pH regulation during ischaemia-reperfusion of isolated rat hearts, and metabolic effects of 2,3-butanedione monoxime

] Mol Cell Cardiol 27, 1703-1713 (1995)

pH Regulation During Ischaemia-Reperfusion of Isolated Rat Hearts, and Metabolic Effects of 2,3-Butanedione Monoxime Guy Bauza 1'2, Laurence Le Moyec 2 and Michel Eugbne 2 1Fabrique d'hnplants et d'Instruments chirurgicaux (ELI.), 17 route de Jonzieux, 4 3 2 4 0 Saint-JustMalmont, France and 2Laboratoire de RMN, Universit~ Paris 7, 27 rue Juliette Dodu, 7 5 0 1 0 Paris, France (Received 7 April 1994, accepted in revisedform 20 January 1995) G. BAUZA,L. LE MOYEC,ANDM. EUGENE.pH Regulation During Ischaemia-Reperfusion of Isolated Rat Hearts, and Metabolic Effects of 2,3-Butanedione Monoxime. Journal of Molecular and Cellular Cardiology (1995) 27, 1703-1713. We investigated changes in pH~ during ischaemia-reperfusion of isolated rat hearts using phosphorus nuclear magnetic resonance spectroscopy (np NMR). Hearts were separated into three groups according to the perfusion buffer: bicarbonate-buffered Krebs solution, HEPES-buffered Krebs solution, or bicarbonate-buffered Krebs solution plus 10 -6 M 5-(N-ethyl-N-isopropyl) amiloride (EIPA). In HEPES buffer and in bicarbonate buffer plus EIPA, pH at the end of 30 min of ischaemia and pH oscillations observed during early reperfusion were lower than in bicarbonate buffer. Thus, the presence of two pH regulation mechanisms (Na+-H + antiport and Na+-HCO; symport) was confirmed in the isolated rat heart, while in HEPES buffer, pH was regulated by Na+-H + antiport, and in bicarbonate buffer plus EIPA, by Na+-HCO7 symport. When cardiac contraction was inhibited by 10 mM 2,3-butanedione 2-monoxime (BDM), we observed, in all • cases, a less pronounced decrease in pH~ at the end of ischaemia, and in pH~ oscillations at the onset of reperfusion. These effects were similar to those observed with 150 x 10 -s M verapamil and might thus be related to a decrease in intraceilular calcium. However, with BDM, a greater reduction in the pH recovery rate was observed only in HEPES buffer, suggesting a possible phosphatase-like effect affecting the Na+-H + exchange. Whatever the buffer used, the protective effect of BDM was reflected by an increase in the rate pressure product, which was not observed with verapamil. © 1995 Academic Press Limited Kv.yWORDS:np Nuclear magnetic resonance spectroscopy; Langendorffheart perfusion; Na+-HCOi symport; Na+-H + exchange; Intracellnlar pH; 2,3-Butanedione monoxime; 5-(N-ethyl N-isopropyl) amiloride.

doudi et al., 1990). Historically, the main intracellular pH regulator system identified was the N a + - H + antiport, sensitive to acidosis (Cingolani et al., 1990; Khandoudi et al., 1990) and inhibited either by amiloride (1 raM) (Benos 1982) or the more potent analogue of amiloride which exhibits 100- to 200-fold greater potency t h a n amiloride:

Introduction Intracellular pH drops quickly during 3 0 m i n of cold ischaemia in isolated rat hearts. The pill recovery rate during early reperfusion, achieved by N a + - H + exchange and Na+-HCO7 symport, can affect metabolic and functional recovery (Khan-

Please address allcorrespondenceto: Guy Bauza, Laboratoirede RMN, 27 rue IulietteDODU, 75010 Paris,France.

0022-2828/95/081703 + 11 $12.00/0

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@ 1995 Academic Press Limited

