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Am J Physiol Heart Circ Physiol 293: H3517-H3523, 2007. First published September 28, 2007; doi:10.1152/ajpheart.00483.2006
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Na+/H+ exchanger inhibitor cariporide attenuates the mitochondrial Ca2+ overload and PTP opening

Takako Toda,1,* Toshie Kadono,1,* Minako Hoshiai,1 Yu Eguchi,2 Shinpei Nakazawa,1 Hiroe Nakazawa,2 Naoko Higashijima,2 and Hideyuki Ishida2

1Department of Pediatrics, Yamanashi University School of Medicine, Yamanashi; and 2Department of Physiology, Tokai University School of Medicine, Kanagawa, Japan

Submitted 11 May 2006 ; accepted in final form 18 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Na+/H+ exchanger (NHE) inhibitor cariporide has a cardioprotective effect in various animal models of myocardial ischemia-reperfusion. Recent studies have suggested that cariporide interacts with mitochondrial Ca2+ overload and the mitochondrial permeability transition (MPT); however, the precise mechanisms remain unclear. Therefore, we examined whether cariporide affects mitochondrial Ca2+ overload and MPT. Isolated adult rat ventricular myocytes were used to study the effects of cariporide on hypercontracture induced by ouabain or phenylarsine oxide (PAO). Mitochondrial Ca2+ concentration ([Ca2+]m) and the mitochondrial membrane potential ({Delta}{Psi}m) were measured by loading myocytes with rhod-2 and JC-1, respectively. We also examined the effect of cariporide on the MPT using tetramethylrhodamine methyl ester (TMRM) and oxidative stress generated by laser illumination. Cariporide (1 µM) prevented ouabain-induced hypercontracture (from 40 ± 2 to 24 ± 2%, P < 0.05) and significantly attenuated ouabain-induced [Ca2+]m overload (from 149 ± 6 to 121 ± 5% of the baseline value, P < 0.05) but did not affect {Delta}{Psi}m. These results indicate that cariporide attenuates the [Ca2+]m overload without the accompanying depolarization of {Delta}{Psi}m. Moreover, cariporide increased the time taken to induce the MPT (from 79 ± 11 to 137 ± 20 s, P < 0.05) and also attenuated PAO-induced hypercontracture (from 59 ± 3 to 50 ± 4%, P < 0.05). Our data indicate that cariporide attenuates [Ca2+]m overload and MPT. Thus these effects might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.

calcium; mitochondria; Na+/H+ exchange; permeability transition pore; adenosine 5'-triphosphate potassium channel

THE CARDIAC SARCOLEMMAL Na+/H+ exchanger (NHE) regulates intracellular pH via proton extrusion driven by the transmembrane Na+ gradient. Inhibition of NHE results in cardioprotective effects in various animal models of myocardial ischemia-reperfusion with consistent improvement in functional recovery, metabolic status, and attenuation of arrhythmias (2, 3, 34). Inhibition of NHE reduces intracellular Na+ ([Na+]i) accumulation and prevents excessive Ca2+ influx via the Na+/Ca2+ exchanger during ischemia-reperfusion; this has been proposed as the mechanism of cardioprotection resulting from NHE inhibition (1, 9).

Mitochondria play a key role in the regulation of apoptotic and necrotic cell death. It is well known that the mitochondrial permeability transition (MPT) induces the release of cytochrome c and results in apoptosis. Mitochondrial Ca2+ ([Ca2+]m) overload and oxidative stress are the major triggers of the MPT, which may be a central coordinating event of apoptotic and necrotic cell death (18, 20). Under stress conditions (e.g., death signals), the MPT opens to allow the passage of molecules <1.5 kDa, including protons and water. The proton gradient and electrical potential across the inner mitochondrial membrane collapses, leading to uncoupling of oxidative phosphorylation. Cariporide inhibits cardiomyocyte apoptosis, and an increase in [Ca2+]m has been reported (29, 31).

