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(Circulation. 2003;108:2275.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Cariporide (HOE642), a Selective Na⁺-H⁺ Exchange Inhibitor, Inhibits the Mitochondrial Death Pathway

From the Institute of Molecular Cardiobiology, Johns Hopkins University, Baltimore, Md.

Correspondence to Eduardo Marbán, MD, PhD, FACC, Institute of Molecular Cardiobiology, Johns Hopkins University, 720 Rutland Ave, 844 Ross Building, Baltimore, MD 21205. E-mail marban{at}jhmi.edu

Received May 6, 2003; revision received July 15, 2003; accepted July 16, 2003.

	Abstract

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Background— The Na⁺-H⁺ exchanger figures prominently in cardiac ischemia-reperfusion injury. Several experimental and clinical studies have demonstrated a cardioprotective effect of Na⁺-H⁺ exchanger inhibition; however, the precise mechanisms have not been established.

Methods and Results— We examined the effects of cariporide (HOE642, 10 µmol/L) on cell death induced by oxidative stress (H₂O₂, 100 µmol/L) in cultured neonatal rat cardiomyocytes. Cariporide significantly suppressed markers of cell death, such as TUNEL positivity and caspase-3 cleavage, at 8 or 16 hours after H₂O₂. The early phase of cell death, reported by increases in phosphatidylserine exposure and propidium iodide uptake, was also inhibited by cariporide. To explore the mechanisms of cell protection, we examined the effects of cariporide on increases in intracellular Na⁺ and Ca²⁺ induced by oxidative stress. Cariporide remarkably suppressed cytosolic Na⁺ and Ca²⁺ accumulation. Next, we investigated the effects of cariporide on mitochondria-associated death process. Mitochondrial Ca²⁺ overload induced by H₂O₂ was remarkably suppressed by cariporide. Loss of mitochondrial membrane potential is a critical step of the death pathway; cariporide prevented mitochondrial membrane potential loss induced by H₂O₂.

Conclusions— Cariporide protects cardiomyocytes against oxidant-induced cell death by preserving intracellular ion homeostasis and mitochondrial integrity.

Key Words: ion channels • apoptosis • prevention • calcium • sodium

	Introduction

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The cardiac sarcolemmal Na⁺-H⁺ exchanger (NHE) regulates intracellular pH via proton extrusion driven by the transmembrane Na⁺ gradient. Inhibition of NHE produces cardioprotective effects in various animal models of myocardial ischemia-reperfusion with consistent improvement in functional recovery, metabolic status, and attenuation of arrhythmias.^1,2 Furthermore, a clinical study revealed that NHE inhibition during reperfusion therapy preserved cardiac function in patients with acute myocardial infarction.³ NHE is relatively quiescent under basal conditions but becomes activated during ischemia in response to intracellular acidosis. Inhibition of intracellular Na⁺ ([Na⁺]_i) accumulation induced by increased Na⁺-H⁺ exchange and prevention of excessive Ca²⁺ influx via Na⁺-Ca²⁺ exchanger working in reverse mode have been proposed as the mechanisms of cardioprotection by NHE inhibition.^2,4 However, the precise mechanisms of cardioprotection by NHE inhibition remain unclear.

Mitochondria play key roles in cell death. The mitochondrial death pathway features the sequential loss of mitochondrial membrane potential (_m), the release of toxic proteins into the cytoplasm, and caspase activation. We have recently reported that _m loss in response to oxidative stress is accompanied by the opening of the mitochondrial permeability transition pore (PTP) and is irreversible,⁵ indicating _m loss is a critical step to cell death. Mitochondrial Ca²⁺ ([Ca²⁺]_mito) overload is a major trigger of PTP opening.^6,7 We had previously shown that activation of mitochondrial ATP-sensitive potassium (mitoK_ATP) channels prevents _m loss and inhibits apoptosis,⁸ presumably by inhibition of [Ca²⁺]_mito overload.⁹ Based on these observations, we hypothesized that NHE inhibition protects cardiomyocytes against cell death by inhibiting [Ca²⁺]_mito overload and preserving _m. We investigated the precise mechanisms of cardioprotection by NHE inhibition using cariporide, a specific blocker of the NHE-1 isoform, in cultured cardiomyocytes.

	Methods

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All procedures were performed in accordance with the Johns Hopkins University animal care guidelines, which conform to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.

