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Acidification reduces mitochondrial calcium uptake in rat cardiac mitochondria

¹Division of Cardiovascular Medicine, University of California-Davis, Davis 95616; and ²Cardiology Section, Department of Veteran Affairs Northern California Health Care System, Sacramento, California 95655

Submitted 12 April 2004 ; accepted in final form 29 July 2004

	ABSTRACT

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Cardiac ischemia-reperfusion (I/R) injury is accompanied by intracellular acidification that can lead to cytosolic and mitochondrial calcium overload. However, the effect of cytosolic acidification on mitochondrial pH (pH_m) and mitochondrial Ca²⁺ (Ca_m²⁺) handling is not well understood. In the present study, we tested the hypothesis that changes in pH_m during cytosolic acidification can modulate Ca_m²⁺handling in cardiac mitochondria. pH_m was measured in permeabilized rat ventricular myocytes with the use of confocal microscopy and the pH-sensitive fluorescent probe carboxyseminaphthorhodafluor-1. The contributions of the mitochondrial Na⁺/H⁺ exchanger (NHE_m) and the K⁺/H⁺ exchanger (KHE_m) to pH_m regulation were evaluated using acidification and recovery protocols to mimic the changes in pH observed during I/R. Ca_m²⁺transport in isolated mitochondria was measured using spectrophotometry and fluorimetry, and the mitochondrial membrane potential was measured using a tetraphenylphosphonium electrode. Cytosolic acidification (pH 6.8) resulted in acidification of mitochondria. The degree of mitochondrial acidification and recovery was found to be largely dependent on the activity of the KHE_m. However, the NHE_m was observed to contribute to the recovery of pH_m following acidification in K⁺-free solutions as well as the maintenance of pH_m during respiratory inhibition. Acidification resulted in mitochondrial depolarization and a decrease in the rate of net Ca_m²⁺uptake, whereas restoration of pH following acidification increased Ca_m²⁺uptake. These findings are consistent with an important role for cytosolic acidification in determining pH_m and Ca_m²⁺handling in cardiac mitochondria under conditions of Ca²⁺ overload. Consequently, interventions that alter pH_m can limit Ca_m²⁺overload and injury during I/R.

mitochondrial pH; mitochondrial calcium; sodium/hydrogen exchange; potassium/hydrogen exchange; mitochondrial membrane potential

Ischemia results in cytosolic acidification, followed by a recovery of pH during reperfusion. Cytosolic acidification during ischemia may serve as one mechanism for the elevated cytosolic Ca²⁺ observed during I/R. Specifically, cytosolic acidification can stimulate the sarcolemmal Na⁺/H⁺ exchanger (NHE), resulting in increased cytosolic sodium and a subsequent increase in cytosolic Ca²⁺ via activity of the Na⁺/Ca²⁺ exchanger. However, the effects of cytosolic acidification on mitochondrial pH (pH_m) and Ca_m²⁺handling are not thoroughly understood. An important role for pH_m was suggested by a recent study in permeabilized HL-1 cells showing that reduced mitochondrial acidification resulted in increased levels of Ca_m²⁺during simulated ischemia (27). Mitochondrial acidosis has also been shown to affect Ca_m²⁺release via the MPT (3, 16, 24) as well as the mitochondrial Na⁺/Ca²⁺ exchanger (2).

Under physiological conditions, mitochondria generate and maintain an alkaline matrix pH as a result of proton transport by the electron transport chain. However, the pH_m gradient could be altered under conditions of metabolic inhibition when proton generation by the mitochondria is compromised, resulting in mitochondrial acidification. In addition, mitochondria have been shown to express several proton antiporters (6), including a mitochondrial NHE exchanger (NHE_m) and a mitochondrial K⁺/H⁺ exchanger (KHE_m) (14). Both of these exchangers could potentially regulate pH_m under conditions of cytosolic acidification via coupling of Na⁺ and K⁺ fluxes to the proton gradient. However, the relative roles of these exchangers in the regulation of pH_m under conditions of cytosolic acidification or metabolic inhibition have not been evaluated.