Opening of the mitochondrial ATP-sensitive K+ (mito-KATP) channels is thought to provide cardioprotection as one of the end effectors of ischemic preconditioning. Furthermore, recent studies have suggested that the opening of the mito-KATP channels is involved in pharmacological cardioprotection (22, 27). Murata et al. (23) have reported that the opening of mito-KATP channels resulted in the attenuation of the [Ca2+]m overload as a consequence of partial mitochondrial membrane depolarization. We have previously shown that the opening of mito-KATP channels by diazoxide and nicorandil depolarized the mitochondrial membrane potential ({Delta}{Psi}m), attenuated the [Ca2+]m overload, and inhibited the hypercontracture of myocytes during ouabain application (13, 14). Miura et al. (21) have reported that mito-KATP channel blockers, namely, 5-hydroxydecanoate (5-HD) and glibenclamide, inhibited the cardioprotective effects on infarct size and myocardial stunning resulting from NHE inhibition. In contrast, Hale et al. have reported that 5-HD did not inhibit the cardioprotective effects resulting from NHE inhibition (8). Recently, Teshima et al. (31) have reported that the NHE inhibitor cariporide remarkably inhibited the increase in [Ca2+]m and the loss of {Delta}{Psi}m induced by oxidative stress and prevented cell death by preserving mitochondrial integrity.

Thus it is suggested that cariporide interacts with the mitochondrial Ca2+ overload and permeability transition pore (PTP) opening; however, the precise mechanisms remain unclear. Therefore, in this study, we investigated whether the NHE inhibitor cariporide directly attenuates the mitochondrial death pathway (i.e., mitochondrial Ca2+ overload and/or induction of the MPT). In addition, the relationship between NHE inhibition and mito-KATP channel activity was assessed in isolated adult rat ventricular myocytes.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. Cariporide was gifted from Aventis Pharma. Ouabain, sodium 5-HD, glibenclamide, diazoxide, strophanthidin, monensin sodium salt, and phenylarsine oxide (PAO) were purchased from Sigma Chemicals. Rhod-2 acetoxymethyl ester, JC-1, sodium green, gramicidin D, and tetramethylrhodamine methyl ester (TMRM) were purchased from Molecular Probes. Cyclosporin A (CsA) was purchased from Research Biochemicals International. Other chemicals were purchased from Wako Chemical Industries.

Preparation of fresh adult rat cardiomyocytes. Isolated ventricular myocytes were prepared from the hearts of adult male Sprague-Dawley rats (CLEA, Tokyo, Japan). All procedures complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, Revised 1996) and were approved by the Institutional Animal Care and Use Committee of Tokai University School of Medicine.

Adult rat ventricular myocytes were isolated using collagenase digestion, as previously described (12). Briefly, a Sprague-Dawley rat was anesthetized with pentobarbital sodium (250 mg/kg ip). The heart was rapidly excised and perfused retrogradely via the aorta using the Langendorff method. The heart was first perfused with a Ca2+-free solution (solution A) containing the following (in mM): NaCl, 137; KCl, 5.4; MgCl2, 1; HEPES, 5; dextrose, 22; taurine, 20; creatine, 5; and sodium pyruvate, 5. The solution was adjusted to pH 7.4 with NaOH (37°C). After 5 min of initial perfusion, the heart was perfused for ~30 min with solution A and 0.1 mM CaCl2 with 0.5 mg/ml collagenase. The enzyme was then washed off by perfusion with solution A and 0.1 mM CaCl2 for 5 min. The left ventricle was excised from the heart, chopped into small pieces, and then shaken at 37°C for 10 min in a glass conical flask containing 30 ml of solution A and 0.1 mM CaCl2; the cell suspension was filtered (200-µm mesh) and sedimented in a 50-ml glass beaker for 5 min, and the supernatant was then replaced with a solution containing a higher concentration of Ca2+. The Ca2+ concentration was increased to 1 mM in three steps. Once isolated, the cells were resuspended in a culture medium composed of 5% fetal calf serum, 47.5% M199, and 47.5% F-12 and stored at room temperature until use.

Quantification of myocyte hypercontracture. Isolated adult rat myocytes along with the drugs were observed using an inverted phase-contrast microscope (Nikon) at 37°C. After a 60-min exposure to the drugs, myocyte hypercontracture, defined as a shortening of the long axis by >50% of the initial length, was expressed as a percentage of the cells before treatment. Individual experiments in each group were performed using myocytes isolated from at least three different hearts.

