Effect of Vacuolar Proton ATPase on pH_i, Ca²⁺, and Apoptosis in Neonatal Cardiomyocytes During Metabolic Inhibition/Recovery

From the Research Service (E.K.-P., J.A.N., H.L.L., R.L.E., R.A.G.), Veterans Affairs San Diego Healthcare System, San Diego, Calif; the Division of Biochemistry (R.A.G.), Department of Molecular & Experimental Medicine, The Scripps Research Institute, La Jolla, Calif; and the Department of Medicine (E.K.-P., H.L.L., R.L.E.), University of California, San Diego, School of Medicine, San Diego, Calif.

Correspondence to Robert L. Engler, MD, ACOS/Research, VA San Diego Health Care System (151), 3350 La Jolla Village Dr, San Diego, CA 92161. E-mail rengler{at}ucsd.edu

	Abstract

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Abstract—Recently, we found that vacuolar proton ATPase (VPATPase) operates in cardiomyocytes as a complementary proton-extruding mechanism. Its activity was increased by preconditioning with resultant attenuation of intracellular acidification during ischemia. In this study, we examined whether VPATPase-mediated proton efflux during metabolic inhibition/recovery may spare Na⁺ overload via Na⁺-H⁺ exchange, attenuate Na⁺-Ca²⁺ exchange, and decrease apoptosis. Neonatal rat cardiomyocytes were subjected to 2- to 3-hour metabolic inhibition with cyanide and 2-deoxyglucose and 24-hour recovery. The effect of VPATPase inhibition by 50 nmol/L bafilomycin A₁ on apoptosis, pH_i, and [Ca²⁺]_i was studied by flow cytometry with propidium iodide, seminaphthorhodafluor (SNARF)-1-AM, and indo-1-AM staining, respectively. VPATPase inhibition increased the amount of apoptosis measured after 24 hours of recovery and abrogated the protective effect of inhibition of Na⁺-H⁺ exchange by (5-N-ethyl-N-isopropyl)amiloride (EIPA). Dual blockade of VPATPase and Na⁺-H⁺ exchange was additive in effect with EIPA on pH_i during metabolic inhibition/recovery and recovery from the acid challenge with sodium propionate. VPATPase blockade increased the rate of accumulation of intracellular Ca²⁺ at the beginning of metabolic inhibition and abrogated the delaying effect of EIPA on intracellular Ca²⁺ accumulation. These results indicate that VPATPase plays an important accessory role in cardiomyocyte protection by reducing acidosis and Na⁺-H⁺ exchange–induced Ca²⁺ overload.

Key Words: apoptosis • vacuolar proton ATPase • bafilomycin A₁ • metabolic inhibition • cardiomyocyte

	Introduction

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Intracellular acidification is a general feature of the ischemic myocardium and develops as a result of anaerobic metabolism, net hydrolysis of ATP, and CO₂ retention.¹ Interventions that limit myocardial ischemic injury, eg, preconditioning, also attenuate intracellular acidosis,^2–4 which suggests that the latter may be harmful for the ischemic myocardium. There is now a growing number of observations suggesting that acidification is a common characteristic of apoptotic cells.^5–7 We⁸ and others^9,10 have previously demonstrated that ischemia/reperfusion induces death of cardiomyocytes primarily by apoptosis.

Cardiomyocytes possess various mechanisms for regulating pH_i. Commonly known are two alkalinizing ionic transporters, a Na⁺-H⁺ exchange¹¹ and Na⁺-HCO₃^- cotransporter,¹² and one acidifying Na⁺-independent Cl^--HCO₃^- exchanger.¹³ Although Na⁺-H⁺ exchange is considered to play the most important role in the recovery of pH_i from acute acid load (such as that induced by ischemia), in fact, its beneficial role for the ischemic/reperfused myocardium is limited. Paradoxically, the inhibition, not the activation, of the Na⁺-H⁺ exchange is beneficial for the ischemic/reperfused myocardium. Inhibition of the exchanger or low pH_o reduces the activity of the Na⁺-H⁺ exchanger and the detrimental Ca²⁺ overload via consequent Na⁺-Ca²⁺ exchange and protects myocardium.^14–17