In the present study, we tested the hypothesis that cytosolic acidification could regulate Ca_m²⁺handling via alterations in pH_m in cardiac mitochondria. Changes in pH_m in permeabilized myocytes were measured in response to a cytosolic acidification and recovery protocol, and the role of the mitochondrial proton transporters was evaluated under these conditions. pH_m was also measured under conditions of respiratory inhibition as seen during myocardial ischemia. To evaluate the effect of mitochondrial acidification on the mitochondrial membrane potential (_m) and Ca_m²⁺transport, these variables were measured in isolated cardiac mitochondria exposed to a Ca²⁺ bolus at varying pH.

	MATERIALS AND METHODS

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Animals. Cardiac myocytes and mitochondria were isolated from the left ventricle of adult male Sprague-Dawley rats. Rats were anesthetized using pentobarbital sodium (100 mg/kg ip) before the heart was excised from the chest cavity. The procedures for isolation were approved by the Institutional Animal Care and Use Committee of the University of California, Davis.

Isolation of rat ventricular myocytes. Left ventricular myocytes were isolated with the use of a standard enzymatic digestion procedure using collagenase and protease (10). Freshly isolated ventricular myocytes were stored at room temperature and used within 8 h of isolation.

Isolation of mitochondria from rat ventricles. Mitochondria were isolated from the left ventricle using mechanical disruption followed by a differential centrifugation procedure as described previously (17). Briefly, left ventricular tissue was minced and homogenized using two 10-s cycles in a polytron followed by a 10-min centrifugation at 2,000 rpm. After resuspension and further centrifugation at 10,500 rpm, mitochondria were resuspended in storage buffer containing (in mmol/l) 250 sucrose and 5 Tris·HCl, and pH was adjusted to 7.4 using KOH. Mitochondria were used within 3 h of isolation and kept stirred on ice until use. Mitochondrial protein concentration was determined using the Bradford assay and calibrated using bovine serum albumin (BSA).

Measurement of pH_m in permeabilized myocytes using carboxy-SNARF-1 and confocal microscopy. Freshly isolated rat ventricular myocytes were loaded with 5 µmol/l 5- (and 6-) carboxyseminaphthorhodafluor-1 (carboxy-SNARF-1) AM acetate for 30 min at room temperature in an extracellular medium containing (in mmol/l) 118 NaCl, 4.6 KCl, 1.2 MgCl₂, 1.2 KH₂PO₄, 10 HEPES, 1 CaCl₂, and 10 dextrose; pH was adjusted to 7.2 with NaOH. For imaging experiments, the loaded myocytes were plated on glass coverslips coated with Cell-tak (BD Biosciences; Bedford, MA).

SNARF-1 AM-loaded myocytes were washed in extracellular medium containing 0 Ca²⁺ and 50 µmol/l EGTA before permeabilization. Myocytes were permeabilized using 0.001% digitonin for 3 min in an intracellular medium containing (in mmol/l) 110 KCl, 10 3-(N-morpholino)propanesulfonic acid (MOPS), 5 MgCl₂, 5 Na₂ATP, and 0.5 EGTA, plus BSA 2 mg/ml; the pH was adjusted to either 6.8 or 7.2 using Tris base (10). KCl (110 mmol/l) was replaced with 110 mmol/l choline chloride for choline chloride-containing solutions. Oligomycin (4 µg/ml) and KCN (2 mmol/l) were added to the intracellular medium in experiments involving respiratory inhibition. 5-(N-ethyl-N-isopropyl) amiloride (EIPA) was prepared as a 100 mmol/l stock solution in DMSO, and 0.1% DMSO was used as a vehicle control.

