INTRODUCTION

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PROTOCOLS OF REPERFUSION of the ischemic myocyardium that prevent a rapid renormalization of intracellular pH have been found to protect myocardial structure and function (12, 17, 27). We showed recently using an isolated cell model of simulated ischemia-reperfusion that a low intracellular pH (pH_i) can be beneficial because it prevents reoxygenation-induced hypercontracture of cardiomyocytes (17). Reoxygenation-induced hypercontracture represents a major cause for acute lethal cell injury in reperfused myocardium (2). The present study was undertaken to define a treatment of cardiomyocytes applied solely during reoxygenation that provides protection against reoxygenation-induced hypercontracture by blockade of the main routes of H⁺ extrusion.

Normal myocardial cells possess two major sarcolemmal transport mechanisms for H⁺ extrusion. One is the Na⁺/H⁺ exchanger (NHE) and the other is the HCO₃-dependent H⁺ outward transport, commonly called the Na⁺-HCO₃ symporter (NBS) (18, 35). The relative importance of either of these mechanisms in cardiomyocytes after an extended period of ischemic energy depletion is unknown. Experimental studies in vivo and in the isolated heart in which NHE inhibitors were administered solely during the reperfusion period (4, 10, 14, 20, 22, 23, 25, 29) have produced controversial results with respect to myocardial protection against reperfusion injury. This is in contrast to the robust protection found when NHE inhibitors were applied during the ischemic period.

In the present study the hypotheses was tested that it may be required to inhibit on reoxygenation the NBS in addition to NHE to protect energy-depleted myocardial cells against reoxygenation-induced injury. The study was performed with isolated cardiomyocytes (from adult rats) exposed to anoxia in medium with pH 6.4 and subsequent reoxygenation in medium with pH 7.4, simulating important parts of ischemia-reperfusion conditions in the whole heart. This model was characterized previously with respect to cellular energy metabolism, ion homeostasis (Na⁺, H⁺, Ca²⁺), and cell morphology (16, 17, 30, 32, 33). It has been shown that reoxygenation-induced hypercontracture is provoked by the recovery of aerobic energy production in the state of cytosolic Ca²⁺ overload, which results from the disturbances of cellular cation control under ischemic conditions (33).

Recently, specific inhibitors of the NHE have become available. In the present study, the benzoylguanidine derivative HOE-642 was used, which was shown to inhibit with great selectivity the prevalent myocardial subtype of NHE, i.e., NHE-1 (26, 28). The tool most frequently used to inhibit the bicarbonate-linked transporters is 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). This irreversible anion channel inhibitor is not as selective for the NBS as HOE-642 is for the NHE, but more specific inhibitors are currently not available. Because of these limitations the experiments with DIDS were compared with another set of experiments in which bicarbonate-dependent sarcolemmal H⁺ extrusion in reoxygenated cardiomyocytes was prevented by omission of bicarbonate from media.

	METHODS

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Isolation of cardiomyocytes. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1985). Ventricular heart muscle cells were isolated from 200 to 250 g adult male Wistar rats and plated in medium 199 with 4% fetal calf serum on glass coverslips, which had been preincubated overnight with 4% fetal calf serum (24). Four hours after plating was completed, the coverslips were washed with medium 199. As a result of the wash, damaged cells were removed, leaving a homogeneous population of rod-shaped quiescent cardiomyocytes (>95%) attached to the coverslip.

Loading of fura 2, SBFI, and BCECF. To measure cytosolic Ca²⁺, Na⁺, or H⁺ concentrations, cardiomyocytes were loaded at 35°C with fura 2, Na⁺-binding benzolfuran isophthalate (SBFI), or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), respectively. For loading, cells attached to the glass coverslips were incubated for 30 min in medium 199 with the acetoxymethyl ester of fura 2 (5 µmol/l), SBFI (10 µmol/l), and for 15 min with BCECF (1.25 µmol/l). After loading, the cells were washed twice with medium 199. This washing step was followed by a 30-min postincubation period in medium 199 to allow hydrolysis of the acetoxymethyl esters within the cell. The fluorescence from dye-loaded cells was 20-30 times higher than background fluorescence, i.e., fluorescence from cells not loaded with the dye. The loading protocols used were selected from a number of variations because they provided the highest yield in fluorescence and minimal dye compartmentation (16).