[Ca2+]m and {Delta}{Psi}m measurements. The Ca2+ fluorophore rhod-2 was used to measure changes in [Ca2+]m. Myocytes were loaded with 10 µM rhod-2 acetoxymethyl ester for 120 min at 4°C and then for 30 min at 37°C in the culture medium. This two-step cold loading-warm incubation protocol achieves exclusive loading of rhod-2 into the mitochondria (33). The {Delta}{Psi}m was monitored with a fluorescent probe JC-1. The myocytes were loaded with 0.5 µM JC-1 for 10 min at 37°C (6). Rhod-2 was excited at 488 nm by argon ion laser, and the emission was collected above 515 nm through a long-pass barrier filter. JC-1 was excited at 488 nm, and the red fluorescence emission was detected using a long-pass filter of 580 nm.

Intracellular Ca2+ concentration and [Na+]i measurements. Intracellular Ca2+ concentration ([Ca2+]i) measurement was performed by a previously described method (12). Cells were exposed to 10 µM fluo-3 AM for 30 min at 37°C. Myocytes were exited at 488 nm with light from an argon laser, and fluorescence at 530 nm was detected via a barrier filter.

The Na+ fluorophore sodium green was used to measure changes in [Na+]i. Myocytes were loaded with 6.5 µM sodium green for 30 min at 37°C in the culture medium. Sodium green was excited by argon ion laser at 488 nm, and the emission was collected above 515 nm through a long-pass barrier filter. After recording of the emission intensities, an in vivo calibration was performed using a modified method of Yao et al. (36). For calibration, the myocytes were sequentially exposed to three calibration solutions of 5, 10, and 15 mM Na+ containing the following (in mM): gramicidin D, 2; monensin, 40; and strophanthidin, 100. In each solution, [Na+]i was equilibrated to the extracellular Na+ concentration, and the stable fluorescence at each [Na+]i was then obtained. The calibration solutions were prepared using appropriate mixtures of a high-Na+ solution and a high-K+ solution. The former consisted of the following (in mM): NaCl, 30; sodium gluconic acid, 110; EGTA, 2; and HEPES, 10. The latter was similar in composition, except that Na+ was replaced with K+. The pH of the high-Na+ and high-K+ solutions was adjusted to 7.2 with NaOH and KOH, respectively.

Confocal fluorescence imaging. Myocytes loaded with each of the fluorescent dyes were perfused with a HEPES-buffered Tyrode's solution (37°C) and imaged using a Nipkow disk confocal system (CSU22, Yokogawa), as previously described (12). An objective lens of x100 (numerical aperture 1.3, oil immersion lens, Zeiss) and x40 was used for [Ca2+]m and {Delta}{Psi}m, and [Na+]i, respectively. The emission light was imaged through a relay lens to an intensified charge-coupled device camera (SR UB GEN III+, Solamere). Images were recorded on a computer (Macintosh G3) at video rate and analyzed with NIH Image 1.62f software.

Detection of the MPT with TMRM. Induction of the MPT opening in myocytes was detected using the fluorescent dye TMRM, as described by Hausenloy et al. (10). The myocytes were exposed to 6 µM TMRM for 15 min at 37°C; this resulted in TMRM accumulation in the mitochondria at a concentration that was sufficiently high to cause autoquenching of fluorescence. Laser illumination of TMRM causes generation of reactive oxygen species (ROS) in the mitochondria, which induce the MPT. The induction of the MPT opening is initiated by the loss of TMRM from the mitochondria into the cytosol, where TMRM fluorescence (excitation, 488 nm; fluorescence, 525 nm) was quantitated by the Nipkow disk confocal system (CSU22) as described above. The MPT induction also resulted in hypercontracture, since it was associated with ATP depletion.