Primary Culture of Neonatal Rat Cardiac Ventricular Myocytes
Cardiac ventricular myocytes were prepared from 1- to 2-day-old Sprague-Dawley rats (Zivic Laboratories, Inc, Pittsburgh, Pa) and cultured as previously described.⁸

Experimental Protocol
Neonatal rat cardiomyocytes were randomly assigned to 1 of the following 3 experimental groups: (1) control group, (2) incubation with 100 µmol/L hydrogen peroxide (H₂O₂), or (3) pretreatment with 10 µmol/L cariporide for 20 minutes followed by 100 µmol/L H₂O₂. Cariporide was a gift from Aventis Pharma (Frankfurt, Germany).

Terminal Deoxynucleotidyl Transferase-Mediated TUNEL Staining
TUNEL staining was performed according to the manufacturer’s protocol (Roche) at 16 hours after 100 µmol/L H₂O₂ application. Fluorescein labels incorporated in nucleotide polymers were detected by laser-scanning confocal microscopy.

Caspase-3 Activity Assay
Caspase-3 activity was measured as described previously¹⁰ by detection of the cleavage of a colorimetric caspase-3 substrate, N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline, using an assay kit, ApoAlert CPP32 (Clontech), at 8 and 16 hours of 100 µmol/L H₂O₂ stimulation.

Loading of Cells With Fluorescent Indicators
For quantification of cellular viability, cells were double-stained with annexin V and propidium iodide (PI) according to manufacturer’s instructions (Roche). To monitor _m, cells were loaded with 100 nmol/L tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes) at 37°C for 20 minutes. CoroNa Red (1 µmol/L) was used to examine [Na⁺]_i level. To monitor intracellular Ca²⁺ ([Ca²⁺]_i) and [Ca²⁺]_mito levels, cells were loaded with 1 µmol/L fluo-4 and 2 µmol/L rhod-2 AM, respectively (Molecular Probes) at 37°C for 30 minutes.

Confocal Imaging
Cells plated on glass-bottom dishes were loaded with fluorescent probes as described above. After loading with each dye, cells were placed in phenol red-free DMEM supplemented with 25 mmol/L HEPES (pH 7.4). Cells were illuminated (488- and 568-nm line of a krypton/argon laser), and images were taken by confocal microscopy (UltraVIEW, Perkin-Elmer). Time-lapse confocal imaging was carried out with various intervals between frames using a x20 objective lens.

Image Analysis
Fluorescence intensity was assessed at mitochondria-rich lesions randomly selected around the nucleus in each scan. Quantitative image analysis was performed using image analysis software (ImageJ; http://rsb.info.nih.gov/ij/).

FACS Analysis
For FACS analysis of _m, TMRE-loaded cells were harvested by trypsinization at the end of experimental protocols and analyzed using FACScan (20 000 cells per sample) (Becton Dickinson). The fluorescence intensity of TMRE was monitored at 582 nm (FL-2 channel). FACS data were analyzed using WinMDI software (http://facs.scripps.edu).

Statistical Analysis
Data are presented as mean±SEM. Multiple comparisons among groups were carried out by one-way ANOVA with Fisher’s least-significant difference as the post-hoc test. A level of P<0.05 was accepted as statistically significant.

	Results

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Effects of Cariporide on Cell Death
First, we examined the long-term effects of cariporide on cell death. We performed TUNEL staining 16 hours after H₂O₂ application. Representative images and quantitative data are shown in Figures 1A and 1B, respectively (n=4 for each group). In the control group, 11.7±1.4% of the cells showed TUNEL-positive nuclei, whereas exposure to H₂O₂ increased the number of TUNEL-positive nuclei to 33.9±0.8% (P<0.001). This increase of TUNEL positivity was suppressed by cariporide (18.6±0.7%) (P<0.001 versus H₂O₂). Activation of caspase-3 is a critical step in the process of cell death. Figure 1C shows relative caspase-3 activity in each group at 8 and 16 hours after H₂O₂ application. Caspase-3 activity was increased in H₂O₂ compared with control at each time point (P<0.01), and cariporide suppressed these increases (P<0.05 versus H₂O₂ at each time point) (n=3 for each data point).

View larger version (36K): [in this window] [in a new window]   Figure 1. Effects of cariporide on cell death. A, Representative images of TUNEL staining in each group. B, Quantitative percentages of the cells with TUNEL-positive nuclei. Cariporide suppressed the increase of TUNEL positivity induced by H2O2. Data are mean±SEM (n=4 for each group from 2 independent experiments). *P<0.001 vs CON, #P<0.001 vs H2O2. C, Effects of cariporide on caspase-3 activity at 8 and 16 hours after H2O2 application. Average of control value at each time point is defined as 100%. Data at each time point are relative percentage of caspase-3 activity to control at the same time point. There are no actual data at 0 hours. Cariporide attenuated the activation of caspase-3 induced by H2O2 at each time point. Data are mean±SEM (n=3 for each group). *P<0.05 vs CON, #P<0.05 vs H2O2.