Ca²⁺, Na⁺, pH, and cell length measurements. The cover-slip with the loaded cells was introduced into a gas-tight, temperature-controlled (37°C), transparent perfusion chamber positioned in the light path of an inverted microscope (Diaphot, Nikon, Düsseldorf, Germany). Alternating excitation of the fluorescent dye at wavelengths of 340 and 380 nm for fura 2 and SBFI, respectively, and 440 and 490 nm for BCECF was performed with an AR-Cation Measurement System adapted to the microscope (ISA, Grasbrunn, Germany). Light emitted (500-520 nm for fura 2 and SBFI and 520-560 nm for BCECF) from an area of 10 × 10 µm within a single fluorescent cell was collected by the photomultiplier of the system. The light signal was recorded and analyzed by an IBM PC/AT-based data analysis system (model DM3000CM, ISA, Grasbrunn, Germany). Simultaneously to the measurement of fluorescence, the microscopic image of the cell was recorded with a video camera, stored on tape, and printed with a video printer. Changes in cell length were determined from these recordings. In the case of hypercontracted cells, the cell dimension along its previous longitudinal axis was determined.

In vivo calibration of fura 2, SBFI, and BCECF. Because of the saturation limit of fura 2, Ca²⁺ data were usually expressed in arbitrary units of fluorescence ratios of the emitted light of the two corresponding excitation wavelengths. To facilitate understanding, calibration protocols were performed to obtain numerical relationships between selected ratio values and ion concentrations. The fura 2 signal was calibrated according to the method described by Li et al. (19). For this purpose, the cells were exposed to 5 µmol/l ionomycin in modified Tyrode solution (pH 7.4; composition see below) containing either 3 mmol/l Ca²⁺ or 5 mmol/l EGTA to obtain the maximum (R_max) and the minimum (R_min) ratio of fluorescence, respectively. To prevent morphological alterations during calibration, cells were depleted of ATP with 1 mmol/l KCN. The free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was calculated according to Grynkiewicz et al. (8) with the use of a dissociation constant (K_d) for fura 2 of 312 nmol/l, determined in intact cardiomyocytes by constructing a calibration curve (16). For the first 10 min of reoxygenation the integral of the fura 2 ratio was determined as the area between the actual trace of the fura 2 ratio and the ratio of 0.5, which is the normoxic value of the fura 2 ratio. SBFI ratio was calibrated according to Harootunian et al. (9) with 6 µmol/l gramicidin D and incubation in media containing various Na⁺ concentrations and pH values. Calibration of the BCECF ratio signal was performed, as previously described (15), with 10 µg/ml nigericin, a K⁺/H⁺ ionophore, and incubation media with various pH values.

Media. The normoxic bicarbonate-buffered medium contained (in mmol/l) 118.0 NaCl, 2.6 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 5.0 glucose, 1.0 CaCl₂, and 22.0 NaHCO₃; the medium was gassed with 5% CO₂-95% O₂, and the resulting intracellular pH (pH_o) was 7.4. This medium was used for the preanoxic period and for reoxygenation after anoxia. For anoxia, the medium was changed as follows: glucose was omitted and the bicarbonate concentration was reduced to 2.2 mmol/l, resulting in a pH of 6.4 when medium was gassed with 5% CO₂-95% N₂. For preparation of the anoxic media the water had been autoclaved to remove oxygen to <0.01 Torr (1). NaCl concentration of the anoxic medium was elevated to 137.8 mmol/l to equalize the Na⁺ concentrations of anoxic and normoxic media. In one series of experiments, cells were reoxygenated in bicarbonate-free, HEPES-buffered medium of the following composition (in mmol/l): 125 NaCl, 2.6 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 5.0 glucose, 1.0 CaCl₂, and 25.0 HEPES; the pH of this medium was adjusted with 1 N NaOH to pH 7.4. HOE-642 and DIDS were administered during the reoxygenation period in a final concentration of 3 µmol/l and 0.5 mmol/l, respectively.