Data analysis. Data are represented as means ± SE, and the number of experiments is designated as n. Intergroup comparisons for the two groups were performed by use of Student's t-test and ANOVA, followed by Tukey's test for multiple groups. A value of P < 0.05 was regarded as significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of cariporide on ouabain-induced hypercontracture. The exposure of myocytes to a toxic concentration of ouabain causes a great increase in the [Ca2+]i concentration, arrhythmic contraction, and eventually contracture (32). Therefore, to evaluate the cardioprotective effects of cariporide, we examined whether cariporide inhibits myocyte hypercontracture when exposed to ouabain.

As shown in Fig. 1, the exposure of myocytes to 1 mM ouabain for 60 min greatly increased the fraction of hypercontracted cardiomyocytes to 40.4 ± 2.0% compared with 7.8 ± 1.1% in controls (P < 0.01). Treatment with 1 µM cariporide decreased the percentage of hypercontracted cardiomyocytes on exposure to ouabain (23.5 ± 1.6%; P < 0.05 vs. ouabain). This effect of cariporide was abolished by both 500 µM 5-HD (40.9 ± 3.7%) and 10 µM glibenclamide (35.0 ± 3.7%), although neither 5-HD nor glibenclamide alone significantly affected the cardiomyocyte hypercontracture.


Figure 1
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  		Fig. 1. Effect of cariporide on ouabain-induced hypercontracture. Myocyte hypercontracture 60 min after ouabain exposure (1 mM) was plotted as a percentage of the total viable cells before application of the drug. Cont, control; Ouab, ouabain (1 mM); Caripo, cariporide (1 µM); 5HD, 5-hydroxydecanoate (500 µM); Glib, glibenclamide (10 µM). *P < 0.05, vs. ouabain.

 
These results are consistent with a previous study reporting that mito-KATP channel blockers (i.e., 5-HD and glibenclamide) inhibited the cardioprotective effects of cariporide on infarct size and myocardial stunning (21).

Effects of cariporide on ouabain-induced [Ca2+]i, [Ca2+]m, and {Delta}{Psi}m. Cariporide inhibited the ouabain-induced hypercontracture; however, this cardioprotective effect was abolished by the mito-KATP channel blockers. Therefore, to investigate the effect of cariporide on [Ca2+]i, [Ca2+]m, and {Delta}{Psi}m, we measured the changes in the fluorescence intensities of fluo-3, rhod-2, and JC-1 and compared these with those obtained in the presence of the mito-KATP channel opener.

Figure 2 shows the representative confocal images of myocytes loaded with rhod-2. Treatment of myocytes with 1 mM ouabain increased the intensity of rhod-2 fluorescence, suggesting that the Ca2+ overload in the mitochondria was induced (Fig. 2A). Treatment with cariporide (1 µM) attenuated the mitochondrial Ca2+ overload during ouabain exposure (Fig. 2B). Figure 3A shows the relative changes in rhod-2 fluorescence measured 30 min after the drug treatment. The intensity of rhod-2 fluorescence after the 30-min exposure to ouabain significantly increased to 149.3 ± 6.2% of the baseline level (P < 0.01 vs. baseline). This data confirmed that ouabain induced the accumulation of [Ca2+]m. Addition of 1 µM cariporide or 0.1 mM diazoxide, the mito-KATP channel opener, attenuated the [Ca2+]m overload during ouabain exposure (120.7 ± 5.4 or 113.0 ± 5.0% of the baseline level, respectively; P < 0.05 vs. ouabain).


Figure 2
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  		Fig. 2. Effect of cariporide on ouabain-induced mitochondrial Ca2+ overload. Typical 2-dimensional confocal images of rhod-2 fluorescence before (baseline) and after 30-min treatment with ouabaine (Ouab, 1 mM; A) and/or cariporide (Caripo, 1 µM; B) are shown.

 

Figure 3
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  		Fig. 3. Effects of cariporide on ouabain-induced mitochondrial Ca2+ overload and mitochondrial Ca2+ concentration ([Ca2+]m). Summarized data of changes in rhod-2 fluorescence as [Ca2+]m (A) and the relative changes in JC-1 as mitochondrial potential ({Delta}{Psi}m; B) after 30-min treatment with ouabain and/or drugs. Ouab, ouabain (1 mM); Caripo, cariporide (1 µM); Diaz, diazoxide (100 µM). *P < 0.05 vs. ouabain; #P < 0.05 vs. baseline.

