 µmol/L) alone significantly reduced the intensity of JC-1 fluorescence to 89±2% of baseline (P<0.05). Moreover, NS1619 (30 µmol/L) significantly reduced the intensity of JC-1 fluorescence during application of ouabain (1 mmol/L) to 81±5% of baseline (P<0.05 versus ouabain), and such effect was antagonized by 2 µmol/L paxilline (102±2% of baseline; P<0.05 versus ouabain plus NS1619). These results suggest that opening of mitoK_Ca channels by NS1619 depolarized _m. As shown in Figure 2C, measurement of [Ca²⁺]_c by fluo-3 fluorescence indicated that exposure to ouabain (1 mmol/L) for 30 minutes caused a marked rise in [Ca²⁺]_c, and the intensity of fluo-3 fluorescence significantly increased to 170±19% of baseline (P<0.0001 versus control). NS1619 (30 µmol/L) did not significantly alter the [Ca²⁺]_c in the presence and absence of ouabain.

The [Ca²⁺]_m increase induced by ouabain eventually resulted in cell death. Figure 3 plots the fraction of cells stained by 40-minute exposure to ouabain (1 mmol/L) as a percentage of the total number of viable cells before treatment. Exposure to ouabain significantly increased the percentage of stained cells to 42±5% (P<0.0001) compared with 5±2% in control. Although NS1619 (30 µmol/L) did not affect the cell death in the absence of ouabain, treatment with NS1619 significantly decreased the percentage of stained cells during exposure to ouabain (25±2%; P<0.05). The cardioprotective effect of NS1619 was abolished by 2 µmol/L paxilline (44±4%; P<0.05 versus ouabain plus NS1619).

View larger version (47K): [in this window] [in a new window]   Figure 3. Summarized effects of NS1619 on cell death during application of ouabain. Cells stained 40 minutes after exposure to ouabain (1 mmol/L) were plotted as a percentage of total viable cells before application of drugs. CONT indicates control; OUAB, ouabain (1 mmol/L); NS(30), NS1619 (30 µmol/L); and PX, paxilline (2 µmol/L). *P<0.05 vs OUAB; ¶P<0.05 vs OUAB+NS(30).

To study the protein kinase-dependent modulation of mitoK_Ca channels, we examined the effects of activators of PKA and PKC on flavoprotein oxidation. Figure 4A shows a representative effect of the cell-permeable cAMP analogue 8Br-cAMP on NS1619-induced flavoprotein oxidation. 8Br-cAMP (0.5 mmol/L) enhanced the oxidative effect of NS1619 (30 µmol/L) when applied after the effect of NS1619 had reached steady state. Although 8Br-cAMP alone had no significant effect on flavoprotein fluorescence (Figure 4E), the drug significantly increased the NS1619-induced flavoprotein oxidation (NS1619 of 23±4% versus NS1619 plus 8Br-cAMP of 41±7%; P<0.05; Figure 4B). Figure 4E further shows that forskolin, a direct activator of adenylate cylcase,¹⁷ mimicked the potentiating effect of 8Br-cAMP, and exposure to 10 µmol/L forskolin significantly increased NS1619-induced flavoprotein oxidation from 24±4% to 38±5% (P<0.05). In contrast, as shown in Figure 4C and 4D, PKC-activating phorbol ester PMA (100 nmol/L) did not alter the oxidative effect of NS1619 (NS1619 of 22±5% versus NS1619 plus PMA of 21±4%; P=NS). Furthermore, 8Br-cAMP did not augment the flavoprotein oxidation induced by the mitoK_ATP channel opener diazoxide (100 µmol/L) (diazoxide of 34±6% versus diazoxide plus 8Br-cAMP of 31±6%; P=NS). These results indicate that mitoK_Ca channels are modulated by PKA but not by PKC.

View larger version (40K): [in this window] [in a new window]   Figure 4. Effects of PKA and PKC activators on NS1619-induced flavoprotein (FP) oxidation. A and C, Representative data showing effects of 8Br-cAMP (0.5 mmol/L) and PMA (100 nmol/L) on NS1619 (30 µmol/L)-induced oxidation. Bars indicate periods when cells were exposed to each drug. Flavoprotein fluorescence was calibrated by exposing the cells to DNP (100 µmol/L) at the end of experiments. B and D, Summarized data for percentage of NS1619-induced flavoprotein oxidation measured in the absence and presence of 8Br-cAMP or PMA. *P<0.05 vs NS1619 alone. E, Summarized data for percentage of flavoprotein oxidation. Forskolin (10 µmol/L) potentiated the NS1619-induced flavoprotein oxidation, whereas 8Br-cAMP (0.5 mmol/L) failed to augment the oxidative effect of diazoxide (100 µmol/L). NS(30) indicates NS1619 (30 µmol/L); DZ(100), diazoxide (100 µmol/L).

