Mitochondrial Ca2+-Activated K+ Channels in Cardiac Myocytes
http://www.100md.com
循环学杂志 2005年第1期
the Department of Pharmacology, Chiba University Graduate School of Medicine, Chiba, Japan.
Abstract
Background— The large-conductance Ca2+-activated K+ (BKCa) channel in the cardiac inner mitochondrial membrane (mitoKCa channel) has been shown to protect the heart against ischemic injury. However, questions about the cardioprotective mechanism and the kinase-mediated regulation of mitoKCa channels remain to be answered.
Methods and Results— Flavoprotein fluorescence in guinea pig ventricular myocytes was measured to assay mitoKCa 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 BKCa 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 BKCa channel blocker paxilline (2 μmol/L) but not by the mitochondrial ATP-sensitive K+ (mitoKATP) channel blocker 5-hydroxydecanoate (500 μmol/L). Paxilline, however, failed to block the oxidative effect of diazoxide (100 μmol/L), a mitoKATP 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 mitoKCa channels, which is modulated by cAMP-dependent protein kinase, depolarizes the m and attenuates the mitochondrial Ca2+ overload. Our study further indicates that mitoKCa channel activation confers cardioprotection in a manner similar to but independent of mitoKATP channel activation.
Key Words: potassium channels, calcium-activated ; myocytes ; mitochondria ; cyclic AMP-dependent protein kinases
Introduction
A large amount of evidence has implicated mitochondrial ATP-sensitive K+ (mitoKATP) channels as a key element of cardioprotection.1,2 A recent study by Xu et al3 has revealed that, besides the mitoKATP channels, large-conductance Ca2+-activated K+ (BKCa) channels are present on the mitochondrial inner membrane (mitoKCa channels) of guinea pig ventricular myocytes. They have further demonstrated that pharmacological preconditioning of rabbit heart with the BKCa channel opener NS1619 causes a significant reduction in infarct size, and such effect can be antagonized by the BKCa channel blocker paxilline.3 Despite these novel exciting observations, there are still many open questions about the nature of the mitoKCa channels. First, it remains to be established whether the primary target for BKCa channel-selective agents4,5 in cardiac myocytes is the mitoKCa channels, although sarcolemmal BKCa channels have not been found in ventricular myocytes.6 Second, the mechanism by which opening of mitoKCa channels protects the heart against ischemic damage remains elusive. Third, although BKCa channel in smooth muscle cells can be activated by cAMP-dependent protein kinase (PKA),7 the regulatory mechanism of cardiac mitoKCa 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 mitoKATP channels in intact cardiac myocytes.2,8,9 It is to be expected that K+ entry through mitoKCa channels leads to net oxidation of the mitochondria in the same manner as mitoKATP channels. To determine whether NS1619 and paxilline can selectively act on mitoKCa 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 mitoKATP channel activation.10–12 To explore the mechanism of cardioprotection, we determined whether opening of mitoKCa channels attenuates the mitochondrial Ca2+ overload. A previous study has shown that, with the use of the flavoprotein fluorescence method, mitoKATP channel activation is potentiated by protein kinase C (PKC).9 In the present study we investigated the effects of PKA and PKC activators on mitoKCa channel activity.
Methods
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
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 mitoKATP 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 mitoKATP 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 mitoKCa channels, independently of the mitoKATP channels.
We then examined whether opening of mitoKCa 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).
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 mitoKCa 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).
To study the protein kinase-dependent modulation of mitoKCa 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 mitoKATP channel opener diazoxide (100 μmol/L) (diazoxide of 34±6% versus diazoxide plus 8Br-cAMP of 31±6%; P=NS). These results indicate that mitoKCa channels are modulated by PKA but not by PKC.