 
Figure 3B shows the relative changes in JC-1 fluorescence measured 30 min after drug treatment. Cariporide did not affect JC-1 fluorescence (101.8 ± 6.3% of the baseline level), although the mito-KATP channel opener diazoxide reduced the intensity of JC-1 fluorescence to 89.0 ± 2.0% (P < 0.05 vs. baseline). These results indicate that cariporide attenuates the [Ca2+]m overload without the depolarization of {Delta}{Psi}m; moreover, cariporide affects the rise in [Ca2+]m in a manner different from that of the mito-KATP channel opener.

Ouabain induced [Ca2+]i overload (224.2 ± 31% of the baseline level, n = 14). However, cariporide did not affect ouabain-induced [Ca2+]i overload (216 ± 37% of the baseline level, n = 15). This result suggests that an excessive [Ca2+]i overload is regulated by mitochondrial Ca2+ buffering.

Effect of cariporide on ouabain-induced [Na+]i accumulation. To investigate whether cariporide affects [Na+]i, we examined the effect of cariporide on [Na+]i accumulation during ouabain exposure.

Resting [Na+]i was measured using the fluorescent indicator sodium green. In isolated rat myocytes, the resting [Na+]i, as determined by the calibration method, was 8.3 ± 0.6 mM. As shown in Fig. 4, ouabain exposure increased [Na+]i to 12.6 ± 0.3 mM (P < 0.01 vs. baseline). Cariporide (1 µM) attenuated the rise in [Na+]i during ouabain exposure (11.6 ± 0.4 mM; P < 0.05 vs. ouabain). Neither ouabain nor cariporide influenced the cytosolic pH (data not shown).


Figure 4
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  		Fig. 4. Effect of cariporide on ouabain-induced intracellular Na+ accumulation. Summarized data for the time courses of change in intracellular Na+ concentration ([Na+]i). Cont, control; Ouab, ouabain (1 mM); Caripo, cariporide (1 µM). *P < 0.05 vs. ouabain; #P < 0.01 vs. control.

 
Effect of cariporide on induction of the MPT with TMRM. Confocal fluorescence imaging of myocytes loaded with TMRM revealed mitochondria as fluorescent bands oriented with the longitudinal axis of the cell (Fig. 5A). TMRM localizes selectively in the mitochondria according to the {Delta}{Psi}m. Figure 5, A–C, shows the representative images extracted from a time sequence in which a myocyte was loaded with TMRM (Fig. 5A). The myocyte has undergone global mitochondrial membrane depolarization, which is usually observed as an increase in TMRM fluorescence resulting from laser-induced oxidative stress (Fig. 5B). After depolarization, the cell ultimately progresses to hypercontracture (Fig. 5C). The time taken to induce the global mitochondrial membrane depolarization (Fig. 6A) and hypercontracture (Fig. 6B) was noted, and in the control group, the MPT was routinely induced after 78.5 ± 11.4 s of laser-induced oxidative stress. An additional 30 s were required for progression to an irreversible contraction, and hypercontracture occurred after a total of 107.7 ± 15.2 s of laser-induced oxidative stress (Fig. 6B).


Figure 5
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  		Fig. 5. Representative time series showing confocal fluorescence images of a representative adult rat myocyte loaded with tetramethylrhodamine methyl ester (TMRM) and subjected to laser-induced oxidative stress over time. A: 0 s, before oxidative stress. B: 79 s, the whole myocyte has now undergone global mitochondrial depolarization. C: 108 s, after the collapse of {Delta}{Psi}m, and the cell undergoes hypercontracture.

 

Figure 6
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  		Fig. 6. Effect of cariporide and CsA on the time taken to induce mitochondrial permeability transition (MPT; A) and hypercontracture (B) in TMRM-loaded myocytes. Cont, control; Caripo, cariporide (1 µM); CsA, cyclosporin A (0.4 µM). *P < 0.05 and **P < 0.01 vs. control.