In the normoxic experiments where hypercontracture was elicited by Na⁺ withdrawal, a HEPES buffer of the above-specified composition was used in which NaCl was replaced by isoosmolar N-methyl-D-glucamine. HOE-642 (3 µmol/l) or DIDS (0.5 mmol/l) was administered 5 min before the Na⁺ withdrawal.

Experimental protocols. During anoxia cardiomyocytes were superfused (0.5 ml/min) with the anoxic glucose-free bicarbonate-buffered medium (pH 6.4). After 70 min of anoxia, cells were reoxygenated for 10 min with bicarbonate-containing or bicarbonate-free, HEPES-buffered media (each pH 7.4). Six experimental conditions for reoxygenation were compared: 1) bicarbonate-buffered medium without additions, 2) bicarbonate-buffered medium with addition of HOE-642, 3) bicarbonate-buffered medium with addition of DIDS, 4) bicarbonate-buffered medium with addition of HOE-642 and DIDS, 5) HEPES-buffered medium without additions, and 6) HEPES-buffered medium with addition of HOE-642.

Materials. Medium 199 was purchased from Boehringer, fetal calf serum from GIBCO, and acetoxymethyl esters of fura 2, SBFI, and BCECF from Paesel and Lorey. HOE-642 was a gift from Dr. H. J. Lang from Hoechst. All other chemicals were from Merck or Sigma and of highest purity available.

Statistics. Data are given as means ± SE from n individual cells investigated in separate experiments. Statistical comparisons were performed by one-way ANOVA and use of the Student-Newman-Keuls test for post hoc analysis. Differences with P < 0.05 were regarded as statistically significant.

	RESULTS

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Changes in pH_i, [Ca²⁺]_i, [Na⁺]_i, and cell length during anoxia and reoxygenation. The time course of changes in pH_i, [Ca²⁺], cytosolic Na⁺ concentration, and cell length was monitored in individual cardiomyocytes submitted to anoxia (superfusion of the cells in media with pH_o = 6.4) and reoxygenation (superfusion in media with pH_o = 7.4). Figure 1 depicts a synopsis of the investigated parameters in original recordings from individual cardiomyocytes: 1) pH_i, 2) the fura 2 ratio indicating changes in cytosolic Ca²⁺ concentration, and 3) cell length. Table 1 provides the statistical data. In cardiomyocytes incubated under normoxic control conditions in medium with pH 7.4, a pH_i of 7.1 was observed. When the cells were submitted to anoxia in medium with pH 6.4 in bicarbonate-containing buffer, pH_i decreased to 6.4. During reoxygenation in bicarbonate-containing medium with pH 7.4, pH_i returned to the control value within <10 min. During the investigated 70-min anoxia, 95 ± 4% of all cells underwent rigor shortening (n = 47), which reduces cell length by approximately one-third. Rigor shortening is a rapid process completed within 1 min. It occurred after 35 ± 7 min of anoxic incubation (n = 16). Simultaneously to rigor shortening the fura 2 ratio started to rise. After 70 min of anoxia the cardiomyocytes reached a fura 2 ratio of 3.5, corresponding to an intracellular [Ca²⁺]_i of 1.8 µM/l. This is to be compared with the preanoxic fura 2 ratio of 0.5, corresponding to an [Ca²⁺]_i of 0.08 µM/l. The SBFI ratio rose from an initial value of 0.61 to a value of 0.77 during anoxia, corresponding to a rise of intracellular Na⁺ concentration from 15 to 84 mmol/l. On reoxygenation it returned to the control level. During anoxia cardiomyocytes underwent a partial rigor contracture; on reoxygenation they shortened to ~30% of their initial length, i.e., they developed hypercontracture. As typical for the isolated cell model (30, 33), reoxygenated cardiomyocytes were able to reestablish a normal cytosolic Ca²⁺ and Na⁺ control after the development of hypercontracture.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Time course of intracellular pH (pHi) (A) and fura 2 ratio indicating changes in cytosolic Ca2+ concentrations (B; continuous trace of original recording) and cell length in percentage of initial normoxic value (B; open circles) in single cardiomyocyte during simulated ischemia and reoxygenation. Anoxic incubations were performed in medium with extracellular pH (pHo) 6.4, reoxygenation in medium with pHo 7.4. au, Arbitrary units.

