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Mitochondrial Ca²⁺-activated K⁺ channels more efficiently reduce mitochondrial Ca²⁺ overload in rat ventricular myocytes

¹Mitochondrial Signaling Laboratory, Mitochondria Research Group, Department of Physiology and Biophysics, College of Medicine, Biohealth Products Research Center, Cardiovascular and Metabolic Disease Research Center, Inje University, Busan, Korea; and ²Department of Medicine, University of California, San Diego, La Jolla, California

Submitted 24 July 2006 ; accepted in final form 21 February 2007

	ABSTRACT

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We investigated the role of the mitochondrial ATP-sensitive K⁺ (K_ATP) channel, the mitochondrial big-conductance Ca²⁺-activated K⁺ (BK_Ca) channel, and the mitochondrial permeability transition pore (MPTP) in the ouabain-induced increase of mitochondrial Ca²⁺ in native rat ventricular myocytes by loading cells with rhod 2-AM. To overload mitochondrial Ca²⁺, we pretreated cells with ouabain before applying mitochondrial K_ATP or BK_Ca channel and/or MPTP opener. Ouabain (1 mM) increased the rhod 2-sensitive fluorescence intensity (160 ± 5.0% of control), which was dramatically decreased to the control level on application of diazoxide and NS-1619 in a dose-dependent manner (half-inhibition concentrations of 78.3 and 7.78 µM for diazoxide and NS-1619, respectively). This effect was reversed by selective inhibition of the mitochondrial K_ATP channel by 5-hydroxydecanoate, the mitochondrial BK_Ca channel by paxilline, and the MPTP by cyclosporin A. Although diazoxide did not efficiently reduce mitochondrial Ca²⁺ during prolonged exposure to ouabain, NS-1619 reduced mitochondrial Ca²⁺. These results suggest that although mitochondrial BK_Ca and K_ATP channels contribute to reduction of ouabain-induced mitochondrial Ca²⁺ overload, activation of the mitochondrial BK_Ca channel more efficiently reduces ouabain-induced mitochondrial Ca²⁺ overload in our experimental model.

mitochondrial BK_Ca channel; mitochondrial K_ATP channel; mitochondrial permeability transition pore; mitochondrial Ca²⁺; cardioprotection

During I/R injury, mitochondrial Ca²⁺ overload occurs via Ca²⁺ uniporter. The developed Ca²⁺ gradient between the cytosol and the mitochondrial matrix can be counteracted by mitochondrial Na⁺/Ca²⁺ exchange, Ca²⁺ uniporter reverse mode, and the MPTP. Recent studies have proposed that the MPTP is also a candidate for mitochondrial Ca²⁺ modulation (2, 4, 11, 12, 15), but this mechanism is unclear.

In the present study, mitochondrial Ca²⁺ overload was evoked by exposure to ouabain. Elevation of cytosolic Ca²⁺ concentration during ischemia eventually results in mitochondrial Ca²⁺ accumulation. Diazoxide could decrease mitochondrial Ca²⁺ when the resting mitochondrial Ca²⁺ level is raised (17). In this study, we used the mitochondrial Ca²⁺-sensitive fluorescent dye rhod 2-AM to determine the role of mitochondrial K_ATP and/or BK_Ca channel openers in attenuation of ouabain-induced increase of mitochondrial Ca²⁺. Additionally, we used the MPTP inhibitor cyclosporin A to examine the role of the MPTP in mitochondrial Ca²⁺ release. This study may be the first to compare the efficiency with which mitochondrial BK_Ca and K_ATP channels reduce mitochondrial Ca²⁺ during short- and long-duration exposure to ouabain under the same experimental conditions.