	Discussion

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The existence of mitoK_Ca channels has been found in a patch-clamp study performed on mitoplasts prepared from cardiac myocytes,³ although proteomic analysis of mitochondrial inner membrane did not allow identification of mitoK_Ca channels.¹⁸ A role for mitoK_Ca channel in K⁺ transport into the matrix has been proposed, and such K⁺ influx can be augmented by the BK_Ca channel opener NS1619.³ We therefore reasoned that, in a manner similar to that for the mitoK_ATP channel, K⁺ influx through mitoK_Ca channels accelerates electron transfer by the respiratory chain and leads to net oxidation of mitochondria if uncompensated by electron donors.^2,8 As expected, the BK_Ca channel opener NS1619 reversibly oxidized flavoproteins in a concentration-dependent manner; this effect was antagonized by the BK_Ca channel blocker paxilline. The specificity of NS1619 and paxilline for mitoK_Ca channel is further supported by the following results. The oxidative effect of NS1619 was unaffected by the mitoK_ATP channel blocker 5HD, consistent with previous study in patch-clamping mitoplasts.² Paxilline failed to inhibit the flavoprotein oxidation induced by the mitoK_ATP channel opener diazoxide. Taken together, these results indicate that NS1619 activates whereas paxilline inhibits the mitoK_Ca channels, without affecting cardiac mitoK_ATP channels.

Previous studies failed to demonstrate the oxidation of flavoproteins by diazoxide, presumably because measurements of flavoprotein fluorescence were performed in the presence of glucose with the use of freshly isolated cardiomyocytes.^19,20 In our experiments, cells were kept in culture medium to stabilize the mitochondrial redox state. Moreover, to minimize the production of electron donors and thereby amplify the small signal due to opening of mitoK_Ca and/or mitoK_ATP channels,^1,2 cells were perfused with glucose-free Tyrode’s solution. Consequently, we have succeeded in detecting flavoprotein oxidation induced not only by diazoxide but also by NS1619. Notably, we found that oxidation of flavoproteins by both NS1619 and diazoxide was additive. These results suggest that mitochondrial oxidation induced by opening of mitoK_Ca and mitoK_ATP channels occurs independently of each other and further indicate that the flavoprotein fluorescence is a useful index to assay not only mitoK_ATP but also mitoK_Ca channel activity in intact cardiomyocytes.

Ishida et al^11,21 previously reported that opening of mitoK_ATP channels by diazoxide and nicorandil depolarizes the _m and attenuates the mitochondrial Ca²⁺ overload during application of ouabain. Using the same experimental design, in the present study we examined the effect of NS1619, a potent mitoK_Ca channel opener, on the ouabain-induced mitochondrial Ca²⁺ overload. Elevation of [Ca²⁺]_c occurs during application of ouabain, as evidenced by increased fluo-3 fluorescence and which eventually results in the mitochondrial Ca²⁺ overload. NS1619 attenuated the mitochondrial Ca²⁺ overload, and the increase in rhod-2 fluorescence was blunted, without significant changes in fluo-3 fluorescence. This effect of NS1619 was antagonized by the mitoK_Ca channel blocker paxilline. The independence of mitoK_ATP channels was confirmed by ensuring that the effect of NS1619 was unaffected by 5HD, and paxilline failed to prevent the effect of diazoxide. Furthermore, we found that NS1619 produced a small reduction of _m that was blocked by paxilline. Because Ca²⁺ uptake into mitochondria is driven primarily by the _m,²² the decrease in _m can reduce the driving force for Ca²⁺ influx into mitochondria. It seems that even a small decrease in the electrochemical driving force for Ca²⁺ entry leads to a large reduction in [Ca²⁺]_m over long periods of time. Thus, our results indicate that opening of mitoK_Ca channels by NS1619 attenuates the mitochondrial Ca²⁺ overload with accompanying depolarization of _m.