Permeabilized myocytes were imaged using a x63 oil-immersion objective of an inverted microscope (Carl Zeiss) equipped with laser-scanning confocal unit (Pascal LSM 510). SNARF was excited using the 514-nm line of an argon laser, and the emitted fluorescence at 580 nm and 650 nm was captured using a dichroic mirror at 615 nm. With the use of an optical slice of <0.7 µm, full-frame images (512 x 512 pixel) were acquired, and the mean fluorescence intensities of the mitochondrial regions were analyzed at the two emission wavelengths. pH_m is expressed as the SNARF (580 nm/650 nm) emission ratio, and an increase in the SNARF (580 nm/650 nm) ratio represents a decrease in pH.

Ca²⁺ transport measurements in isolated mitochondria. Ca²⁺ transport in isolated mitochondria was measured using the metallochromic indicator antipyrylazo III (APIII), which exhibits a strong increase in absorbance at 720 nm when bound to Ca²⁺ (28). APIII does not enter mitochondria, and hence the changes in APIII absorbance are directly proportional to changes in extramitochondrial Ca²⁺. For measurement of Ca²⁺ transport, mitochondria (0.5 mg/ml protein) were suspended in a buffer containing (in mmol/l) 100 KCl, 5 Na₂ATP, 10 MOPS, 0.01 EGTA, and 0.1 APIII; pH was adjusted to various values between 6.4 and 7.4 using Tris base. Thapsigargin (0.5 µmol/l) was added to prevent any potential interference from the sarcoplasmic reticulum during Ca_m²⁺measurements. Mitochondria were magnetically stirred, and measurements were made at 25°C using a dual-beam spectrophotometer (Hewlett-Packard). After a period of incubation (3 min), mitochondria were exposed to a Ca²⁺ bolus (300 nmol·mg protein^–1·ml^–1), and Ca²⁺ transport was monitored for 900 s using a 720- to 790-nm wavelength pair. The mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 0.2 µmol/l) was added during the last 60 s of each measurement to induce maximal Ca²⁺ release.

Net Ca²⁺ uptake rate was calculated from the linear portion of the net Ca²⁺ uptake phase and is expressed as arbitrary absorbance units per second (AU/s).

[Ca²⁺]_m was also measured using the fluorescent Ca²⁺ indicator indo-1 AM. Isolated mitochondria were incubated with 5 µM indo-1 AM in storage buffer for 30 min at room temperature (15). Mitochondria were then centrifuged and resuspended in fresh, indo-1-free storage buffer. Fluorimetric measurements of indo-1 AM fluorescence were made every 2 s using a modified spectrofluorometer (SLM Instruments; Rochester, NY) in a buffer containing (in mmol/l) 100 KCl, 5 Na₂ATP, 10 MOPS, and 0.01 EGTA (pH adjusted to 6.8 or 7.4 using Tris base). Indo-1 was excited at 350 nm, and the emitted fluorescence at 385 nm and 465 nm was recorded. Ca_m²⁺is expressed as the indo-1 (385 nm/465 nm) fluorescence ratio, and an increase in the ratio represents an increase in intramitochondrial Ca²⁺.

Measurements of mitochondrial membrane potential in isolated mitochondria. Quantitative measures of _m were made as described earlier (8) using tetraphenylphosphonium (TPP⁺) electrodes that exhibited a linear, Nernstian response connected to a high-impedence pH meter (model 290A, Orion; Boston, MA). Briefly, mitochondria (1 mg protein) were incubated in buffer containing (in mmol/l) 100 KCl, 5 Na₂ATP, 10 MOPS, 0.01 EGTA, and 0.003 TPP⁺Cl (pH adjusted to various values between 6.4 and 7.4 using Tris base). Measurements were made every 5 s, and _m was calculated using the formula (8):

where _m is measured in millivolts, v is the mitochondrial matrix volume (1.1 µl/mg protein), V is the volume of the incubation medium (1,200 µl), and E is the deflection of the electrode potential from baseline.

Statistics. A two-way ANOVA followed by a Bonferroni post test was used to analyze differences between groups over time. Data are expressed as means ± SE. Differences were considered significant at P < 0.05.