	METHODS

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Isolation of single ventricular cardiomyocytes. All experimental procedures were reviewed and approved by the Institutional Review Board (IRB) of Animal, Inje University College of Medicine, and the procedure was carried out in accordance with the guidelines of the IRB on the ethical use of animals. Single ventricular myocytes were isolated from rat heart. At 8 wk of age, Sprague-Dawley rats (280–320 g body wt) were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt) and heparin (300 U/ml). The heart was rapidly removed by thoracotomy, and the aorta was cannulated. The whole heart was mounted on a Langendorff apparatus and perfused with oxygenated normal Tyrode solution at 37°C for 5–6 min to flush blood. The heart was perfused with Ca²⁺-free Tyrode solution for 5 min and then with Ca²⁺-free Tyrode solution containing 0.01% collagenase (1 mg/10 ml; Yakult) for 25–30 min and, finally, with Kraft-Brühe (KB) solution for 5–10 min. After separation of the left ventricle from the perfused heart, the single ventricular myocytes were isolated by gentle agitation in KB solution, stored at 4°C, and used within 12 h.

Solutions and drugs. Normal Tyrode solution contained (mM) 143 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 5.5 glucose, and 5 HEPES, with pH adjusted to 7.4 with KOH. The modified KB solution contained (mM) 25 KCl, 10 KH₂PO₄, 16 KOH, 80 glutamic acid, 10 taurine, 14 oxalic acid, 10 HEPES, and 11 glucose, with pH adjusted to 7.4 with KOH.

The fluorescent dye rhod 2-AM was purchased from Molecular Probes, prepared as 1 mM stock solutions in DMSO, and diluted into test solution before each experiment. Diazoxide, 5-hydroxydecanoate (5-HD), ouabain, NS-1619, paxilline, atractyloside, and cyclosporin A were purchased from Sigma (St. Louis, MO). The final concentration of DMSO in the bath solution (<0.1%) was previously shown not to affect the recorded intensities.

Mitochondrial Ca²⁺ measurement. The mitochondrial Ca²⁺-sensitive fluorescent dye rhod 2-AM was used to trace the change in mitochondrial Ca²⁺. A cold-warm incubation protocol (25, 31) was used to exclusively load the mitochondria with rhod 2-AM. Briefly, myocytes were incubated with rhod 2-AM for 120 min at 4°C and then incubated for 10 min at 37°C.

Fluorescence measurement. Rhod 2-AM-loaded cells were excited at 514 nm with a neon laser, and emitted signals were collected through a 580-nm long-pass filter. Fluorescence was recorded every 10 s. Experiments were performed with a laser scanning confocal microscope (510 META, Carl Zeiss, Jena, Germany) coupled to an inverted microscope (Axiovert 200M, Carl Zeiss) with a x40 water-immersion objective lens, optimal laser lines, and filter. Cells were excited with a 514-nm argon laser, and images were acquired using a >560-nm long-pass filter. Fluorescence of rhod 2 was excited at 514 nm and emitted at 588 nm. Fluorescence was monitored every 10 s via time-series image capture. Images were digitized at eight bits and analyzed using LSM-510 META software (Carl Zeiss).

Image processing and statistical analysis. Fluorescent images were processed with LSM-510 META software, and the imaging systems enabled independent recording from several cells in the field of view. To represent changes in fluorescence intensity over time, regions of interest in the cells were outlined/highlighted, and the intensity of the chosen region in the image was calculated. In all cases, the fluorescence intensity was normalized to the initial value (F₀) recorded in normal Tyrode solution and compared with the change of intensity as the relative fluorescence (F₁/F₀).