 
To verify that the observed global mitochondrial membrane depolarization induced by laser-induced oxidative stress represents the MPT, it is necessary to demonstrate whether it is sensitive to the MPT inhibitor CsA (30). CsA was shown to extend the time required to induce both MPT and hypercontracture to 235.7 ± 18.4 and 314.3 ± 22.4 s, respectively (P < 0.01 vs. control; Fig. 6). In this model, cariporide also extended the time taken to induce both MPT and hypercontracture to 136.9 ± 20.4 and 204.4 ± 33.4 s, respectively (P < 0.05 vs. control; Fig. 6). These results indicated that cariporide decreases the susceptibility of mitochondria to the MPT in a manner similar to CsA.

Effect of cariporide on PAO-induced hypercontracture. To evaluate whether cariporide directly inhibits the MPT, we examined whether cariporide inhibits the hypercontracture induced by 5 µM PAO (an MPT inducer). As shown in Fig. 7, cariporide, diazoxide, and CsA attenuated the ouabain-induced hypercontracture (23.1 ± 1.6, 20.5 ± 2.6, and 21.0 ± 1.5%, respectively; P < 0.05 vs. ouabain). Moreover, cariporide and CsA attenuated the PAO-induced hypercontracture (from 59.0 ± 2.9 to 50.3 ± 3.6 and to 26.5 ± 3.5%, respectively; P < 0.05 vs. PAO); however, diazoxide did not inhibit the PAO-induced hypercontracture. These results suggest that cariporide directly affects the MPT in a manner similar to CsA.


Figure 7
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  		Fig. 7. Effect of cariporide on PAO-induced hypercontracture. Myocyte hypercontracture 60 min after ouabain (1 mM) or PAO (5 µM) exposure was plotted as a percentage of the total viable cells before application of the drug. Cont, control; Ouab, ouabain (1 mM); Caripo, cariporide (1 µM); Diazo, diazoxide (100 µM); PAO, phenylarsine oxide (5 µM); CsA, cyclosporin A (0.4 µM). *P < 0.05 vs. ouabain; +P < 0.05 vs. PAO.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have demonstrated the following. 1) Cariporide attenuates ouabain-induced hypercontracture, which is abolished by the mito-KATP channel blockers. 2) Cariporide attenuates the ouabain-induced [Ca2+]m overload without the accompanying depolarization of the {Delta}{Psi}m. 3) Cariporide and CsA inhibit TMRM-induced MPT and PAO-induced hypercontracture, but diazoxide does not.

Contribution of the mito-KATP channel to cardioprotection resulting from NHE inhibition. The mito-KATP channel is thought to provide cardioprotection as one of the end effectors of ischemic preconditioning. Furthermore, recent studies have suggested that the opening of the mito-KATP channel is involved in pharmacological cardioprotection (22). The opening of mito-KATP channels induced attenuation of the [Ca2+]m overload as a consequence of partial depolarization of {Delta}{Psi}m (13, 14, 23). Recent studies have demonstrated that the mito-KATP channel blocker 5-HD did not change the infarct size, which was limited by cariporide in the canine heart in situ (8), and that 5-HD and glibenclamide did not abolish the protective effect of cariporide in response to H2O2-induced dissipation of {Delta}{Psi}m (31). In contrast, it has been shown that 5-HD and glibenclamide, but not a sarcolemmal KATP channel blocker (HMR1098), abolished the cardioprotective effect of cariporide (21). In this study, we found that cariporide attenuates the ouabain-induced hypercontracture, and this cardioprotection by cariporide is abolished by 5-HD and glibenclamide (Fig. 1). Although we do not have a clear explanation for this discrepancy, there are at least two possibilities. First, it may be due to the differences in the experimental conditions and preparations. Another possibility is that NHE inhibition indirectly activated the mito-KATP channel, not directly.