	RESULTS

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Measurement of pH_m in permeabilized myocytes. Figure 1A is a confocal image of a rat ventricular myocyte loaded with SNARF and permeabilized in an intracellular-like medium. The mitochondrial localization of the SNARF fluorescence is supported by the precise overlap of the SNARF fluorescence image with that obtained in intact myocytes loaded with the mitochondrial-selective dye tetramethylrhodamine methyl ester (Fig. 1B). Further support for mitochondrial localization comes from calibration experiments using nigericin and FCCP showing that the pH value of the SNARF fluorescence signal in permeabilized myocytes was >7.8, in agreement with observed values of pH in respiring mitochondria (20). Figure 1C shows that the mitochondrial uncoupler FCCP restored pH in permeabilized myocytes to that of the extramitochondrial medium. In addition, the SNARF fluorescence signal was also sensitive to oligomycin and KCN (see Fig. 4), confirming the mitochondrial origin of the fluorescence signal.

View larger version (40K): [in this window] [in a new window]   Fig. 1. Selective mitochondrial loading of carboxyseminaphthorhodafluor-1 (carboxy-SNARF) in permeabilized cardiomyocytes. A: confocal image of a permeabilized rat ventricular myocyte loaded with carboxy-SNARF. B: SNARF fluorescence is localized to punctate mitochondrial regions, similar to that seen in an intact myocyte loaded with the selective mitochondrial probe tetramethylrhodamine methyl ester (TMRM). C: calibration of the SNARF (580 nm/650 nm) fluorescence ratios at various pH values. Dotted line, SNARF fluorescence value obtained following treatment of permeabilized myocytes with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP).

View larger version (22K): [in this window] [in a new window]   Fig. 4. EIPA enhances mitochondrial acidification under conditions of respiratory inhibition. The time course of the changes in pHm during incubation of mitochondria in KCl medium containing KCN and oligomycin at pH 6.8 (circles) or pH 7.2 (triangles) is plotted for myocytes treated with 0.1% DMSO (Control) (closed symbols) or 100 µmol/l EIPA (open symbols) throughout the incubation. *SNARF fluorescence ratios are significantly different from the corresponding control. n = 7–8 myocytes from 2 to 3 independent isolations.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Mitochondria exhibit increased acidification and delayed recovery in the absence of extramitochondrial potassium. Mitochondrial SNARF fluorescence ratios are plotted for myocytes exposed to an acidic pH (pH 6.8 or pH 5.7) for 5 min followed by recovery to normal extramitochondrial pH (pH 7.2) for 15 min. Experiments were carried out in myocytes incubated in either KCl-containing or choline chloride-containing intracellular-like medium. *Mitochondrial SNARF fluorescence was significantly different from myocytes exposed to pH 6.8 medium in KCl. n = 6 myocytes from 3 independent isolations.

Role of the NHE_m in mitochondrial acidification and recovery. The role of the NHE_m in mitochondrial acidification and recovery was examined using the Na⁺/H⁺ exchange inhibitor EIPA at a concentration of 100 µmol/l. This concentration was chosen based on previous reports in isolated mitochondria, where 100 µmol/l EIPA was shown to inhibit the NHE_m by 50% (4). In KCl-containing medium, addition of EIPA did not significantly alter either the magnitude of acidification or recovery compared with vehicle-treated myocytes (Fig. 3A). EIPA also had no significant effect in control experiments, where myocytes were maintained at pH 7.2 throughout the duration of the experiment. These data suggest that the NHE_m is not a significant contributor to pH_m regulation either under normal physiological conditions or conditions of cytosolic acidification in KCl medium.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Mitochondrial Na+/H+ exchanger (NHEm) inhibition with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) significantly affects mitochondrial pH (pHm) in choline chloride but not KCl medium. Permeabilized myocytes were exposed to either 100 µmol/l EIPA (open symbols) or 0.1% DMSO (control) (closed symbols) in KCl (A) or choline chloride (B), and SNARF fluorescence ratios were measured at the indicated time points. Myocytes were exposed to buffers at pH 6.8 (circles) or pH 7.2 (triangles) during the first 5 min of the protocol. *SNARF fluorescence ratio was significantly different versus the corresponding DMSO control. n = 4–6 myocytes from 3 independent isolations.