Experimental protocol. Rat ventricular myocytes were loaded with rhod 2-AM and then pretreated with ouabain to overload mitochondrial Ca²⁺. The myocytes were exposed to ouabain for 10 min, which is the minimum time required for effective increase in mitochondrial Ca²⁺. In some experiments (see Figs. 5 and 6), however, duration of ouabain exposure was increased to 30 min to more overload mitochondrial Ca²⁺. In this condition, mitochondrial K_ATP and BK_Ca channel and/or MPTP openers were applied.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Effect of diazoxide on mitochondrial Ca2+ (mitoCa2+) overload in rhod 2-AM-loaded rat ventricular myocytes. A: representative recording of relative rhod 2 fluorescence intensity (F1/F0) from a single myocyte. Ouabain (OUA, 1 mM), diazoxide (DIA, 100 µM), and 5-hydroxydecanoate (5-HD, 500 µM) were applied as indicated by horizontal bars. B: confocal images of rhod 2 fluorescence before (a) and after treatment with ouabain (b), ouabain + diazoxide (c), and ouabain + diazoxide + 5-HD (d). Scale bar, 20 µm. C: summary of changes in rhod 2 fluorescence intensity before (control) and after treatment with ouabain, diazoxide, and 5-HD. *P < 0.05 vs. control. P < 0.05 vs. ouabain. #P < 0.05 vs. ouabain + diazoxide. D: concentration-response curve for effect of diazoxide on ouabain-induced mitochondrial Ca2+ overload. Hill's equation was used for curve fitting.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Effect of NS-1619 on mitochondrial Ca2+ overload in rat ventricular myocytes. A: representative recording of relative rhod 2 fluorescence from a single myocyte before (a) and after application of 1 mM ouabain (b), ouabain + NS-1619 (c), and ouabain + NS-1619 + paxilline (d). B: confocal images of rhod 2 fluorescence for corresponding arrowheads in A. Scale bar, 20 µm. C: summary of changes in rhod 2 fluorescence intensity before (control) and after treatment with ouabain, NS-1619, and paxilline. *P < 0.05 vs. control. P < 0.05 vs. OUA. #P < 0.05 vs. OUA + NS-1619. D: concentration-response curve for effect of NS-1619 on ouabain-induced mitochondrial Ca2+ overload. Hill's equation was used for curve fitting.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Effect of cyclosporin A on mitochondrial Ca2+ overload with diazoxide and NS-1619 in rhod 2-AM-loaded rat ventricular myocytes. A: representative recording of relative rhod 2 fluorescence intensity from a single myocyte before (control) and after treatment with 1 mM ouabain, 100 µM diazoxide, and 2 µM cyclosporin A (CysA). B: confocal image of rhod 2 fluorescence for corresponding arrowheads in A. Scale bar, 20 µm. C: summary of changes in rhod 2 fluorescence intensity of A (n = 6). Horizontal bar, 20 µm. D: representative recording of relative rhod 2 fluorescence intensity from a single myocyte before (control) and after treatment with ouabain, NS-1619, and cyclosporin A. E: confocal image of rhod 2 fluorescence for corresponding arrowheads in D. Scale bar, 20 µm. F: summary of changes in rhod 2 fluorescence intensity in D (n = 6). *P < 0.05 vs. control. P < 0.05 vs. OUA. #P < 0.05 vs. OUA + DIA or OUA + NS.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Effect of atractyloside on mitochondrial Ca2+ overload in rhod 2-AM-loaded rat ventricular myocytes. A: representative recording of relative rhod 2 fluorescence intensity from single myocyte before (control) and after treatment with ouabain, atractyloside, and cyclosporin A. B: confocal image of rhod 2 fluorescence for corresponding arrowheads in A. Scale bar, 20 µm. C: summarized of changes in rhod 2 fluorescence intensity in A (n = 6). *P < 0.05 vs. control. P < 0.05 vs. OUA. #P < 0.05 vs. OUA + atractyloside.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Effect of diazoxide and NS-1619 on mitochondrial Ca2+ overload with prolonged exposure to ouabain. A and C: representative recordings of relative rhod 2 fluorescence intensity in a single myocyte. Ouabain (1 mM), diazoxide (100 µM), and NS-1619 (NS, 10 µM) were applied as indicated on horizontal bars. B and D: summaries of A and C, respectively. *P < 0.05 vs. control. P < 0.05 vs. DIA or NS.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Effect of diazoxide, NS-1619, and cyclosporin A on mitochondrial Ca2+ overload. A and C: representative recording of rhod 2 fluorescence intensity in a single ventricular myocyte. B and D: summaries of A and C, respectively. *P < 0.05 vs. control. P < 0.05 vs. OUA. #P < 0.05 vs. OUA + NS. P < 0.05 vs. OUA + DIA + NS, or OUA + NS + DIA.