Effects of Cariporide on Early Phase of Cell Death
To assess the effects of cariporide in the early phase of cell death, we investigated the alterations of annexin V and PI fluorescence in each group. Time-lapse confocal microscopy at 5-minute intervals began immediately after the application of 100 µmol/L H₂O₂. Annexin V fluorescence started to increase approximately 30 minutes after H₂O₂ application and gradually increased to a plateau at approximately 100 minutes (Figure 2A). Cariporide remarkably suppressed this increase of annexin V fluorescence (Figure 2A). PI fluorescence was increased after 70 minutes of latency and reached a plateau at approximately 120 minutes by H₂O₂ stimulation (Figure 2B). This increase of PI fluorescence was also abrogated by cariporide (Figure 2B). After 150 minutes of scanning, cells were permeabilized with saponin (Sigma) to calculate the percentage of PI-positive cells in each group. At the end of scanning, 92.1±3.0% of cells were PI positive by H₂O₂ stimulation. In contrast, only 19.9±9.5% of cells were PI positive in cariporide-treated cells (n=3, P<0.001). Because annexin V is an indicator of apoptosis and PI of necrosis, these results suggest that cariporide reduces both types of cell death.

View larger version (31K): [in this window] [in a new window]   Figure 2. Effects of cariporide on the early phase of cell death. A, Representative images and quantitative results of sequential changes of annexin V fluorescence using time-lapse confocal microscopy in response to H2O2 application in each group. B, Representative images and quantitative results of sequential changes of PI fluorescence using time-lapse confocal microscopy in response to H2O2 application in each group. Each plot is the average of 10 areas from 1 scanning. Similar results were obtained from 3 independent experiments. Increases of annexin V and PI fluorescence induced by H2O2 were abrogated by cariporide.

Effects of Cariporide on Cytosolic Na⁺ and Ca²⁺ Overload
Preservation of intracellular ion homeostasis is a natural potential mechanism of cardioprotection by NHE inhibition. We examined the effects of cariporide on [Na⁺]_i and [Ca²⁺]_i accumulation using coroNa-red and fluo-4. Time-lapse confocal microscopy began with the addition of H₂O₂. Fluorescence did not change during 60 minutes of scanning in controls. CoroNa-red fluorescence increased gradually approximately 10 minutes after H₂O₂ application. Cariporide mitigated the increase in coroNa-red fluorescence induced by H₂O₂ (Figures 3A and 3B). Fluo-4 fluorescence also increased approximately 20 minutes after H₂O₂ application and reached a plateau at approximately 40 minutes. Cariporide also attenuated the increase in fluo-4 fluorescence induced by H₂O₂ (Figures 4A and 4B).

View larger version (54K): [in this window] [in a new window]   Figure 3. Time-lapse analysis of cytosolic Na+ level. A, Representative sequential images of coroNa-red fluorescence in each group. B, Quantitative data of coroNa-red fluorescence. Data are mean±SEM from 3 independent experiments. Cariporide abrogated the increases of coroNa-red fluorescence in response to H2O2.

View larger version (55K): [in this window] [in a new window]   Figure 4. Time-lapse analysis of cytosolic Ca2+ level. A, Representative sequential images of fluo-4 fluorescence. B, Quantitative data of fluo-4 fluorescence. Data are mean±SEM from 3 independent experiments. Cariporide abrogated the increases of fluo-4 fluorescence in response to H2O2.

Effects of Cariporide on Mitochondrial Ca²⁺ Overload
Ca²⁺ overload in mitochondria is one of the critical triggers of cell death and is known to open the PTP.⁶ We examined the effects of cariporide on mitochondrial Ca²⁺ level using the mitochondrial Ca²⁺-sensitive dye rhod-2. Rhod-2 fluorescence was remarkably augmented by H₂O₂ (Figures 5A and 5B). Cariporide completely inhibited the Ca²⁺ increase observed in H₂O₂-stimulated cells (Figures 5A and 5B).

View larger version (50K): [in this window] [in a new window]   Figure 5. Time-lapse analysis of mitochondrial Ca2+ level. A, Representative sequential images of rhod-2 fluorescence, an indicator of mitochondrial Ca2+ level, in each group. B, Quantitative data of rhod-2 fluorescence. Data are mean±SEM from 3 independent experiments. Cariporide abrogated the increase of rhod-2 fluorescence in response to H2O2.