Effect of EIPA during respiratory inhibition. Because the NHE has been shown to play a critical role under conditions of ischemia and reperfusion (1, 23), we examined the role of the NHE_m in pH_m regulation under conditions of respiratory inhibition (Fig. 4). In these experiments, 2 mmol/l KCN and 4 µg/ml oligomycin were included during acidification to inhibit electron transport and to block proton translocation via the F₁F_O ATPase, respectively. Respiratory inhibition during acidification in a KCl-based medium resulted in mitochondrial acidification that was maintained over the 20 min of monitoring. However, myocytes underwent a significantly greater mitochondrial acidification in the presence of 100 µmol/l EIPA. A similar effect of EIPA was observed at pH 7.2 in the presence of oligomycin and KCN. These experiments suggest that the NHE_m is responsible for maintenance of pH_m under conditions of respiratory inhibition when proton translocation out of the mitochondrial matrix is compromised.

Effect of pH_m on Ca_m²⁺ handling and _m. To evaluate the effect of mitochondrial acidification on Ca_m²⁺handling, Ca²⁺ transport was measured in isolated mitochondria incubated at varying pH levels. Ca²⁺ transport in isolated mitochondria was monitored using the indicator APIII, which measures extramitochondrial Ca²⁺ (28). Isolated cardiac mitochondria were incubated in KCl-based buffer containing ATP, a solution similar to that used in the permeabilized myocyte experiments. Addition of a Ca²⁺ bolus (300 nmol·mg mitochondrial protein^–1·m^–1) at pH 7.4 resulted in 50% net uptake of Ca²⁺, followed by net release. With increasing acidification, the rate and the magnitude of maximal uptake of the Ca²⁺ bolus were greatly reduced and essentially no net uptake of Ca²⁺ was observed at pH values of 6.6–6.4 (Fig. 5A). As shown in Fig. 5B, there was a direct linear correlation between extramitochondrial pH and the rate of net Ca²⁺ uptake. Thus extramitochondrial acidification, which also results in acidification of the mitochondrial matrix (Fig. 2), reduces net Ca_m²⁺uptake in isolated mitochondria.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Acidification decreases mitochondrial membrane potential and Ca2+ uptake in isolated cardiac mitochondria. A: representative plot for the changes in the antipyrylazo (APIII) absorbance (extramitochondrial Ca2+) versus time for mitochondria that were incubated in KCl medium at varying external pH values. A 300 nmol·mg protein–1·ml–1 Ca2+ bolus and 0.2 µmol/l FCCP were added at the times indicated. AU, artibrary units. B: mitochondrial membrane potential before addition of Ca2+ (right y-axis) and Ca2+ uptake rates following addition of a Ca2+ bolus (left y-axis) are plotted as a function of extramitochondrial pH. n = 4–6 measurements from 4 independent isolations.

Because acidification was found to limit Ca_m²⁺uptake, we next investigated whether recovery of pH could restore increased Ca²⁺ uptake in mitochondria. These studies were carried out using fluorescence measurements in indo-1 AM-loaded mitochondria, because restoration of pH resulted in an artifact in the APIII absorbance signal. As shown in Fig. 6, [Ca²⁺]_m was significantly lower at pH 6.8 following addition of a Ca²⁺ bolus, consistent with the results in Fig. 5A. However, normalization of pH to 7.4 resulted in a rapid increase in intramitochondrial Ca²⁺ to levels similar to that observed in mitochondria incubated at pH 7.4 alone. Measurements of mitochondrial membrane potential using a TPP⁺ electrode showed that pH normalization resulted in a reestablishment of _m to values similar to that observed at pH 7.4 (data not shown). Furthermore, Ca²⁺ uptake at pH 7.4 (or following pH normalization to 7.4) was significantly blocked in the presence of ruthenium red (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 6. Recovery of pH following acidification results in restoration of increased mitochondrial Ca2+ uptake. Representative plot for the changes in indo-1 fluorescence (intramitochondrial Ca2+) for mitochondria that were incubated in KCl-containing medium at pH 7.4 (triangles) or pH 6.8 followed by normalization of pH to 7.4 (circles) are plotted. A 300 nmol/mg protein Ca2+ bolus was added where indicated, and pH normalization was achieved by addition of 4 mM KOH. Similar results were obtained in 5 independent experiments.