Furthermore, our previous studies reported that the mito-KATP channel openers diazoxide and nicorandil depolarized the {Delta}{Psi}m and attenuated the [Ca2+]m overload during ouabain application (13, 14). In this study, cariporide did not affect the {Delta}{Psi}m, although diazoxide caused depolarization during ouabain exposure (Fig. 3). These results indicate that cariporide attenuates the [Ca2+]m overload without the accompanying depolarization of {Delta}{Psi}m. In this manner, both cariporide and diazoxide attenuated the ouabain-induced [Ca2+]m overload, but the effect of cariporide on {Delta}{Psi}m was different from that of diazoxide. Therefore, we suggest that cariporide does not have a direct effect on the mito-KATP channel. However, cariporide may indirectly affect the mito-KATP channel activity, because the cardioprotective effect of cariporide was abolished by mito-KATP channel blockers (Fig. 1). This hypothesis is supported by a previous study by Miura et al. (21) reporting that NHE inhibitors did not significantly induce mitochondrial flavoprotein oxidation, indicating the opening of the mito-KATP channel. In summary, a mito-KATP channel may indirectly contribute to the cardioprotective effect of NHE inhibitors; however, it might not play the central role in cardioprotection resulting from NHE inhibition.

Effect of cariporide on [Ca2+]m overload. Figure 8 shows a proposed mechanism of cardioprotection by cariporide. Normally, the mitochondrial Ca2+ influx occurs via a uniporter, and efflux occurs via the Na+/Ca2+ exchanger (inhibited by high cytosolic Ca2+). Under physiological conditions, it appears that the role of the mitochondrial Ca2+ transporters is to relay the change in cytosolic Ca2+ to the mitochondrial matrix, which results in increased ATP production. When the cytosolic Ca2+ exceeds the resting levels, significant uptake of Ca2+ from the cytosol to the mitochondria is expected to occur during ischemia and reperfusion, and it eventually results in the [Ca2+]m overload (7). The mitochondrial Ca2+ overload can lead to enhanced generation of ROS, triggering of MPT, and, finally, cell death (5). Therefore, among putative mechanisms of cardioprotection, the hypothesis of mitochondrial Ca2+ handling appears plausible.


Figure 8
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  		Fig. 8. Proposed mechanism of cardioprotection by cariporide. The dashed line explains the possibility of effect points.

 
We have reported previously that, in rat cardiomyocytes, the mito-KATP channel openers diazoxide and nicorandil depolarize {Delta}{Psi}m and attenuate the [Ca2+]m overload experimentally induced by ouabain (13, 14). Ouabain, an Na+/K+-ATPase inhibitor, impairs Na+ extrusion and consequently prevents Ca2+ extrusion via Na+/Ca2+ exchange (15). Here, we demonstrate the ouabain-induced [Na+]i accumulation (Fig. 4) and [Ca2+]m overload (Figs. 2 and 3). Although we used ouabain in an attempt to experimentally induce the [Ca2+]m overload, it may be pertinent to apply our results to ischemic myocardium.

In this study, cariporide significantly attenuated the ouabain-induced [Ca2+]m overload (Fig. 3) and the ouabain-induced [Na+]i accumulation (from 8.3 to 12.6 and to 11.6 mM, respectively; Fig. 4). Reduction in [Na+]i accumulation resulted from sarcolemmal NHE inhibition; [Na+]i accumulation gives rise to [Ca2+]i overload via the Na+/Ca2+ exchanger (15). An increase in [Ca2+]i leads to [Ca2+]m accumulation, because the mitochondria act as a spatial Ca2+ buffer (7). Recently, Macck et al. (19) have shown the accumulation of diastolic [Ca2+]m, even though diastolic [Ca2+]i remained constant over the course of the experiment using the measurement of [Ca2+]i and [Ca2+]m in the same cell (19). Therefore, inhibition of [Na+]i accumulation by cariporide may contribute to the attenuation of the [Ca2+]m overload, even if the inhibition is to a small extent (only 25%, Fig. 4).

In this study, cariporide inhibits [Ca2+]m overload and hypercontracture on exposure to ouabain. However, the inhibition of [Na+]i accumulation by cariporide is to a small extent (only 25%, Fig. 4). Moreover, cariporide did not affect ouabain-induced [Ca2+]i overload. Previous studies have reported that cariporide remarkably suppressed cytosolic Na+ and Ca2+ accumulation in isolated perfused hearts (9, 28) and in cultured neonatal rat cardiomyocytes (31) using bicarbonate buffer. In isolated cardiomyocyte studies, it has been reported that the protective effect of cariporide is independent of changes in cytosolic Na+ or Ca2+ concentration in isolated cardiomyocytes using HEPES buffer (25, 26). However, Baartscheer et al. (4) have shown that cariporide remarkably suppressed cytosolic Na+ and Ca2+ accumulation in isolated cardiomyocytes (4) using bicarbonate buffer. Therefore, the difference in the effect of cariporide on cytosolic Na+ and Ca2+ accumulation may be associated with HCO3–-Na symport. Nevertheless, cariporide attenuates [Ca2+]m overload and cell injury under both buffer solutions (31).