Changes in pH_i during reoxygenation in presence of H⁺ transport inhibition. Inhibition of the NBS or the NHE, either alone or together, significantly influenced the recovery of pH_i in cardiomyocytes reoxygenated in media with pH 7.4. In Fig. 2, recovery of pH_i during the reoxygenation period is shown for the various conditions tested. When reoxygenation was carried out under bicarbonate-buffered conditions in the absence of the inhibitors, pH_i returned to the control value within 5 min. Inhibition of the NBS with DIDS slowed down the initial speed of recovery by ~30%. A more pronounced deceleration of pH_i recovery was found when the NHE was inhibited by HOE-642. When both inhibitors were administrated simultaneously, recovery of pH_i was prevented. When reoxygenation was carried out in bicarbonate-free media, the pH_i recovered with the same rate as when the NBS was blocked with DIDS. When cardiomyocytes were reoxygenated in bicarbonate-free media with HOE-642, a pH_i recovery no longer occurred. The reversibility of H⁺ transport during reoxygenation was tested with cardiomyocytes reoxygenated in bicarbonate-free medium supplemented with HOE-642. When this medium was replaced after 10 min of reoxygenation by the normoxic bicarbonate medium without additions, pH_i recovered within another 10 min to 7.00 ± 0.04 (n = 8, not significant vs. initial normoxic value).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   pHi during reoxygenation under control conditions of reoxygenation, reoxygenation in presence of 0.5 mmol/l DIDS or in HEPES-buffered media, in presence of 3 µmol/l HOE-642 (HOE), and reoxygenation in simultaneous presence of both inhibitors or in HEPES-buffered media with HOE-642 (HEPES + HOE). Dashed line indicates pHi in normoxic cells. Experimental protocol was as specified for Fig. 1. Data are means ± SE, n = 8 cells; *P < 0.05 vs. control.

Changes in [Ca²⁺]_i control during reoxygenation in presence of H⁺ transport. In Fig. 3, original recordings of the first 10 min of reoxygenation under control bicarbonate-buffered conditions are shown in extended time resolution. In the absence of inhibitors (Fig. 3), the fura 2 ratio dropped from the plateau value of 3.5 obtained during anoxia to a ratio of ~1.5. This initial drop was instantaneously followed by a period of oscillations of the fura 2 signal. These oscillations have been shown to represent repetitive shifts of Ca²⁺ between cytosol and sarcoplasmic reticulum (32, 33). Finally, the fura 2 ratio returned to the initial control level and the oscillations of the fura 2 ratio ceased. In the absence of DIDS and HOE-642, hypercontracture developed gradually during the phase of Ca²⁺ oscillations. Statistical data for Ca²⁺ oscillations and hypercontracture are presented in Figs. 4 and 5. When pH_i recovery during reoxygenation was completely inhibited by DIDS and HOE-642 or in bicarbonate-free media with HOE-642 (Figs. 3 and 4), recovery of the fura 2 signal also occurred. Its course was altered, however, in that the Ca²⁺ oscillations were much less frequent but had prolonged transients. Hypercontracture, developing simultaneously to the oscillatory shifts of Ca²⁺ in the control situation, did not occur in the presence of the inhibitors or in bicarbonate-free media with HOE-642 (Figs. 3 and 5). Under control conditions, oscillations of the fura 2 ratio occurred during the first 10 min of reoxygenation (Fig. 4). The frequency was maximal at the fourth minute, i.e., at the time when pH_i recovery was nearly completed. These oscillations were greatly attenuated in simultaneous presence of HOE-642 and DIDS (Figs. 3B and 4) or under bicarbonate-free conditions in the presence of HOE-642 (Fig. 4). The inhibition of only one pH-regulating system reduced the oscillations to a smaller but still significant extent. To estimate net changes on the cytosolic Ca²⁺ balance during reoxygenation, the integral of the fura 2 signal was determined. The integral values of eight individual cells were calculated from the beginning of reoxygenation to complete recovery of the fura 2 signal (after 10 min of reoxygenation). The integral of fura 2 (in arbitrary units, au) during reoxygenation was in the control cells 1,188 ± 124 au, the same as in the HOE-treated cells (1,381 ± 102 au), in the DIDS-treated cells (1,222 ± 145 au), in the HOE + DIDS-treated cells (1,182 ± 140 au), in the HEPES-buffered cells (1,277 ± 92 au), and in the HEPES + HOE-treated cells (1,365 ± 174 au). Despite the clear differences in Ca²⁺ oscillations, there were no significant differences in the integral values of the fura 2 ratio during 10 min of reoxygenation characterizing the removal of bulk Ca²⁺ from the cytosol. These data indicate that the overall rate of Ca²⁺ removal from reoxygenated cells was similar under all experimental conditions tested.