Recently, we found that a third proton-extruding process, VPATPase, may operate in cardiomyocytes.⁴ VPATPases have been described in a variety of cell types, being responsible for the acidification of the intracellular compartment, such as synaptic vesicles, lysosomes, and endosomes.¹⁸ In addition to their endomembrane distribution, VPATPases have also been detected in the plasma membranes of some types of cells (eg, macrophages), thereby maintaining the cytosolic pH by extruding protons out of the cells.¹⁹ Taking into account the sensitivity of VPATPase to bafilomycin A₁, a potent and specific macrolide antibiotic inhibitor of VPATPase,²⁰ we previously found that in adult rabbit cardiomyocytes the VPATPase is activated by preconditioning, with resultant attenuation of intracellular acidification during MI.⁴ Bafilomycin A₁ was also found to induce apoptosis in neonatal rat cardiomyocytes when present in culture medium for 24 hours.²¹ These findings suggest that VPATPase activity may be important to cardiomyocyte pH homeostasis.

We hypothesized that VPATPase-mediated proton efflux may spare Na⁺ overload via Na⁺-H⁺ exchange and thereby attenuate Ca²⁺ influx via Na⁺-Ca²⁺ exchange. To test this hypothesis, we exposed neonatal rat cardiomyocytes to simulated ischemia (MI with cyanide and 2-deoxyglucose) and studied the effect of inhibition of VPATPase by bafilomycin A₁ on pH_i, [Ca²⁺]_i, and the amount of apoptosis observed after 24 hours of recovery.

	Materials and Methods

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Cell Culture
Neonatal rat ventricular myocytes were cultured from 1- to 2-day-old Sprague-Dawley rats as previously described²² with minor modifications. Briefly, ventricles were minced in a nominally Ca²⁺-free HEPES-buffered salt solution containing (mmol/L) NaCl 116, KCl 5.4, MgSO₄ 0.8, NaH₂PO₄ 1.0, HEPES 20, and glucose 5.5, and the myocardial cells were dispersed by the addition of 1 mg/mL pancreatin (GIBCO BRL) and 0.5 mg/mL type II collagenase (Worthington Biochemical Corp). Cells were stirred for 3 minutes at 37°C, and the supernatant was removed and discarded. Cells were incubated with fresh pancreatin-collagenase for 16 minutes at 37°C, the supernatant was collected, and cells were isolated by centrifugation for 6 minutes at 1100 rpm. Cells were resuspended in 4 mL newborn calf serum (Omega Scientific, Inc) and kept at 37°C. The digestion step was repeated 4 times, and cell suspensions from each digestion were combined and centrifuged at 1200 rpm for 6 minutes. Cells were then resuspended in Ca²⁺-free HBSS, layered onto a Percoll density gradient (density, 1.059/1.082), and centrifuged at 3000 rpm for 30 minutes. The band between the two Percoll layers containing the myocyte fraction was carefully removed and centrifuged twice in Ca²⁺-free HBSS. Cells were then resuspended in culture medium: 4:1 DMEM/medium 199 (GIBCO BRL), supplemented with 10% horse serum (Omega Scientific, Inc), 5% fetal calf serum (GIBCO BRL), and 1% antibiotics (10 000 U/mL penicillin and 10 000 µg/mL streptomycin). Cells were preplated for 1 hour to remove additional nonmyocytes. The myocytes were plated at a density of 6x10⁶ cells, 2x10⁶ cells, or 1x10⁶ cells in 100-mm, 65-mm, or 6-well tissue culture dishes, respectively, precoated with 1% gelatin (Sigma). Cells were incubated at 37°C in humidified atmosphere containing 5% CO₂. Culture medium was changed after 24 hours. Within 3 days, a confluent monolayer of spontaneously beating cells was formed. As established by visual determination, >90% of the cells manifested spontaneous contractions. Cells were used for experiments on days 4 and 5.