	DISCUSSION

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Comparison of our results with previous studies. The role of mitochondrial proton transporters, particularly the NHE_m, in regulating pH_m and Ca_m²⁺has been examined in a recent study (27). In an atrial cell line that was subjected to simulated ischemia, inhibition of the NHE_m with cariporide resulted in reduced mitochondrial acidification. The discrepancy of their findings and those of the present study could possibly be explained by differences in conditions under which NHE_m activity was measured, i.e., the presence of high concentrations of Ca²⁺ (10 µmol/l), which was significantly greater than in our studies, and could result in mitochondrial acidification secondary to Ca²⁺ influx. Under conditions similar to those used by Ruiz-Meana et al. (27), experiments performed in this laboratory using rat ventricular myocytes resulted in irreversible contracture, suggesting important differences between freshly isolated rat ventricular myocytes and the atrial cell line. Interestingly, however, a greater mitochondrial acidification was associated with lower Ca_m²⁺, which is in agreement with the results obtained in our study. The exact mechanism for elevated Ca²⁺_m was not addressed in the study by Ruiz-Meana et al. (27), although this effect was not reversed by the mitochondrial mitochondrial Na⁺/Ca²⁺ exchanger inhibitor clonazepam, suggesting that the Na⁺/Ca²⁺ exchanger was not involved in Ca_m²⁺loading.

Role of NHE_m and KHE_m in pH_m regulation. Incubation of permeabilized myocytes in an acidic medium resulted in an immediate decrease in pH_m. The KHE_m appeared to play an important role in maintaining pH_m under conditions of mitochondrial acidification and recovery from acidification. Previous studies characterizing the KHE_m in isolated mitochondrial preparations suggest that the KHE_m is latent under normal, physiological conditions (14) and is primarily activated by depletion of matrix Mg²⁺, by increase in matrix volume, and by alkaline matrix pH (5). However, our studies show that the KHE_m could also be active under conditions of mitochondrial acidification and during recovery following mitochondrial acidification.

In contrast to the KHE_m, the NHE_m did not appear to play a significant role in regulating pH_m during acidification or recovery in the presence of a functional KHE_m. However, in the absence of extramitochondrial K⁺, the NHE_m appeared to be important in regulating recovery from acidification. Studies in isolated mitochondria have shown that the NHE_m is not involved in the maintenance of pH_m under normal physiological conditions (14, 19). Our results support and extend this observation by demonstrating that the NHE_m did not play an active role in the regulation of pH_m under either normal or acidified conditions in the presence of K⁺ fluxes. However, the NHE_m appeared to be important in maintaining pH_m in respiratory inhibited mitochondria when proton extrusion by mitochondria was compromised. Under these conditions, inhibition of the NHE_m with 100 µmol/l EIPA resulted in a greater mitochondrial acidification, suggesting that the NHE_m was functioning to extrude protons from the matrix in exchange for extramitochondrial Na⁺. Altogether, these results suggest that the NHE_m does not regulate pH_m in energized mitochondria but can maintain pH_m in mitochondria under conditions of respiratory inhibition.

pH_m and Ca_m²⁺ regulation. The present study shows that extramitochondrial acidification results in mitochondrial depolarization and decreased Ca_m²⁺uptake in cardiac mitochondria. Furthermore, recovery of pH_m in previously acidified mitochondria resulted in restoration of increased Ca_m²⁺uptake, suggesting an important role for pH_m in determining [Ca²⁺]_m uptake. Because Ca²⁺ influx via the mitochondrial Ca²⁺ uniporter depends on _m (7), it is likely that the reduced Ca²⁺ uptake on acidification is a consequence of decreased _m. These findings are in agreement with previous reports where acidosis was shown to reduce Ca_m²⁺accumulation (13) via the ruthenium red-sensitive uniporter (11).