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Time course of fura 2 ratio (continuous trace of original recording) and cell length in percentage of initial cell length (open circles) in single cardiomyocytes during reoxygenation after simulated ischemia under control conditions of reoxygenation (A) and reoxygenation in presence of 0.5 mmol/l DIDS and 3 µmol/l HOE-642 (B). Experimental protocol was as specified for Fig. 1.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   A: oscillation frequency of fura 2 ratio in reoxygenated cardiomyocytes under control conditions, in simultaneous presence of 0.5 mmol/l DIDS and 3 µmol/l HOE-642, and bicarbonate-free conditions with HOE-642. B: oscillation frequency of fura 2 ratio in presence of 0.5 mmol/l DIDS, in presence of 3 µmol/l HOE-642, or in presence of bicarbonate-free media. Experimental protocol was as specified for Fig. 1. Data are means ± SE, n = 8 cells; *P <0.05 vs. control; #P < 0.05 vs. DIDS + HOE and HEPES + HOE.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Cell length in percentage of initial normoxic value of single cardiomyocytes during reoxygenation under control conditions, in presence of 0.5 mmol/l DIDS, or in HEPES-buffered media, in presence of 3 µmol/l HOE-642, and in simultaneous presence of both inhibitors or in HEPES-buffered media with HOE-642. Experimental protocol was as specified for Fig. 1. Data are means ± SE, n = 8 cells; *P < 0.05.

Changes in cell length during reoxygenation in presence of H⁺ transport inhibition. At the end of anoxia, the length of cardiomyocytes was reduced to ~70% of its initial value, which represents the end-ischemic cell length due to rigor shortening (Fig. 5). When left in the state of rigor shortening for more than 5 min, cells do not relengthen when aerobic energy production is restored (34). During the first 10 min of subsequent reoxygenation, the cells developed hypercontracture to 35.2 ± 5.3% of the initial cell length in bicarbonate-containing medium (Fig. 5). Presence during reoxygenation of either DIDS (cell length after 10 min of reoxygenation: 38.2 ± 5.3%, not significant vs. control; n = 8) or HOE-642 (cell length after 10 min of reoxygenation: 45.3 ± 4.1%, not significant vs. control; n = 8) in bicarbonate-buffered media did not prevent hypercontracture. However, hypercontracture was completely prevented when both inhibitors were applied simultaneously (cell length after 10 min of reoxygenation: 65.0 ± 3.5%, P > 0.5 vs. control; n = 8). This protection could also be achieved when the cardiomyocytes were reoxygenated under bicarbonate-free condition in the presence of HOE-642 (Fig. 5) but not with bicarbonate-free media alone. The stability of protection against hypercontracture by conditions inhibiting both sarcolemmal H⁺ transport mechanisms at the onset of reoxygenation was tested with cardiomyocytes reoxygenated in bicarbonate-free medium supplemented with HOE-642. When this medium was replaced by the normoxic bicarbonate-buffered medium without additions after 10 min of reoxygenation, hypercontracture remained absent (cell length after another 10 min: 64.2 ± 5.2%; P < 0.05 vs. control, n = 8) even though the pH_i returned to 7.0.