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5-(N-ethyl-N-isopropyl) amiloride (EIPA, IO-"M) (Kleyman et al., 1988: Pierce et al., 1993). Stimulation of the exchanger was obtained by direct phosphorylation of either the exchange protein or an associated regulatory protein (Guizouarn et al., 1993). Recently, the efficiency of an Na+-HCO~symport, also sensitive to acidosis, was demonstrated in mmnmalian myocardial cells (LagadicGossmann et al., 1992). The Na ÷ independent C1--HCO7 exchange probably contributes to pH~ recovery from an intracellular alkaline load, but not from an acid load (Vanheel et al., 1984). Recent studies have demonstrated the potent cardiac effects of 2,3-bt~tanedione 2-monoxime (BDM), a low molecular weight molecule with a nucleophilic oxime function and a phosphataselike effect (Wiggins et al., 1980). The negative inotropic effect of BDM was originally attributed to inhibition of the Ca2+ slow inward current, with a decrease in the action potential plateau (Bergey et al., 1981; Blanchard et al., 1990) following dephosphorylation of membrane proteins. Other studies have shown that BDM inhibits Ca2+ release from the sarcoplasmic reticulum and cross-bridge kinetics, and decreases the myofibrillar ATPase sensitivity to Ca2+ (Kurihara et al., 1990). This negative inotropic effect is dose-dependent (Blanchard et al., 1990; Coulombe et al., 1990). There is complete rate tension product recovery of isolated papillary muscle after BDM washout (Wiggins et al., 1980~ Alpert et al., 1989). Due to its effective, quick-acting and reversible inhibition of cardiac contractility, BDM has been proposed as a cardioplegic agent for open heart surgery (Mulieri et al., 1989; Stringham e t a ! . , 1992). In the present study, intracellular pH (pHi) and energetic effects of BDM were studied using 31p NMR spectroscopy on isolated perfused rat hearts during global and total ischaemia and reperfusion. Ischaemia results in tissue acidosis, due to the hydrolysis of high energy phosphates and anaerobic glycolysis, and in lower Ca2+ sensitivity (Orchard and Kentish, 1990). The effects of BDM on post-ischaemic recovery were assessed by contractile function recovery, high energy phosphate metabolite variation and intracellular pH time course when hearts were perfused with different buffers, to selectively inhibit pH~ regulator systems. The negative inotropic effects of BDM were compared to those of a Ca2+ channel blocker (verapamfl) (Nayler, 1992). The BDM cardioprotective effect was demonstrated by improved intracellular pH and contractile function recovery.

Materials and Methods Isolated perfused heart preparation Male Wistar rats weighing 3 5 0 - 4 0 0 g were anesthetized with urethane (16.8 pmol/kg i.p.). Hearts were rapidly excised, rinsed in ice-cold heparinated perfusion medium and perfused through an aortic canula at a constant aortic pressure of l OOmmHg. The pulmonary artery was vented and the left ventricle was drained by incising the apex. A thin latex balloon tied to the end of a polyethylene tube was inserted into the left ventricle (LV) through the mitral valve and connected to a pressure transducer (Statham P 23Gb) to measure isovolumetric pressure. After calibration of the transducer, the balloon volume was set to achieve an end-diastolic pressure of 10 mmHg (range 8 to 14) and kept isovolumetric throughout the experiment. The left ventricular developed pressure and heart rate were recorded each time a spectrum was acquired. Contractile function was assessed by the rate pressure product (RPP) expressed in mmHg x bpm.

Solutions Hearts were separated into three groups according to the perfusion buffer. Hearts from group I were perfused with a phosphate-free bicarbonate-buffered Krebs solution containing (in mmol/l) NaC1118, NaHCO325, KCI 6, MgC12 1.2, CaC121.75, ethylene glycol-bis(fl-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA) 0.5, and D-glucose 11. Hearts from group II were perfused with phosphate free HEPES-buffered Krebs solution containing NaC1143, N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES) 10, KC16, MGC121.2, CaCI21.75, EGTA 0.5, and D-glucose 11. Hearts from group III were perfused with the bicarbonate-buffered Krebs solution, and 5-(N-ethyl-N-isopropyl) amiioride (EIPA) (10 -6 mol/1) was added at t27. Solutions were equilibrated with 95%O2/5%CO2 for bicarbonate buffers and with 100%O2 for HEPES buffer, pH was adjusted to pH7.4 at 37°C. In • each group, six control hearts were perfused without, and six hearts with, addition of BDM (lOmmol/1) before and during ischaemia. In groups I and II, six hearts were perfused with addition of verapamil (150 x 10 -8 mmol/1) before and during ischaemia.