Metabolic Inhibition
Each culture was allowed to equilibrate in HBSS containing (mmol/L) NaCl 125, KCl 4.9, MgSO₄ 1.2, NaH₂PO₄ 1.2, CaCl₂ 1.8, NaHCO₃ 8.0, HEPES 20.0, and glucose 10.0, pH 7.4, for 1 hour at 37°C before the start of MI. MI was induced by incubating the cells with 5 mmol/L NaCN and 20 mmol/L 2-deoxyglucose in glucose-free HBSS (HBSS/MI), pH 6.6, at 37°C. Unless otherwise stated, cells were allowed to recover in culture medium for 24 hours after the MI period.

Flow Cytometry
All experiments were performed on a Coulter Elite flow cytometer equipped with an air-cooled 488 argon laser and a helium-cadmium UV laser. At least 10 000 cells were collected for each sample, and the data were analyzed using the Coulter software package and MultiTime (Phoenix Flow).

Detection of Apoptosis
Before harvesting the cells, their culture medium was collected to save possibly detached apoptotic and necrotic cells. The attached cells were washed 2 times with PBS, lifted from the plates with trypsin/EDTA (Irvine Scientific), and combined with the previously collected detached cells. Cells were washed again 2 times with PBS, resuspended in PBS, and stained with 1 µg/mL PI (Sigma). After 5 minutes of incubation at room temperature, cells were analyzed on a flow cytometer with excitation at 488 nm and emission at 620 nm. This technique identifies normal cells as PI-negative, apoptotic cells as PI^dim, and necrotic cells as PI^bright.²³

In 2 experiments, the cells, stained with PI as described above, were analyzed on both the flow cytometer and an LSCM (CompuCyte Corp).²⁴ Cells were also grown on 8-chamber culture slides for 2 days, and then the cells in 4 chambers were subjected to 3 hours of MI and 24 hours of recovery. The culture medium was removed, and cells were stained with PI (1 µg/mL) and analyzed by LSCM. Similar populations, PI-negative, PI^dim, and PI^bright, were found by using LSCM and flow cytometry. With the LSCM it was possible to simultaneously examine the morphology of the cells. Most of the cells in the PI^bright region were swollen in size and showed very brightly stained intact nuclei. In contrast, cells in the PI^dim region were smaller and contracted in size and showed dim staining throughout the cell and blebbing of the plasma membrane. Some cells even showed the distinctive speckled staining of apoptotic cells. Thus, with the LSCM we confirmed that the PI^dim cells were apoptotic cells, whereas the PI^bright were necrotic and/or possibly apoptotic cells that "converted" to necrotic cells.

Determination of pH_i
Cells, detached with trypsin/EDTA, were resuspended in HBSS. Cells were loaded with 10 µmol/L carboxy-SNARF-1-AM (Molecular Probes) for 30 minutes at room temperature, washed 2 times, and resuspended in HBSS. Experiments started 20 to 30 minutes later to ensure adequate loading of the dye. Cells were analyzed on a flow cytometer with excitation at 488 nm and emission at 575 and 620 nm corresponding to the H⁺-bound and -free forms of carboxy-SNARF-1-AM. pH_i was estimated from the ratio of emission intensities at these 2 wavelengths.²⁵ Conversion of the 620/575-nm ratio into pH_i values was performed from calibration curves established from cells whose pH_i values were fixed by incubation with nigericin (10 µmol/L) in high K⁺ buffers.²⁶ There was a linear correlation between pH_i ranging from 6.6 to 7.4 and the 620/575-nm ratio (r=0.986). Fluorescence was arbitrarily scaled from 0–1024. Cells with fluorescence <50 were excluded to avoid analysis of cells that either were not loaded with the dye or that had lost membrane integrity.