Control experiments for effects of DIDS and HOE-642 on hypercontracture. It was tested in another set of experiments if DIDS and HOE-642 could prevent hypercontracture, provoked by Ca²⁺ overload, in a manner not related to intracellular acidosis. For this purpose, Ca²⁺ overload was induced in nonacidotic normoxic cardiomyocytes (pH_i = 7.06 ± 0.02, n = 5). When exposed to Na⁺-free media, these cells developed Ca²⁺ overload due to activation of the reverse mode of the Na⁺/Ca²⁺ exchanger (Table 2). As a sign of the developing Ca²⁺ overload in these cells, spontaneous oscillations of cytosolic Ca²⁺ appeared with a frequency similar to that in reoxygenated cardiomyocytes (4 min after Na⁺ withdrawal: 62.0 ± 7.9 min¹, n = 5). When these oscillations occurred, cells developed hypercontracture (33.4 ± 1.3% of initial cell length, n = 5). In these normoxic experiments, the presence of DIDS and HOE-642 did not alter either the frequency of Ca²⁺ oscillations (64.0 ± 6.9 min¹) or the extent of hypercontracture (33.0 ± 4.4% of initial cell length) (Table 2). In the investigated time span pH_i did not significantly change under any of the investigated conditions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Main finding and model features. It was the central question of the present study whether inhibition of both the NHE and the NBS are required for protection by intracellular acidosis of cardiomyocytes against reoxygenation-induced hypercontracture. The results demonstrate that this is indeed the case.

The experimental model of isolated cardiomyocytes exposed to anoxia in medium with pH_o 6.4 and reoxygenation in medium with pH_o 7.4 was characterized in previous studies (16, 17, 30, 32, 33). In this model several basic aspects of the cellular pathophysiology of ischemic reperfused myocardium can be imitated and investigated in great detail. During anoxia the cardiomyocytes lose their energy reserves and, consequently, undergo rigor shortening. They develop marked cytosolic overload with Ca²⁺, Na⁺, and H⁺. The disturbance can be rapidly reversed when the cells are reoxygenated in media with normal extracellular pH (17, 33). The recovery to the [Ca²⁺]_i control level can be divided into three phases: 1) a rapid fall of the [Ca²⁺]_i due to sequestration into the sarcoplasmatic reticulum (SR) (phase 1); 2) a period of Ca²⁺ oscillations due to cycles of transient release and reuptake of Ca²⁺ by the SR (16) (phase 2); and 3) reestablishment of a normal resting level of [Ca²⁺]_i (phase 3) due to extrusion of excess Ca²⁺ across the sarcolemma (31). Transsarcolemmal extrusion of Ca²⁺ from the reoxygenated cells is mediated by a tandem action of the Na⁺ pump, creating a normal transsarcolemmal Na⁺ gradient, with the Na⁺/Ca²⁺ exchange mechanism, using the Na⁺ gradient for the extrusion of Ca²⁺ (31).

Recovery of pH_i to the normal level is achieved within phase 1 and 2. In this model, activation of the Na⁺-dependent H⁺ extruders at the onset of reoxygenation does not normally impair recovery of cytosolic Na⁺ and Ca²⁺ control unless the activity of the sarcolemmal Na⁺ pump is substantially reduced (17).

During reoxygenation cardiomyocytes with a manifest cytosolic Ca²⁺ overload are jeopardized by the development of hypercontracture. Hypercontracture represents an extreme shortening of the cell, which is caused by strong contractile activation but becomes irreversible by disruption of cytoskeletal structures. We previously reported (16) that cardiomyocytes become increasingly susceptible to hypercontracture after prolonged ischemic conditions and that this seems to be due to weakening of the cytoskeletal architecture. Reoxygenation-induced hypercontracture has been shown to represent an important cause for severe tissue injury in reperfused myocardium (2). During the recovery from oxygen depletion and acidosis, the full extent of reoxygenation-induced hypercontracture develops gradually in cardiomyocytes. At the beginning of phase 1 of reoxygenation the pH_i of 6.4 is too low to allow strong contractile activation, probably due to the acidotic desensitization of the myofibrils to Ca²⁺ (21). Shortly after the onset of reoxygenation, the pH_i transiently decreased even a little further (6.2), probably caused by phosphocreatine resynthesis (31). Hypercontracture developed progressively during the second phase of reoxygenation, when a spontaneous recovery of pH_i was permitted.