In addition to effects on Ca_m²⁺uptake, mitochondrial acidification has also been shown to affect Ca_m²⁺release via irreversible opening of the MPT. In nonenergized mitochondria, an acidic pH_m has been shown to reduce the open probability of the MPT (16) via inhibitory effects of matrix protons on this transporter (3). However, Kristian et al. (24) have shown that acidification promotes MPT opening in energized mitochondria via alterations in mitochondrial phosphate transport. This effect may be relevant under reperfusion conditions when mitochondria are energized under conditions of high cytosolic Ca²⁺.

Protective effects of NHE_m inhibition. The decreased pH_m observed with EIPA during respiratory inhibition, coupled with the observation that increased mitochondrial acidification can limit Ca_m²⁺uptake, suggests that inhibition of NHE_m may be one mechanism to limit Ca_m²⁺during I/R. This mechanism may also partially account for the protective effects of NHE_m blockers observed in several recent studies, where selective inhibition of the NHE_m limited the increases in [Ca²⁺]_m and resulted in improvement of cardiac function during I/R (18), (31). Separation of a direct effect of EIPA on Ca_m²⁺loading from that resulting from limitation of cytosolic Ca²⁺ secondary to plasmalemmal NHE inhibition has not yet been achieved.

Limitations. In the absence of a specific inhibitor of the NHE_m, 100 µmol/l EIPA was used as a NHE_m inhibitor in our studies. The selective NHE-1 inhibitor cariporide was not an effective inhibitor of the NHE_m in our permeabilized rat myocytes, consistent with data that the NHE_m may be a different subtype than the sarcolemmal NHE-1 (26) and exhibit a pharmacological profile distinct from the NHE-1 (4, 22). EIPA (100 µmol/l) has been shown to inhibit the NHE_m by 50% in previous studies (4). Preliminary studies indicated that lower concentrations of EIPA (10 µmol/l), while effective in inhibiting NHE-1, did not significantly inhibit NHE_m. In addition to having known inhibitory effects on the NHE, EIPA has also been shown to be an inhibitor of mitochondrial Na⁺/Ca²⁺ exchanger (21). However, inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger by EIPA would not be relevant in our study in permeabilized myocytes because no Ca²⁺ was added to the intracellular medium and 0.5 mmol/l EGTA was included to chelate any adventitious Ca²⁺. EIPA has also been reported to have protonophoric effects in bilayers (9), an effect that could lead to mitochondrial acidification. However, no effect of EIPA on pH_m was observed in KCl medium, arguing against a significant role for protonophoric effects in our study.

Our results indicate a key role for mitochondrial acidification in limiting mitochondrial Ca²⁺ and suggest that recovery of pH may allow for greater Ca_m²⁺overload. However, extrapolation of these results to cardiac I/R may be limited because our experiments involving pH changes in isolated mitochondria under fixed ionic conditions may not accurately model the more complex changes that occur during I/R in the whole heart.

In summary, our studies in rat cardiac mitochondria suggest that increased mitochondrial acidification under simulated ischemic conditions can result in decreased uptake of Ca_m²⁺. Reduced levels of Ca_m²⁺during I/R have been shown to strongly correlate to improved recovery of cardiac function following I/R. The results from our studies suggest that strategies that could enhance the magnitude of mitochondrial acidification during ischemia and/or reperfusion could potentially limit Ca_m²⁺loading and enable functional recovery of mitochondria on reperfusion.

	GRANTS

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This study was supported by the American Heart Association, National Grant-in-Aid and the Philip Morris External Research Program Grant to S. Schaefer.

	ACKNOWLEDGMENTS

 
We thank the University of California, Davis Health System Confocal Microscopy Facility for use of the confocal microscope for this study.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. Schaefer, One Shields Ave., TB 172, Bioletti Way, Univ. of California, Davis, Davis, CA 95616 (E-mail: sschaefer{at}ucdavis.edu)

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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