Determination of [Ca²⁺]_i
Cells, detached with trypsin/EDTA and resuspended in HBSS, were loaded for 45 minutes at room temperature in the dark with 10 µmol/L indo-1-AM (Molecular Probes) initially dissolved in DMSO containing 2% [wt/vol] Pluronic F-127 (Molecular Probes). Cells were then washed 2 times and resuspended in HBSS. Experiments started 30 minutes later to ensure adequate loading of the dye. Cells were analyzed on a flow cytometer with excitation at 325 nm and emission at 405 and 525 nm corresponding to the peak emission of the Ca²⁺-bound and Ca²⁺-free forms of the indicator, respectively. The 405/525 ratio was used as an index of [Ca²⁺]_i. Cells with fluorescence <50 were excluded. No autofluorescence of unloaded cells was detected in any of the experimental conditions. Neither of the drugs (EIPA and bafilomycin A₁) used affected the 405/525 ratio measured in the cells loaded with indo-1 during control conditions.

Drugs
Bafilomycin A₁ (Sigma) and EIPA (Research Biochemicals Intl) were kept at -20°C as stock solutions in DMSO at 0.2 mmol/L and 50 mmol/L, respectively. The working solutions were freshly prepared before each experiment. KB-R7943, the inhibitor of the Na⁺-Ca²⁺ exchanger, was kindly provided by Kanebo, Ltd, through the generosity of Munekazu Shigekawa of the National Cardiovascular Center Research Institute, Osaka, Japan. KB-R7943 was dissolved in DMSO at 10 mmol/L and diluted further in buffer to a final concentration of 10 µmol/L.

Statistical Analysis
Results are expressed as mean±SEM. Statistical comparisons between groups were carried out by a one-way ANOVA with a post hoc Tukey-Kramer test for multiple comparisons or unpaired Student t test. An level of 0.05 was considered indicative of a statistically significant difference.

	Results

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Role of VPATPase in the Regulation of pH_i in Neonatal Rat Cardiomyocytes
To determine whether the VPATPase is active in neonatal rat cardiomyocytes and plays a role in the recovery of pH_i after intracellular acidification, we challenged carboxy-SNARF-1-AM–loaded cells with 50 mmol/L sodium propionate in the presence of bafilomycin A₁ and/or EIPA. The pH_i was monitored by flow cytometry. In control (untreated) cells, sodium propionate loading immediately decreased pH_i from 7.49±0.08 to 6.92±0.07 (P<0.05). Neither bafilomycin A₁ nor EIPA significantly affected these values. During the first 5 minutes after acid loading, pH_i recovered by 0.266±0.02 pH units (Figure 1). EIPA decreased the recovery of pH_i by 77%. Bafilomycin A₁ alone decreased this recovery by only 21% (P=NS) but showed an additive effect with EIPA: the combination of EIPA+bafilomycin A₁ decreased the recovery of pH_i by 93% (Figure 1). These results suggest that VPATPase is present in neonatal rat cardiomyocytes, but its activity may be masked by more active Na⁺-H⁺ exchange. However, when the Na⁺-H⁺ exchanger is inhibited, the VPATPase seems to be able to partially compensate for the increased acid load.

View larger version (33K): [in this window] [in a new window]   Figure 1. pHi during 5 minutes of recovery from acute intracellular acid load. Cells were loaded with carboxy-SNARF-1-AM in HBSS, and pHi was monitored by flow cytometry. Untreated cells (C) were compared with cells pretreated for 30 minutes before acid load (50 mmol/L sodium propionate) with bafilomycin A1 (BAF), EIPA, or both compounds together (BAF+EIPA). Results are expressed as mean±SEM (n=4). *P<0.05 for EIPA or BAF+EIPA vs control (C); #P<0.05 for BAF+EIPA vs EIPA.