Discussion
The existence of mitoKCa 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 mitoKCa channels.18 A role for mitoKCa channel in K+ transport into the matrix has been proposed, and such K+ influx can be augmented by the BKCa channel opener NS1619.3 We therefore reasoned that, in a manner similar to that for the mitoKATP channel, K+ influx through mitoKCa 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 BKCa channel opener NS1619 reversibly oxidized flavoproteins in a concentration-dependent manner; this effect was antagonized by the BKCa channel blocker paxilline. The specificity of NS1619 and paxilline for mitoKCa channel is further supported by the following results. The oxidative effect of NS1619 was unaffected by the mitoKATP channel blocker 5HD, consistent with previous study in patch-clamping mitoplasts.2 Paxilline failed to inhibit the flavoprotein oxidation induced by the mitoKATP channel opener diazoxide. Taken together, these results indicate that NS1619 activates whereas paxilline inhibits the mitoKCa channels, without affecting cardiac mitoKATP 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 mitoKCa and/or mitoKATP 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 mitoKCa and mitoKATP channels occurs independently of each other and further indicate that the flavoprotein fluorescence is a useful index to assay not only mitoKATP but also mitoKCa channel activity in intact cardiomyocytes.
Ishida et al11,21 previously reported that opening of mitoKATP 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 mitoKCa 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 mitoKCa channel blocker paxilline. The independence of mitoKATP 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 mitoKCa 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 mitoKCa 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 mitoKATP channel opening prevents apoptosis, presumably by inhibiting the activation of the mitochondrial permeability transition pore.24 Further investigations are required to demonstrate whether mitoKCa channel opening exerts its protective effect by inhibiting both necrosis and apoptosis, as is the case with the mitoKATP channel.
Modulation of the mitoKATP 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 mitoKATP channel activation by adenosine via a PKC-mediated pathway was demonstrated with the same method.25 In the present study mitoKCa 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 mitoKCa channel is modulated by PKA, as is the case for the BKCa 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 mitoKCa 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 BKCa channel opener NS1619 significantly reduces infarct size, and such effect can be antagonized by the BKCa 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 mitoKCa channel-mediated cardioprotection, ie, mitoKCa 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 mitoKCa 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 mitoKCa channels, (2) opening of mitoKCa channels attenuates mitochondrial Ca2+ overload with accompanying depolarization of m, and (3) mitoKCa 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 mitoKCa channel is likely to face the mitochondrial matrix,3 the attenuation of matrix Ca2+ overload by opening of mitoKATP channels may rather impede mitoKCa 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 mitoKCa and mitoKATP channels in cardioprotection.
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.
References
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Abstract
Background— The large-conductance Ca2+-activated K+ (BKCa) channel in the cardiac inner mitochondrial membrane (mitoKCa channel) has been shown to protect the heart against ischemic injury. However, questions about the cardioprotective mechanism and the kinase-mediated regulation of mitoKCa channels remain to be answered.
Methods and Results— Flavoprotein fluorescence in guinea pig ventricular myocytes was measured to assay mitoKCa 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 BKCa 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 BKCa channel blocker paxilline (2 μmol/L) but not by the mitochondrial ATP-sensitive K+ (mitoKATP) channel blocker 5-hydroxydecanoate (500 μmol/L). Paxilline, however, failed to block the oxidative effect of diazoxide (100 μmol/L), a mitoKATP 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 mitoKCa channels, which is modulated by cAMP-dependent protein kinase, depolarizes the m and attenuates the mitochondrial Ca2+ overload. Our study further indicates that mitoKCa channel activation confers cardioprotection in a manner similar to but independent of mitoKATP channel activation.