pH Regulation of Rat Hearts

NMR measurements Perfused hearts immersed in the perfusate were placed in 20 mm NMR tubes and introduced into the vertical magnet (tO). During the experiment, the magnet bore temperature was maintained at 20°C. The coronary effluent was removed from the NMR tube by an aspiration line and recycled. The coronary flow was measured every 10 rain by time collection of buffer in a volumetric container. Global ischaemia was applied to the preparation by clamping the aortic inflow tubes. Phosphorus (3,p) NMR experiments were performed on a Bruker AM400WB spectrometer (9.4 T) at 162 MHz. Field homogeneity was achieved by shimming the water signal. The free induction decay signals (FID) were acquired using 60 ° pulses on 2K data points and a 20 000 Hz spectral width. The repetition delay was 2.5 s, and 128 or 32 scans were accumulated. Exponential multiplication (corresponding to 15 Hz line broadening) was applied to the FID before Fourier transformation on 4K data points. The chemical shifts were expressed in ppm and referenced to the phosphocreatine resonance assigned to 0 ppm. These parameters of data collection were chosen to optimize the signal-to-noise ratio in any given time. Under these conditions (interpulse delay of 2.5 s and 60 ° pulse angle), partial saturation of the signal occurred but no corrections for saturation were made. Since changes in phosphocreatine (PCr) and ATP were assessed relative to the initial value for each heart, and if we considered that the relaxation time T1 did not vary significantly during the experiments, saturation was not a problem in interpretation of the data. The signal intensities of PCr and ATP were estimated by measuring the peak intensity on plotted spectra and were compared to the value obtained for the first spectrum of the experiment.

pH measurements In the rest of this paper, we will focus on the intracellular pH in ischaemia and reperfusion, calculated from the frequency of the Pi peak. Intracellular pH was measured by using the chemical shift of the Pi (t~ Pi) according to the formula: pH, = 6.7 + log [ (& P i - 3.148)/(5.695 Pi)] (Kost, 1990). The maximal pH, recovery rate (d(pH)/dt max) was calculated during the initial rapid upstroke of pH, and expressed in pH unit/ rain. The pH, oscillations (pH oscill.) were quantified

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as the sum of the squared pH, derivatives during recovery (from t60 to t70).

Schedule In each experiment, 128 scan spectra (total acquisition time 6 min) were acquired at tlO (initial), t20, t30, t40, t50, t70 and t80 (min after tO), and 32 scan spectra (total acquisition time 90s) wre acquired at t28, t58, t60, t62, t64, and t66. For treated hearts, BDM or verapamil was administered at t20. For group [LI, EIPA was added to the perfusion buffer at t27 and BDM at t28. In all experiments, ischaemia was performed from t30 to t60 and reperfusion without BDM and verapamil from t60 to t90.

Statistical analysis Data for each parameter were expressed as a mean (m) _+standard error of the mean (S.E.M.). The data were analysed using a two-way variance analysis and the t-test for multiple comparison.

Results Typical 31p NMR spectra of a rat heart acquired during ischaemia and reperfusion are shown in Figure 1. The peaks from PCr, Pi and the three phosphates of ATP are clearly identified in all spectra. Spectrum 1A was collected during initial perfusion (tlO) of the heart with bicarbonate buffer (group I). After BDM addition (spectrum 1B), ATE Pi and PCR peak intensities were unchanged. A decrease in PCr and ATP and in increase in Pi (with a change in the chemical shift of Pi) were evident at the end of ischaemia (spectrum 1C). Spectrum 1D shows partial recovery of PCr and ATE a decrease in Pi, and recovery of phi (chemical shift in the Pi peak). For cardiac function, there was no difference in initial (tlO) RPP (range 2 8 2 0 8 . 3 3 m m H g x b p m to 28720.01 m m H g x bpm) or heart rate (range 210 bpm to 235 bpm). Post-recovery heart rates were slightly decreased, but without any significant differences between groups (range 185 bpm to 200 bpm).

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Controlgroups(withoutBDMand verapamil) pH recovery There were no differences in intracellular pH values in groups I, II, and II ( 7 . 0 5 + 0 . 0 2 , 7 . 1 4 + 0 . 0 5 , 7.08-t-0.02 respectively) measured in initial preischaemic conditions; addition of EIPA (10- 6 M) did not alter the pHi (7.03 +0.03). These values were in close agreement with literature data. Figure 2a shows the time course of pH~ decline during ischaemia, and recovery during reperfusion. In groups II and III, zero flow ischaemia induced a drop in pH~ to 6 . 0 6 + 0 . 0 5 and 6 . 0 4 + 0 . 0 2 , respectively. In group I, the fall in pHi was significantly slower (P
group, pH~ oscillations were still diminished, but without overshoot. There was no difference in pH~ between the three groups at the end of the recovery time.

High energyphosphates During ischaemia, PCr and ATP cardiac levels were dramatically reduced (Table 1), and ATP recovery during reperfusion was incomplete. Nevertheless, there were no differences in the time course of high energy phosphates between the three groups.