	RESULTS

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Mitochondrial K_ATP channel opening attenuates overloaded mitochondrial Ca²⁺. Rat ventricular myocytes were loaded with the mitochondrial Ca²⁺-selective fluorescent dye rhod 2-AM, and mitochondrial Ca²⁺ was monitored by confocal microscopy. The Na⁺-K⁺-ATPase inhibitor ouabain was used to produce mitochondrial Ca²⁺ overload. Application of 1 mM ouabain increased the rhod 2-sensitive fluorescence intensity within 12 ± 3 min (n = 10): 1.03 ± 0.02 and 1.62 ± 0.05 F₁/F₀ for control and ouabain, respectively (Fig. 1A). Application of 100 µM diazoxide dramatically decreased ouabain-induced mitochondrial Ca²⁺ overload to control level (Fig. 1A). Subsequent addition of the selective mitochondrial K_ATP channel inhibitor 5-HD (500 µM) (18) reversed the attenuation effect of diazoxide on ouabain-induced mitochondrial Ca²⁺ overload, suggesting that opening of the mitochondrial K_ATP channel reduced the ouabain-sensitive mitochondrial Ca²⁺ overload. Attenuation of ouabain-induced mitochondrial Ca²⁺ is dependent on the dose of diazoxide (Fig. 1D): in the presence of 30, 60, 100, and 300 µM diazoxide, relative fluorescence intensity (F₁/F₀) of rhod 2 evoked by 1 mM ouabain (2.40 ± 0.12) decreased to 2.28 ± 0.11 (n = 6), 2.07 ± 0.10 (n = 6), 1.31 ± 0.07 (n = 8), and 0.97 ± 0.05 (n = 10), respectively. Diazoxide attenuated the ouabain-induced mitochondrial Ca²⁺ overload at a half-inhibition concentration of 78.3 ± 3.36 µM.

Mitochondrial BK_Ca channel opening attenuates mitochondrial Ca²⁺ overload. To test the role of the mitochondrial BK_Ca channel in mitochondrial Ca²⁺ overload, we applied the specific mitochondrial BK_Ca channel agonist 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H) benzimid-axolone (NS-1619) to freshly isolated rat ventricular myocytes loaded with rhod 2-AM. After 10 min of ouabain-induced mitochondrial Ca²⁺ overload (0.90 ± 0.05 and 1.77 ± 0.12 F₁/F₀ for control and ouabain, respectively, n = 8), mitochondrial Ca²⁺ overload was reduced (0.99 ± 0.05 F₁/F₀) by 10 µM NS-1619 (Fig. 2, A and C). The NS-1619-induced reduction of mitochondrial Ca²⁺ was reversed by 2 µM paxilline. Attenuation of ouabain-induced mitochondrial Ca²⁺ was dependent on the dose of NS-1619 (Fig. 2D): in the absence of NS-1619 (ouabain alone), the relative increase in rhod 2 fluorescence intensity (F₁/F₀) was 2.40 ± 0.11 of control (n = 15); however, it decreased to 2.28 ± 0.11 (n = 3), 2.07 ± 0.10 (n = 3), and 1.31 ± 0.07 (n = 7) in the presence of 2, 6, and 10 µM NS-1619, respectively. The half-inhibition concentration of NS-1619 for attenuation of ouabain-induced mitochondrial Ca²⁺ overload was 7.78 ± 0.44 µM.

Role of MPTPs in efflux of mitochondrial Ca²⁺. The role of the MPTPs in ouabain-induced mitochondrial Ca²⁺ overload was evaluated by the MPTP blocker cyclosporin A and the MPTP opener atractyloside. After blockade of ouabain-induced mitochondrial Ca²⁺ overload with 100 µM diazoxide (Fig. 3, A–C) or 10 µM NS-1619 (Fig. 3, D–F), the cells were treated with cyclosporin A. Ouabain-induced mitochondrial Ca²⁺ overload was suppressed by diazoxide and NS-1619, which was antagonized by cyclosporin A (Fig. 3, A and D). To investigate the direct role of MPTPs in ouabain-induced mitochondrial Ca²⁺ overload, we applied 20 µM atractyloside. After ouabain-induced mitochondrial Ca²⁺ overload (1.05 ± 0.01 and 2.25 ± 0.11 F₁/F₀ for control and ouabain, respectively), direct opening of MPTPs induced by atractyloside attenuated the ouabain-induced mitochondrial Ca²⁺ overload (1.64 ± 0.09 F₁/F₀, n = 8), which was antagonized by cyclosporin A (Fig. 4A).