Key Words: potassium channels, calcium-activated ; myocytes ; mitochondria ; cyclic AMP-dependent protein kinases
Introduction
A large amount of evidence has implicated mitochondrial ATP-sensitive K+ (mitoKATP) channels as a key element of cardioprotection.1,2 A recent study by Xu et al3 has revealed that, besides the mitoKATP channels, large-conductance Ca2+-activated K+ (BKCa) channels are present on the mitochondrial inner membrane (mitoKCa channels) of guinea pig ventricular myocytes. They have further demonstrated that pharmacological preconditioning of rabbit heart with the BKCa channel opener NS1619 causes a significant reduction in infarct size, and such effect can be antagonized by the BKCa channel blocker paxilline.3 Despite these novel exciting observations, there are still many open questions about the nature of the mitoKCa channels. First, it remains to be established whether the primary target for BKCa channel-selective agents4,5 in cardiac myocytes is the mitoKCa channels, although sarcolemmal BKCa channels have not been found in ventricular myocytes.6 Second, the mechanism by which opening of mitoKCa channels protects the heart against ischemic damage remains elusive. Third, although BKCa channel in smooth muscle cells can be activated by cAMP-dependent protein kinase (PKA),7 the regulatory mechanism of cardiac mitoKCa 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 mitoKATP channels in intact cardiac myocytes.2,8,9 It is to be expected that K+ entry through mitoKCa channels leads to net oxidation of the mitochondria in the same manner as mitoKATP channels. To determine whether NS1619 and paxilline can selectively act on mitoKCa 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 mitoKATP channel activation.10–12 To explore the mechanism of cardioprotection, we determined whether opening of mitoKCa channels attenuates the mitochondrial Ca2+ overload. A previous study has shown that, with the use of the flavoprotein fluorescence method, mitoKATP channel activation is potentiated by protein kinase C (PKC).9 In the present study we investigated the effects of PKA and PKC activators on mitoKCa channel activity.
Methods
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
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 mitoKATP 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 mitoKATP 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 mitoKCa channels, independently of the mitoKATP channels.
We then examined whether opening of mitoKCa 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).
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 mitoKCa 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).
To study the protein kinase-dependent modulation of mitoKCa 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 mitoKATP channel opener diazoxide (100 μmol/L) (diazoxide of 34±6% versus diazoxide plus 8Br-cAMP of 31±6%; P=NS). These results indicate that mitoKCa channels are modulated by PKA but not by PKC.
Discussion
The existence of mitoKCa 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 mitoKCa channels.18 A role for mitoKCa channel in K+ transport into the matrix has been proposed, and such K+ influx can be augmented by the BKCa channel opener NS1619.3 We therefore reasoned that, in a manner similar to that for the mitoKATP channel, K+ influx through mitoKCa 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 BKCa channel opener NS1619 reversibly oxidized flavoproteins in a concentration-dependent manner; this effect was antagonized by the BKCa channel blocker paxilline. The specificity of NS1619 and paxilline for mitoKCa channel is further supported by the following results. The oxidative effect of NS1619 was unaffected by the mitoKATP channel blocker 5HD, consistent with previous study in patch-clamping mitoplasts.2 Paxilline failed to inhibit the flavoprotein oxidation induced by the mitoKATP channel opener diazoxide. Taken together, these results indicate that NS1619 activates whereas paxilline inhibits the mitoKCa channels, without affecting cardiac mitoKATP 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 mitoKCa and/or mitoKATP 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 mitoKCa and mitoKATP channels occurs independently of each other and further indicate that the flavoprotein fluorescence is a useful index to assay not only mitoKATP but also mitoKCa channel activity in intact cardiomyocytes.
Ishida et al11,21 previously reported that opening of mitoKATP 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 mitoKCa 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 mitoKCa channel blocker paxilline. The independence of mitoKATP 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 mitoKCa 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 mitoKCa 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 mitoKATP channel opening prevents apoptosis, presumably by inhibiting the activation of the mitochondrial permeability transition pore.24 Further investigations are required to demonstrate whether mitoKCa channel opening exerts its protective effect by inhibiting both necrosis and apoptosis, as is the case with the mitoKATP channel.
Modulation of the mitoKATP 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 mitoKATP channel activation by adenosine via a PKC-mediated pathway was demonstrated with the same method.25 In the present study mitoKCa 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 mitoKCa channel is modulated by PKA, as is the case for the BKCa 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 mitoKCa 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 BKCa channel opener NS1619 significantly reduces infarct size, and such effect can be antagonized by the BKCa 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 mitoKCa channel-mediated cardioprotection, ie, mitoKCa 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 mitoKCa 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 mitoKCa channels, (2) opening of mitoKCa channels attenuates mitochondrial Ca2+ overload with accompanying depolarization of m, and (3) mitoKCa 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 mitoKCa channel is likely to face the mitochondrial matrix,3 the attenuation of matrix Ca2+ overload by opening of mitoKATP channels may rather impede mitoKCa 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 mitoKCa and mitoKATP channels in cardioprotection.
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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