Effects of Cariporide on _m
Dissipation of _m is a critical event early in the process of cell death.¹¹ To examine whether preservation of _m is associated with cardioprotective effects by cariporide, we assessed the change of TMRE fluorescence by H₂O₂ in each group using FACS analysis. Incubation with H₂O₂ for 1 hour decreased TMRE fluorescence and shifted the distribution curve leftward, indicating the depolarization of _m (Figure 6A, top). The decrease of TMRE fluorescence was remarkably suppressed by cariporide, indicating the preservation of _m (Figure 6A, bottom). Summarized data from FACS analysis are shown in Figures 6B and 6C. We evaluated the percentage of cells that exhibited a high level of TMRE fluorescence (defined as >1x10² in this analysis). Cariporide preserved _m in a concentration-dependent manner (n=3 for each group) (Figure 6B). Inhibition of mitoK_ATP channels by 5-hydroxydecanoate (5HD) or glibenclamide did not cancel _m preservation by cariporide (n=3 for each group) (Figure 6C). Thus, the effects of cariporide are not attributable to indirect mitoK_ATP channel activation. To examine time-dependent changes of _m, confocal microscopy was performed using cells loaded with TMRE. Time-lapse scanning began immediately after application of H₂O₂. Figure 7A shows sequential images in each group, and Figure 7B shows the average of TMRE fluorescence intensity from 10 randomly selected cells in each group. TMRE fluorescence did not change during 60 minutes of scanning in control. Cells exposed to H₂O₂ progressively lost TMRE fluorescence intensity, indicating irreversible dissipation of _m. Cariporide remarkably preserved TMRE fluorescence.

View larger version (20K): [in this window] [in a new window]   Figure 6. Effects of cariporide on m. A, Representative histograms obtained by FACS analysis of cells loaded with TMRE are shown. Top, Gray area shows H2O2 group and black line shows control (CON). Bottom, Representative histogram from H2O2 plus cariporide. B and C, Quantitative results of FACS analysis. B, Cariporide preserved m in a concentration-dependent manner. C, Effects of 5HD or glibenclamide on m preservation by cariporide. Neither 5HD nor glibenclamide canceled the effects of cariporide. Data are expressed as percentage of cells with high TMRE fluorescence (defined as above 1x102 of fluorescence intensity). Data are mean±SEM (n=3 per group). *P<0.05 vs H2O2.

View larger version (58K): [in this window] [in a new window]   Figure 7. Time-lapse analysis of m. A, Representative sequential images of TMRE fluorescence in each group. B, Time course of TMRE fluorescence in each group. The graph shows mean fluorescence intensity of 10 cells randomly selected in each group. Cariporide prevented rapid decrease of TMRE fluorescence that was observed in H2O2. Similar results were obtained from 3 independent experiments.

	Discussion

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The major findings in the present study are as follows. First, cariporide suppressed markers of cell death, such as increased TUNEL positivity, caspase-3 activity, phosphatidylserine exposure, and PI positivity induced by oxidative stress. Second, [Na⁺]_i and [Ca²⁺]_i accumulation induced by oxidative stress were reduced by cariporide. Third, [Ca²⁺]_mito overload was prevented by cariporide. Fourth, cariporide prevented oxidative stress-induced _m loss in a dose-dependent manner. Fifth, _m preservation by cariporide was not abolished by 5HD or glibenclamide.

Figure 8 shows a proposed mechanism of cardioprotection by cariporide. During ischemia, NHE becomes activated to induce [Na⁺]_i overload in response to intracellular acidosis.^1,2,4 In addition, inhibition of Na⁺/K⁺ ATPase in ischemia prompts the sarcolemmal Na⁺/Ca²⁺ exchanger to work in reverse mode. In the present study, stimulation by H₂O₂ increased [Na⁺]_i, indicating that not only ischemia but also oxidative stress itself can activate NHE.¹² These alterations of intracellular ion dynamics inhibit Ca²⁺ efflux and enhance Ca²⁺ influx, thus producing [Ca²⁺]_i overload.^2,4 Consistent with this theory, we observed [Ca²⁺]_i increase in response to H₂O₂, although we lack direct evidence that [Ca²⁺]_i increase is obligatorily linked to the [Na⁺]_i increase. Cariporide suppressed increases in both [Na⁺]_i and [Ca²⁺]_i in the present study. These results are consistent with previous reports that cariporide preserved [Ca²⁺]_i transients in an ischemia-reperfusion rat heart model¹³ and that cariporide suppressed intracellular acidosis-induced [Na⁺]_i increase.¹⁴ Although the present results are striking, we cannot exclude the possibility that the dramatic effect of cariporide observed in the present study may be modified in a beating heart, in which excitation-contraction coupling may be more robust than it is in cultured cells.