Consistent with the effects of NS1619 on mitochondrial Ca²⁺ overload, NS1619 could blunt the percentage of cell death; this protective effect was abrogated by paxilline. Cell death results, at least in part, from impairment of mitochondrial Ca²⁺ homeostasis.²³ It is therefore reasonable to assume that such an effect of mitoK_Ca channel can be attributed to the mechanism of cardioprotection. Although we used ouabain in an attempt to produce mitochondrial Ca²⁺ overload experimentally, the mode of the ouabain-induced cell death by either necrosis or apoptosis remains to be determined. It has been reported that mitoK_ATP channel opening prevents apoptosis, presumably by inhibiting the activation of the mitochondrial permeability transition pore.²⁴ Further investigations are required to demonstrate whether mitoK_Ca channel opening exerts its protective effect by inhibiting both necrosis and apoptosis, as is the case with the mitoK_ATP channel.

Modulation of the mitoK_ATP channel by PKC was revealed by a previous study that showed that PKC-activating phorbol ester PMA potentiates the mitochondrial oxidation induced by diazoxide in rabbit ventricular myocytes.⁹ Moreover, potentiation of mitoK_ATP channel activation by adenosine via a PKC-mediated pathway was demonstrated with the same method.²⁵ In the present study mitoK_Ca channel activity was indexed by measuring flavoprotein oxidation, and we found that 8Br-cAMP, but not PMA, potentiated the NS1619-induced flavoprotein oxidation. This potentiation could not be due to nonspecific action of 8Br-cAMP because 8Br-cAMP alone did not increase flavoprotein oxidation, nor did treatment with 8Br-cAMP enhance the diazoxide-induced oxidation. Furthermore, the NS1619-induced flavoprotein oxidation was similarly potentiated by another PKA activator, forskolin. Therefore, these findings suggest that the mitoK_Ca channel is modulated by PKA, as is the case for the BK_Ca channel in smooth muscle.⁷ We found that 8Br-cAMP did not significantly increase the intensity of rhod-2 fluorescence (105±4% of baseline; n=8; not shown). Although further study is needed to know how PKA interacts with the mitoK_Ca channel, it seems unlikely that the enhanced response of NS1619 could be secondary to the elevation of [Ca²⁺]_m.

Xu et al³ demonstrated that pharmacological preconditioning of rabbit heart with the BK_Ca channel opener NS1619 significantly reduces infarct size, and such effect can be antagonized by the BK_Ca channel blocker paxilline. However, the mechanism by which preconditioning with NS1619 results in infarct size reduction remains poorly understood. Our findings may provide insight into a mechanism responsible for mitoK_Ca channel-mediated cardioprotection, ie, mitoK_Ca activation blunts mitochondrial Ca²⁺ accumulation during ischemia. Interestingly, ischemic preconditioning increases myocardial cAMP level and activates PKA in rat hearts,²⁶ but a role of PKA in the process of ischemic preconditioning is as yet incompletely understood. Our present results further suggest that mitoK_Ca channel activity can be augmented by PKA and thus may provide a mechanistic link between the signal transduction of PKA-dependent preconditioning and its likely effector.

In conclusion, our data indicate that (1) NS1619 and paxilline target cardiac mitoK_Ca channels, (2) opening of mitoK_Ca channels attenuates mitochondrial Ca²⁺ overload with accompanying depolarization of _m, and (3) mitoK_Ca channels are modulated by PKA. Notably, the combined application of submaximally effective concentrations of NS1619 and diazoxide produced additive effects on flavoprotein oxidation and ouabain-induced mitochondrial Ca²⁺ overload. These combined effects of NS1619 and diazoxide can be rationalized by the model proposed in Figure 5, namely, mitochondrial K⁺ influx through the distinct types of channels occurs independently of each other and then confers cardioprotection in a similar manner. However, some caution may be exercised in extrapolating our results to ischemic myocardium. Because the relevant regulatory site for Ca²⁺ on the mitoK_Ca channel is likely to face the mitochondrial matrix,³ the attenuation of matrix Ca²⁺ overload by opening of mitoK_ATP channels may rather impede mitoK_Ca channel activation during ischemia. In this regard, further studies concerning possible interaction of these 2 K⁺ channels in ischemic myocardium are needed to assign the relative roles for mitoK_Ca and mitoK_ATP channels in cardioprotection.

View larger version (33K): [in this window] [in a new window]   Figure 5. Schematic representation of mechanism of protection mediated by mitoKCa and mitoKATP channels. PKA potentiates mitoKCa channel activation, whereas PKC potentiates mitoKATP channel activation. Opening of mitoKCa channel and mitoKATP channels depolarizes m, which reduces the driving force for Ca2+ influx, thereby attenuating the mitochondrial Ca2+ overload.

	Acknowledgments

 
This study was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science, Mitsui Life Social Welfare Foundation, K. Watanabe Research Foundation, and Vehicle Racing Commemorative Foundation.

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