MI Induces Apoptosis in Cardiomyocytes: Effect of Inhibition of VPATPase and Na⁺-H⁺ Exchange
To test the ability of MI to induce apoptosis in neonatal rat cardiomyocytes, cells were subjected to different periods of MI followed by 24 hours of recovery in culture medium. Apoptosis was measured by staining nonpermeabilized cells with PI followed by flow cytometric analysis. MI-induced apoptosis and the percentage of apoptotic (PI^dim) cells progressively increased with extension of the MI period, reaching 65.9±2.6% of apoptotic cells after 3 hours of MI (Figure 2). The percentage of necrotic cells was similar for all groups studied and did not exceed 10%. This MI-induced apoptosis is cardiomyocyte specific; noncardiomyocytes (ie, cells collected from the upper Percoll gradient and cultured like cardiomyocytes) showed only 7.26±0.86% apoptosis (n=3) when measured after 3-hour MI/24-hour recovery.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of MI on the number of cardiomyocytes undergoing apoptosis after 24 hours of recovery. Apoptosis was detected by PI staining and flow cytometry. Results are expressed as mean±SEM (n=5 to 7). *P<0.05 vs time 0 (control cells not subjected to MI).

To evaluate the role of VPATPase on the MI-induced apoptosis, bafilomycin A₁ (50 nmol/L) was included in the culture medium 15 minutes before, during, and 15 minutes after 2.5-hour MI. Bafilomycin A₁ increased the percentage of apoptotic cells compared with the untreated group in a dose-dependent fashion (Figure 3). Control cells (not subjected to MI) did not show any difference in the amount of apoptosis when treated for the same time period (3 hours) with bafilomycin A₁ (6.96±0.82% versus 6.72±0.28% for untreated [n=5] and bafilomycin A₁–treated [n=4] cells, respectively). This finding suggests that the activity of VPATPase limits the development of apoptosis in the cardiomyocytes subjected to MI and recovery.

View larger version (58K): [in this window] [in a new window]   Figure 3. Dose-dependent effect of bafilomycin A1 on the number of cardiomyocytes undergoing apoptosis after 2.5 hours of MI and 24 hours of recovery. Apoptosis was detected by PI staining and flow cytometry. Cells were treated with bafilomycin A1 15 minutes before, during, and 15 minutes after MI (total, 3 hours). Results are expressed as mean±SEM (n=4). *P<0.05 vs time 0 (cells subjected to MI without bafilomycin A1).

Bafilomycin A₁ also abrogated the protective antiapoptotic effect of inhibition of Na⁺-H⁺ exchange by EIPA during MI/recovery, most probably by blocking the compensatory mechanism of proton elimination after inhibition of Na⁺-H⁺ exchange (Figure 4). Neither 1 µmol/L EIPA alone nor in combination with bafilomycin A₁ affected the amount of apoptosis when applied for 3 hours to control cells not subjected to MI (5.92±0.7% [bafilomycin A₁+EIPA, n=4] and 4.73±0.2% [untreated controls, n=5] versus 5.56±0.5% [EIPA, n=4]). These results suggest that the protective effect of VPATPase during MI/recovery may be due to its ability to decrease the intracellular acid load. To test this possibility, we compared the changes in the pH_i in cells subjected to MI/recovery with or without inhibition of the VPATPase and/or Na⁺-H⁺ exchanger.

View larger version (44K): [in this window] [in a new window]   Figure 4. Effect of EIPA with or without bafilomycin A1 (BAF) on the number of cardiomyocytes undergoing apoptosis after 3 hours of MI and 24 hours of recovery. Apoptosis was detected by PI staining and flow cytometry. Cells were treated with EIPA or EIPA+BAF 15 minutes before, during, and 15 minutes after MI. Results are expressed as mean±SEM (n=6 for all except for EIPA at 10 µmol/L [n=4]). *P<0.05 vs untreated cells (C); #P<0.05 for 1 µmol/L EIPA vs 1 µmol/L EIPA+50 nmol/L BAF.