View larger version (12K): [in this window] [in a new window]   Figure 8. Proposed mechanism of cardioprotection by cariporide.

Reduction of functional cardiomyocytes by apoptosis and necrosis contributes to ischemia-reperfusion injury in the heart.¹⁵ The previous reports showed that NHE inhibitors can attenuate apoptosis in animal models, such as ischemia/reperfusion in isolated heart¹⁶ and in vivo coronary artery occlusion.¹⁷ In the present study, we demonstrated that cariporide reduced TUNEL positivity, caspase-3 activity, and annexin V/ PI fluorescence in cardiomyocytes stimulated by H₂O₂, indicating that cariporide can inhibit both types of cell death, apoptosis and necrosis. Despite nearly complete suppression of intracellular Ca²⁺ overload, the partial effects of cariporide on TUNEL positivity and caspase-3 activity may suggest a contribution of Ca²⁺-independent death pathways, such as Fas, tumor necrosis factor ,^18,19 and Ca²⁺-independent cytochrome c release.²⁰ Time-lapse confocal microscopy using annexin V and PI demonstrated that cariporide prevents the early phase of cell death. Mitochondria are key determinants of cell death. We recently reported that cardiomyocytes exposed to oxidative stress show Ca²⁺-dependent morphological changes in mitochondria, such as swelling and loss of cristae before the opening of PTP.⁵ Increases in [Ca²⁺]_i should lead to [Ca²⁺]_mito accumulation, which triggers PTP opening, accompanied by catastrophic _m loss. Our findings that cariporide remarkably inhibited the [Ca²⁺]_mito increase and loss of _m induced by oxidative stress indicate that cariporide may prevent cell death by preserving mitochondrial integrity.

Despite the proposed mechanisms in Figure 8, we cannot exclude the possible role of NHE isoform located in the mitochondrial inner membrane²¹ in the observed cardioprotective effects by cariporide. Cariporide may directly work on mitochondria and may reduce [Ca²⁺]_mito overload by inhibiting mitochondrial NHE. Indeed, another NHE inhibitor, SM-20550, was reported to inhibit Na⁺ and H⁺ transport in mitochondria,²² and it preserved mitochondrial respiratory function.²³

MitoK_ATP channels have cardioprotective effects against ischemia/reperfusion injury.^24,25 The mitoK_ATP channel opener, diazoxide, prevents apoptosis induced by oxidative stress in cultured cardiomyocytes.⁸ Inhibition of mitochondrial Ca²⁺ overload and subsequent preservation of _m are considered the mechanisms of cardioprotection. A recent study demonstrated that 5HD, a mitoK_ATP channel blocker, did not abolish infarct size limitation by a NHE inhibitor.²⁶ In contrast, another group showed that 5HD and glibenclamide, but not a sarcolemmal K_ATP channel blocker (HMR1098), abolished the cardioprotection by cariporide.²⁷ We find that neither 5HD nor glibenclamide inhibits the effect of cariporide. These results suggest that the cardioprotective effects by cariporide may not be mediated by activation of mitoK_ATP channels. Consistent with the present results, a recent report showed that the cardioprotective effects of cariporide operate through a different pathway from 5HD-inhibitable ischemic preconditioning.²⁸

NHE inhibition is one of the most promising therapeutic strategies for ischemia-reperfusion injury. The classic NHE inhibitor, amiloride, was used in the first report of cardioprotective effects by NHE inhibition.²⁹ Thereafter, NHE inhibitors have attracted much attention, resulting in the development of specific and selective compounds (eg, cariporide). Based on experimental and clinical evidence of cardioprotection by NHE inhibitors, several multicentered international clinical trials have been initiated. In the Evaluation of the Safety and Cardioprotective Effects of Eniporide in Acute Myocardial Infarction (ESCAMI) trial, one of the newly developed compounds did not show any salutary effect.³⁰ However, cariporide significantly reduced combined incidence of death and myocardial infarction in patients with high-risk coronary artery bypass grafting surgery in subgroup analysis in the Guard During Ischemia Against Necrosis (GUARDIAN) trial.³¹ Based on these results, the Na⁺/H⁺ Exchanger Inhibition to Prevent Coronary Events in Acute Cardiac Conditions (EXPEDITION) trial was recently initiated using a modified dosing regimen of cariporide.¹ The results of EXPEDITION, although eagerly awaited, are not yet available.

	Acknowledgments

 
This study was supported by the NIH (R37 HL36957) and the Banyu Fellowship in Cardiovascular Medicine (to Dr Akao). Dr Marbán holds the Michel Mirowski, MD, Professorship of Cardiology.

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