The time course of the pH_i recovery was significantly influenced by the inhibition of the H⁺ transport mechanisms. Application of DIDS to the reoxygenated cardiomyocyte reduced the initial rate of pH_i recovery to ~70%. When HOE-642 was applied instead, the rate of pH_i recovery was retarded to ~30% of the rapidity seen in absence of the inhibitor. The results suggest that the NBS accounts for the minor part of extrusion of H⁺ equivalents during recovery from intracellular acidosis. Simultaneous inhibition of the NHE and the NBS inhibited the realkalinization of the cytosol completely (7, 18, 35). However, our data cannot be used for a quantitative comparison of the two H⁺-extruding mechanisms, because the experimental design required application of the inhibitors not before the onset of reoxygenation, and the buffers differed for some experiments. The apparent kinetics of pH_i recovery, therefore, may contain a kinetic element of drug diffusion to and binding at its cellular site of action. Intracellular buffering was probably not very different because pH_i recovery proceeded at the same pace when HEPES-buffered media were used during reoxygenation as when DIDS and bicarbonate-buffered media were used.

The slowdown of pH_i recovery during phase 2 had a significant impact on the generation of the SR-dependent Ca²⁺ oscillations (17). Reoxygenation-induced spontaneous Ca²⁺ oscillations were less frequent in the presence of either DIDS or HOE-642 or in bicarbonate-free media. When both H⁺-extruding mechanisms were inhibited simultaneously, oscillations were effectively suppressed. Recently, it has been shown that the progressive development of hypercontracture is provoked by these repetitive transient Ca²⁺ elevations in the cytosol during phase 2 (32). If these Ca²⁺ oscillations are suppressed, e.g., by use of ryanodine, a specific inhibitor of the SR Ca²⁺ release channel, the progression of hypercontracture during phase 2 is interrupted. In contrast to the significant differences in oscillatory alterations of the [Ca²⁺]_i under the various experimental conditions, the rapidity of removal of bulk Ca²⁺ from the cytosol seems the same, as indicated by the invariance of the fura 2 ratio integrals of the first 10 min of reoxygenation.

Taken together, the simultaneous inhibition of both H⁺-extruding mechanisms produces two independent causes for protection against hypercontracture: 1) acidotic myofibrilar desensitization that is already effective during phase 1; and 2) acidotic suppression of SR-dependent oscillations of cytosolic Ca²⁺ during phase 2 (17). Inhibition of the H⁺ extruders needs to be maintained only for the first 10 min of reoxygenation (i.e., during phase 1 and 2). When the inhibitors are then removed, hypercontracture remains absent, even though a rapid pH_i recovery takes place. These data indicate that it is only required to prevent a rapid realkalinization of the cytosolic pH in an early vulnerable phase of reoxygenation for a protection against reoxygenation-induced hypercontracture.

It was investigated whether DIDS and HOE-642 would interfere with SR-dependent Ca²⁺ oscillations or the myofibrilar apparatus also in a different way, not related to changes of pH_i. In these experiments normal nonacidotic cardiomyocytes were exposed to Na⁺-free conditions in the extracellular medium, a procedure inducing Ca²⁺ overload, SR-dependent Ca²⁺ oscillations and hypercontracture as in phase 2 of reoxygenation, because in the absence of exogenous Na⁺ the cells take up Ca²⁺ by the reverse mode of the Na⁺/Ca²⁺ exchanger. In these normoxic cells incubated in medium with pH 7.4, presence of the inhibitors did not significantly alter pH_i in the period investigated nor the frequency of Ca²⁺ oscillations nor the extent of hypercontracture. These results demonstrate that DIDS and HOE-642 do not protect cardiomyocytes against Ca²⁺ overload-induced hypercontracture without their effects on pH_i regulation.

In conclusion, the results demonstrate that simultaneous inhibition of NHE and NBS during simulated reperfusion is needed to protect myocardial cells against reoxygenation-induced hypercontracture. Development of hypercontracture has been demonstrated in vitro (5, 11, 34) and in vivo (4) to contribute substantially to lethal reperfusion injury and thus to the postischemic extent of myocardial infarction. The results of the present study may therefore provide an explanation why the sole application of NHE inhibitors to myocardium during reperfusion has not consistently been found effective to reduce infarct size when the myocardium was perfused with bicarbonate-buffered solutions or blood (3, 4, 6, 13, 14).