pH_i Changes During MI and Recovery: Effect of Bafilomycin A₁ and EIPA
In order to monitor pH_i during MI and recovery, cardiomyocytes were loaded with carboxy-SNARF-1-AM and analyzed by flow cytometry. MI induced a rapid decrease in pH_i in cardiomyocytes, so within 10 minutes of initiation of MI, pH_i decreased by 0.46 pH units from 7.44±0.06 to 6.98±0.07. Thereafter, pH_i continued to decrease more slowly, reaching a plateau of 6.73±0.06 after 60 minutes of MI. Extending the period of MI to 3 hours did not significantly change the pH_i. Neither bafilomycin A₁ nor EIPA affected pH_i values during MI. During recovery after 1 hour of MI, pH_i rapidly increased within the first 10 minutes by 0.41 pH units and slowly continued to increase thereafter, reaching a plateau of 7.34±0.04 at 30 minutes of recovery (Figure 5, inset). No difference in recovery of pH_i was observed if MI was extended to 2 or 3 hours (not shown). The recovery of pH_i during 0 to 30 minutes was decreased by EIPA but not affected by bafilomycin A₁ alone (Figure 5). However, the combination of bafilomycin A₁ and EIPA showed an additive effect, decreasing further the recovery of pH_i. These results support our conclusion that VPATPase is constitutively active, although its contribution to pH homeostasis is small relative to the activity of the Na⁺-H⁺ exchange in neonatal rat cardiomyocytes. Note that only cells with intact cell membranes (to retain SNARF) are included in this analysis.

View larger version (53K): [in this window] [in a new window]   Figure 5. pHi during recovery (0 to 30 minutes) from 1 hour of MI in neonatal rat cardiomyocytes. Cells were loaded with carboxy-SNARF-1-AM in HBSS, and pHi was monitored by flow cytometry. Control untreated cells (C) were compared with cells treated 15 minutes before, during, and 15 minutes after MI with bafilomycin A1 (BAF), EIPA (1 µmol/L), or both compounds together (BAF+EIPA). Results are expressed as mean±SEM (n=5). *P<0.05 for EIPA or BAF+EIPA vs control (C); #P<0.05 for BAF+EIPA vs EIPA. Inset shows the time course of pHi change during recovery from MI in untreated neonatal rat cardiomyocytes.

The consequence of Na⁺-H⁺ exchange during ischemia is Na⁺ influx and, as a result, compensation by Na⁺-Ca²⁺ exchange, leading to Ca²⁺ influx. In the next series of experiments, we tested the hypothesis that the VPATPase activity in exporting protons during MI reduces Na⁺ influx via Na⁺-H⁺ exchange and that this attenuates Ca²⁺ influx.

[Ca²⁺]_i Changes During MI: Effect of Bafilomycin A₁ and EIPA
To monitor the [Ca²⁺]_i changes during MI, cardiomyocytes were loaded with indo-1 and analyzed by flow cytometry. We noted that the mean ratio of indo-1 fluorescence (=relative [Ca²⁺]_i) increased as MI proceeded; however, the range of values was quite wide and made statistical analysis difficult. For that reason, we considered only the number of cells that had an elevated indo-1 ratio (=elevated [Ca²⁺]_i). We established the upper limit of normal as the value that included >95% of the control cell population (before MI). In untreated cells, increased [Ca²⁺]_i was first observed 10 minutes after the beginning of MI. The percentage of cells with elevated [Ca²⁺]_i reached 100% by 60 minutes of MI (Figure 6). Bafilomycin treatment resulted in an increased number of cells with elevated [Ca²⁺]_i at every time point examined. The percentage was nearly double compared with control after 15 minutes of MI (Figure 6). Inhibition of Na⁺-H⁺ exchange with EIPA reduced the number of cells with elevated [Ca²⁺]_i at all time points (Figure 6). Blockade of both Na⁺-H⁺ exchange and proton pumping resulted in accumulation of cells with elevated Ca²⁺ at rates similar to those for untreated cells (not shown), suggesting that other modes of Ca²⁺ accumulation exist. These results demonstrate that the VPATPase indirectly reduces Ca²⁺ influx, presumably by attenuating Na⁺-H⁺ exchange and secondary Na⁺-Ca²⁺ exchange.

View larger version (27K): [in this window] [in a new window]   Figure 6. Time course of increase in [Ca2+]i during MI. Cells were loaded with indo-1-AM in HBSS, and [Ca2+]i was monitored by flow cytometry just before (0 minutes) and during MI. Untreated cells (, n=9) were compared with those treated with bafilomycin A1 (, n=11) or with 10 µmol/L EIPA (, n=4). Results are expressed as mean±SEM. Quantification of the percentage of cells with elevated [Ca2+]i was obtained by drawing 2 arbitrary regions on the count/ratio profiles at 0 minutes (see inset). Region A included 90% of cells in the major peak representing those with normal [Ca2+]i; region B included the remaining cells with elevated [Ca2+]i. A shift from region A to B was observed during MI (see inset). *P<0.05 vs 0 minutes of MI.