Comparison of the attenuation effect on mitochondrial Ca²⁺ overload between mitochondrial K_ATP and BK_Ca channels during prolonged exposure to ouabain. To evaluate the efficacy of mitochondrial K_ATP and BK_Ca channels during prolonged mitochondrial Ca²⁺ overload (extended exposure to ouabain), single rat ventricular myocytes loaded with rhod 2-AM were exposed to 1 mM ouabain for 30 ± 5 min. The rhod 2 fluorescence intensity (F₁/F₀) increased to 2.67 ± 0.03 (Fig. 5A) and 2.92 ± 0.11 (Fig. 5C), respectively. Half-inhibition concentrations of diazoxide and NS-1619 decreased mitochondrial Ca²⁺ overload to 2.21 ± 0.15 (Fig. 5B; n = 7) and 1.57 ± 0.09 F₁/F₀ (Fig. 5D; n = 9), respectively.

Additive effect of mitochondrial K_ATP and BK_Ca channels and MPTPs on mitochondrial Ca²⁺ efflux during prolonged mitochondrial Ca²⁺ overload. When cells were exposed to ouabain for an extended time (30 ± 5 min), it was difficult to lower the mitochondrial Ca²⁺ overload to the control value with 100 µM diazoxide alone (Fig. 5A). Although NS-1619 reduced the mitochondrial Ca²⁺ overload by 60%, NS-1619 did not completely lower mitochondrial Ca²⁺ to control level. However, when prolonged ouabain-induced mitochondrial Ca²⁺ overload was treated with diazoxide + NS-1619, mitochondrial Ca²⁺ was effectively reduced to control level. Diazoxide could not attenuate the prolonged mitochondrial Ca²⁺ overload by ouabain (Fig. 6A), whereas subsequent application of NS-1619 significantly reduced the prolonged ouabain-induced increase in mitochondrial Ca²⁺ (Fig. 6B; n = 6). Furthermore, NS-1619 alone lowered the prolonged ouabain-induced increase in mitochondrial Ca²⁺ by 60%. However, subsequent application of diazoxide reduced the mitochondrial Ca²⁺ to control level (Fig. 6C; n = 8). The attenuation effect of diazoxide + NS-1619 or NS-1619 + diazoxide on prolonged ouabain-induced mitochondrial Ca²⁺ overload was reversed by cyclosporin A (Fig. 6, B and D). These results suggest that the mitochondrial BK_Ca channel more efficiently attenuates mitochondrial Ca²⁺ than the mitochondrial K_ATP channel during the prolonged mitochondrial Ca²⁺ overload.

	DISCUSSION

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K_ATP channel opening in inner mitochondrial membranes protects hearts from I/R injury. Opening of the BK_Ca channel is also known to elicit cardiac preconditioning. We investigated the role of pharmacological opening of these channels on the ouabain-induced mitochondrial Ca²⁺ overload. The mitochondrial K_ATP channel opener diazoxide and the mitochondrial BK_Ca channel opener NS-1619 attenuated the early phase of ouabain-induced mitochondrial Ca²⁺ overload. The data revealed that mitochondrial BK_Ca channel opening more efficiently attenuates mitochondrial Ca²⁺ than mitochondrial K_ATP channel opening during prolonged exposure to an ouabain-induced increase in mitochondrial Ca²⁺.

During the early stage of mitochondrial Ca²⁺ overload evoked by 1 mM ouabain, an attenuation effect was induced by both openers. This attenuation, however, was blocked by their specific blockers: the mitochondrial K_ATP channel blocker 5-HD and the mitochondrial BK_Ca channel blocker paxilline. Our results support the suggestion that opening of the two mitochondrial K⁺ channel isoforms can prevent ischemia- or I/R injury-induced mitochondrial Ca²⁺ overload. Each of the mitochondrial K⁺ openers showed dose-dependent responses. The mitochondrial BK_Ca channel opener NS-1619 exhibited a high efficacy for mitochondrial Ca²⁺ attenuation and a sensitive response before diazoxide administration during the early stage or with prolonged exposure to ouabain. O'Rourke et al. (23) also proposed that the mitochondrial BK_Ca channel might be activated at high mitochondrial Ca²⁺ loads, acting as a relief valve to prevent further Ca²⁺ accumulation.