Despite the proposed mechanisms in Fig. 8, we cannot exclude the possible role of the NHE isoform, which is located in the mitochondrial inner membrane, in the observed cardioprotective effects of cariporide (24). Another NHE inhibitor, SM-20550, was reported to inhibit Na+/H+ transport in the mitochondria, and it preserved mitochondrial respiratory function (11, 35). Another possibility is that cariporide affects the mitochondrial Ca2+ transporter or reduces the driving force, which in turn reduces the [Ca2+]m overload. However, there have been no reports of NHE inhibitors that directly block the mitochondrial Ca2+ transporter (i.e., uniporter, rapid mode). Moreover, our results indicate that cariporide attenuated the [Ca2+]m overload without the accompanying depolarization of {Delta}{Psi}m (Fig. 3B), which does not reduce the driving force for Ca2+ influx into the mitochondria.

The mitochondrial Ca2+ overload induces the MPT directly and/or via generation of ROS (5). It has been reported that cariporide suppresses the [Ca2+]m overload and the MPT induction by H2O2 (31). Additionally, Sun et al. (29) have reported that cariporide inhibits cardiomyocyte apoptosis during hypoxic reperfusion.

Effect of cariporide on the MPT. Mitochondrial Ca2+ overload leads to the induction of the MPT directly and/or via enhanced generation of ROS in mitochondria (5). ROS generated within mitochondria have been shown to play a pivotal role in both mediating cell death and inducing the MPT during ischemic reperfusion (17). Here, we examined the effect of cariporide on the MPT using a myocyte model of oxidative stress. Laser illumination generates oxidative stress in cardiomyocytes loaded with TMRM, which induces the MPT and subsequently results in hypercontracture (Figs. 5 and 6). Hausenloy et al. (10) have investigated the effects of hypoxic and pharmacological preconditioning using the mito-KATP channel openers diazoxide and nicorandil, which prolonged the time taken to induce the MPT and hypercontracture, respectively, compared with controls; the MPT inhibitor CsA also increased this time period. In this study, cariporide and CsA increased the time taken to induce MPT and hypercontracture when compared with control (Fig. 6). Thus it was consistent with Hausenloy's results (10).

To examine whether cariporide affects the MPT, we investigated whether cariporide attenuates PAO-induced hypercontracture (PAO is a potent inducer of the MPT). Korge et al. (16) showed that PAO caused severe hypercontracture and irreversible sarcolemmal injury in isolated cardiac myocytes. In this study, cariporide and CsA significantly attenuated PAO-induced hypercontracture (Fig. 7). In contrast, the mito-KATP channel opener diazoxide did not inhibit the PAO-induced hypercontracture (Fig. 7). Thus cariporide inhibits the MPT, and this effect might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.

Although these results indicated that cariporide inhibits the MPT, the mechanism(s) of this effect is unclear. Loss of {Delta}{Psi}m is a critical step of the death pathway. Although cariporide did not have an effect on {Delta}{Psi}m in this study, Theshima et al. (31) reported that cariporide prevented {Delta}{Psi}m loss induced by H2O2. Cariporide may prevent mitochondrial homeostasis, leading to decreased sensitivity for MPT.

In conclusion, these results indicate that cariporide attenuates [Ca2+]m overload and MPT. Thus these effects might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Ishida, FAHA, Dept. of Physiology, School of Medicine, Tokai Univ., Bohseidai, Isehara, Kanagawa 259-1193, Japan (e-mail: ishida{at}is.icc.u-tokai.ac.jp)

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.

* T. Toda and T. Kadono contributed equally to this study. Back


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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