Effect of Inhibition of Na⁺-Ca²⁺ Exchange
To directly test the role of Na⁺-Ca²⁺ exchange, we used the inhibitor KB-R7943, which has been shown to inhibit the reverse mode of the Na⁺-Ca²⁺ exchanger.²⁷ We found that KB-R7943 reduced the number of cells with elevated [Ca²⁺]_i during MI. Although the differences were small at early time points, the effect was significant by 45 minutes (67.0±10.9% versus 54.3±7.4%, P<0.05). Similarly, KB-R7943 reduced the number of cells undergoing apoptosis after MI/recovery (51.5±9.8% versus 29.3±14.2%, P=0.001). These findings again support a link between Na⁺-Ca²⁺ exchange, Ca²⁺ influx, and apoptosis.

	Discussion

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MI with cyanide and 2-deoxyglucose is used as an experimental model to simulate ischemic cell injury.¹⁶ In the present study, we show that when the period of recovery is extended to 24 hours, apoptosis is the dominant form of cardiomyocyte death. The molecular events leading to myocardial apoptosis are still poorly understood. Our observation that inhibition of the VPATPase by bafilomycin A₁ increased the percentage of apoptotic cells after MI/recovery supports the notion of the involvement of VPATPase in defense against ischemia/reperfusion injury. We treated cells with 50 nmol/L bafilomycin A₁, a concentration expected to inhibit >95% of VPATPase activity (K_i for bafilomycin is 5 nmol/L) but not to affect other ion-translocating ATPases, as this requires micromolar concentrations of bafilomycin A₁.²⁰ However, it is important to note that all small-molecule inhibitors have the potential to exert nonspecific effects on enzymes other than the desired targets. Short-term treatment with bafilomycin A₁ did not affect the viability of control cells but only those subjected to MI/recovery, thus excluding the possibility of drug toxicity. Altogether, this observation is consistent with our previous results showing that upregulation of the VPATPase delays apoptosis in neutrophils exposed to granulocyte colony-stimulating factor⁶ and in preconditioned adult rabbit cardiomyocytes subjected to MI/recovery.⁴ Prolonged inhibition (24 to 48 hours) of VPATPase with bafilomycin A₁ was also reported to induce apoptotic cell death in WEHI 231 lymphoma cells,²⁸ pheochromocytoma PC12 cells,²⁹ and neonatal rat cardiomyocytes.²¹ It should be noted that bafilomycin A₁ inhibits the VPATPase at the plasma membrane as well as in association with lysosomal vesicles, which in prolonged culture could interfere with receptor recycling. The short-term effect of bafilomycin treatment is the release of protons from acidic vesicles, as well as a failure of proton extrusion across the plasma membrane, with net cytoplasmic acidification.

These results indicate that the VPATPase is constitutively active but that its contribution to pH homeostasis is masked by more active Na⁺-H⁺ exchange. Bafilomycin A₁ increased the rate of accumulation of intracellular Ca²⁺ during MI. The most probable explanation of this finding is that the activity of VPATPase in exporting protons during MI reduces the contribution of the Na⁺-H⁺ exchanger in this process. The consequence of Na⁺-H⁺ exchange is Na⁺ influx, which is compensated for by the Na⁺-Ca²⁺ exchanger (reverse mode), resulting in intracellular Ca²⁺ accumulation.^14,15 Proton elimination by the VPATPase spares Na⁺-H⁺ exchange and limits Na⁺ influx and secondary Ca²⁺ influx. The relationship between excessive Ca²⁺ influx and cell death has already been established. The interactions between the VPATPase, the Na⁺-H⁺ exchanger, and the Na⁺-Ca²⁺ exchanger are depicted in Figure 7.