Recently, mitochondrial K_ATP and BK_Ca channels have been proposed as cytoprotective mitochondrial channels. The mitochondrial K_ATP channel was first identified by Inoue et al. (16). The conductance of the channel was recorded to be 9.7 ± 1.0 pS with 100 mM K⁺ pipette solution and 33.3 mM K⁺ bathing solution. Diazoxide, nicorandil, and BMS-191095 selectively open mitochondrial K_ATP channels, with minimal effects on the cardiac isoform sarclemmal K_ATP channel, whereas other K⁺ channel openers, including cromakalim, EMD-60480, EMD-57970, pinacidil, RP-66471, minoxidil sulfate, and KRN-2391, nonselectively activate mitochondrial and sarcolemmal K_ATP channels. In the case of mitochondrial K_ATP channel blockers, glibenclamide and glipizide antagonize both isoforms, whereas 5-HD and MCC-134 selectively inhibit the mitochondrial K_ATP channel, with little effect on the sarcolemmal K_ATP channel (1, 3, 7, 30). The mitochondrial BK_Ca channel was first demonstrated by Siemen et al. (27). Using a patch-clamp technique in mitoplasts of the human glioma cell line LN 229, they recorded a K⁺-selective channel showing a conductance of 295 ± 18 pS. Xu et al. (33) first showed the presence of the mitochondrial BK_Ca channel in cardiomyocytes. They also recorded single-channel currents (307 ± 4.6 pS) in mitoplasts from cardiac myocytes. The channel was activated by 512 nM Ca²⁺ and inhibited by 200 nM charybdotoxin. Mitochondrial BK_Ca channel opening led to K⁺ influx into the mitochondrial matrix; this event was activated by NS-1619 and valinomycin and antagonized by charybdotoxin. Although the mechanism is unclear, several investigators proposed that opening of mitochondrial K_ATP and BK_Ca channels functions as a trigger or mediator in ischemia-preconditioning (IPC). Some investigators have shown that diazoxide (5) and NS-1619 (28) trigger the protective effect of IPC and that these events are antagonized by 5-HD and paxilline, respectively. Furthermore, the MPTP is a large nonselective channel that plays a role in the apoptotic pathway by releasing cytochrome c from the intermembrane space (4). Hausenloy et al. (14) showed that transient opening of MPTPs (low-conductance) during diazoxide-induced IPC may play a role in cell survival and that this cytoprotection of IPC is abolished by the MPTP inhibitor cyclosporin A or by reactive oxygen species scavengers. Recently, several reports showed that MPTP opening mediates depolarization, Ca²⁺ efflux from mitochondria, and cardioprotection (20, 26). Consistent with these reports, our results also showed that opening of MPTPs reduced ouabain-induced mitochondrial Ca²⁺ overload.

The present investigation was intended to connect the mitochondrial Ca²⁺ attenuation effect of two mitochondrial K⁺ channel isoforms with the MPTP during ouabain-induced mitochondrial Ca²⁺ overload. Although we cannot confirm the cardioprotective effect of MPTPs on mitochondrial Ca²⁺ overload, the involvement of these nonselective ion channels in mitochondrial Ca²⁺ reduction during ouabain-induced mitochondrial Ca²⁺ overload might indicate MPTP-mediated cardioprotection.

	GRANTS

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This work was supported by Korea Research Foundation Grants KRF-2005-210-E00003, KRF-2005-211-E00006, KRF-2005-037-E00002, KRF-2005-042-E00010, KRF-2006-521-E00013, KRF-2006-312-E00018, and KRF-2006-312-C00601 funded by the Korean Government.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. Han, Mitochondrial Signaling Laboratory, Dept. of Physiology and Biophysics, College of Medicine, Biohealth Products Research Center, Cardiovascular and Metabolic Disease Research Center, Inje Univ. 633-165 Gaegeum-Dong, Busanjin-Gu, Busan 613-735, Korea (e-mail: phyhanj{at}ijnc.inje.ac.kr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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