View larger version (20K): [in this window] [in a new window]   Figure 7. Proposed interactions of two pathways for proton export in myocytes. Protons are eliminated through the Na+-H+ exchanger (NHE-1) with resulting Na+ influx. This causes the Na+-Ca2+ exchanger (NCX) to operate in reverse mode to eliminate Na+ at the expense of Ca2+ influx. Inhibition of NHE-1 by EIPA limits Na+ influx and secondary Ca2+ accumulation, which can also be prevented by inhibition of the reverse mode of the NCX by KB-R7943 (KBR). Proton elimination is also accomplished by activity of the VPATPase, which thereby reduces the activity of NHE-1. Inhibition of the VPATPase by bafilomycin A1 (BAF) causes increased activity of NHE-1, with increased Na+ influx and secondary Ca2+ accumulation.

Our results confirmed that inhibition of Na⁺-H⁺ exchange during MI/recovery was protective. Although the beneficial effect of inhibition of Na⁺-H⁺ exchange on the ischemic/reperfused myocardium is widely reported,^14,15 its mechanism is still a matter of debate. Decrease in intracellular Na⁺ and, consequently, Ca²⁺ loading¹⁵ or beneficial effect of acidosis itself¹⁶ have been considered as potential mechanisms. However, inhibition of Na⁺-H⁺ exchange was not protective if VPATPase was simultaneously inhibited. With both VPATPase and the Na⁺-H⁺ exchanger inhibited, the degree of apoptosis was comparable to that of untreated cells subjected to MI/recovery. The abrogation of the protective effect of EIPA by bafilomycin A₁ was accompanied by an increased rate of accumulation of Ca²⁺ and an even lower pH_i during recovery. Acidification in and of itself may be injurious. Acidification will also increase the levels of free Ca²⁺ through a shift in equilibrium and may also affect other ion transport systems controlling Ca²⁺ influx.

The VPATPase plays an important accessory role in cardiomyocyte protection by reducing acidosis and Na⁺-H⁺ exchange-induced Ca²⁺ overload. Evidence supporting the hypothesis that acidosis contributes to apoptosis are the reports showing that (1) intracellular acidification precedes apoptosis in neutrophils⁶ and Jurkat cells³⁰; (2) apoptotic cells have lower pH_i compared with normal cells⁵; and (3) acidic endonuclease (DNAse II), which becomes active at pH 6.8 and below, is involved in apoptosis.³¹ We have previously observed lower pH_i during MI in bafilomycin A₁–treated adult rabbit cardiomyocytes.⁴ On the other hand, cytosolic pH was only slightly and transiently decreased in PC12 cells cultured with bafilomycin A₁, and attempted alkalinization with NH₄Cl did not prevent apoptosis.²⁹ In neonatal rat cardiomyocytes, prolonged incubation with bafilomycin A₁ resulted in apoptosis, which was accompanied by acidification.²¹ In the present study, however, bafilomycin A₁ alone without Na⁺-H⁺ exchange blockade did not significantly affect the pH_i measured either during MI/recovery or recovery from an acid challenge. When both Na⁺-H⁺ exchange and VPATPase were inhibited, however, the ability of cardiomyocytes to normalize pH_i after an acid load or MI was severely suppressed. Taken together, these results suggest that acidosis, as well as Ca²⁺ overload, contributes to the ischemia/reperfusion injury leading to apoptosis.

	Selected Abbreviations and Acronyms

 

DMSO = dimethyl sulfoxide EIPA = (5-N-ethyl-N-isopropyl)amiloride LSCM = laser scanning cytometer MI = metabolic inhibition PI = propidium iodide SNARF = seminaphthorhodafluor VPATPase = vacuolar proton ATPase

	Acknowledgments

 
This study was supported by Research Service, Veterans Affairs San Diego Health Care System, and Veterans Medical Research Foundation of San Diego. Dr Gottlieb was supported by a Junior Faculty Scholars Award from the American Society of Hematology.

Received December 22, 1997; accepted March 16, 1998.

	References

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