Cardiac function There was no difference in the rate-pressure-product (RPP) of the three groups before ischaemia. The time course of RPP evolution during ischaemia and reperfusion is illustrated in Figure 4a. There was no detectable developed pressure during ischaemia. During reperfusion, marked differences in RPP (expressed in m m H g x bpm) were observed after 5 0 m i n reperfusion, with a recovery of 10616.67+835.29, 13180.05_+1242.41 and

pH Regulation of Rat Hearts

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than in experiments without BDM whatever the buffer used, except at the end of reperfusion for group III (Fig. 2b,c,d). In groups I, I[ and III, pH oscillations during reperfusion decreased (Fig. 3). Only in group II did BDM significantly decrease the rate of change in pH (expressed in pH units/min) during reperfusion (0.129 + 0 . 0 0 2 without BDM, 0.061 + 0 . 0 0 3 with BDM: P<0.001).

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Figure 3 Dynamics of pH recovery at the onset or reperfusion, pH oscillations quantified as the sum of the squared pH derivatives during reperfusion (t60 to t70). ~P<0.08, * <0.05.

18444.76_+ 1545.88 in bicarbonate, HEPES and bicarbonate plus EIPA groups, respectively (but there was no difference in heart rate).

High energyphosphates The inhibition of concentration in the presence of BDM induced no significant changes in PCr and ATP cardiac levels (Table 1). The time course of high energy phosphate levels during ischaemia and reperfusion was unchanged in the three groups when compared to data without BDM.

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pH recovery pHi at the end of ischaemia and at the end of the reperfusion period was always significantly higher

Marked differences in RPP time courses were observed when heart contraction was inhibited using BDM before ischaernia. In all cases, RPP was higher (P<0.05) than the control value at the end of the reperfusion period (Fig. 4b,c,d).

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Figure 4 Rate pressure product (RPP) expressed in "mmHg x bpm" during ischaemia and reperfusion in different solutions. (a) shows the curves obtained in controls groups for bicarbonate buffer (squares), HEPES buffer (triangles) and bicarbonate buffer plus EIPA (circles). (b), (c) and (d) show the differences in RPP evolution with (closed symbols) and without (open symbols) BDM, for bicarbonate buffer (b), HEPES buffer (c) and bicarbonate buffer plus EIPA (d). Each point is the mean value of six experiments_+s.E.M, n.s.: not significant, * P<0.05, § P<0.001.

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from the control groups, but was significantly lower than for BDM-treated hearts (Fig. 5c,d).

pH recovery pH~ at the end of ischaemia was always higher in verapamil-treated hearts than in experiments .without or with BDM whatever the buffer used (Fig. ha,b). Verapamil significantly decreased pH oscillations during reperfusion to the same extent as BDM (Fig. 3), but has no effect on pH recovery rate (express in pH units/min) in group II (0.122 + 0 . 0 0 7 ) . pH at the end of reperfusion was significantly higher than in control groups.

High energy phosphates Before ischaemia, verapamil induced a rise in PCr cardiac level in the two groups. PCr and ATP at the end of reperfusion (for groups I) was significantly higher than for untreated hearts (Table 1).

Discussion In the present experiments, we demonstrated that pH recovery after 30 min of ischaemia in a HC0~-/ C02-buffered medium was achieved by both N a + - H + antiport and Na+-HCO~- symport. In all cases, the addition of BDM improved end-ischaemic pH values, post ishaemic .pH values (for groups I and II) and the inotropic state after 30rain of reperfusion.

Controls groups (without BDM and verapamil)

Na+-H + exchange Cardiac function At the concentration used ( 1 5 0 x IO-SM), verapamil induced a decrease in RPP similar to that of BDM (10 mivi). In the two groups, the RPP at the end of reperfusion was not significantly different

EIPA, which was reported to completely block Na+-H + exchange at a concentration of 10-6M (Pierce et al., 1993), induced no change in pHi, suggesting that in steady-state without EIPA, the exchanger was not functional in the intact heart while the intracellular pH of the cardiomyocytes

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Figure 5 pH evolution (a and b) and RPP (c and d) (expressed in mmHg x bpm) during ischaemia and reperfusion in groups [ (squares) and 1I (triangles). Open symbols: control hearts; closed symbols: verapamil treated hearts. Each point is the mean value of six experiments _+s.E.M.. N.S.: not significant, * P
was approximately 7.1 (Wallert and Frohlich, 1989). The Na+-H + exchange was observed using HEPES-buffered solution, thus eliminating the hypothesis of a contribution to pH regulation via a Na+-HCO~- dependent mechanism. In group II, after 3 0 m i n of ischaemia, pH~ reached a steady level at 6.06___0.05 pH units. At the onset of reperfusion, a large overshoot was observed, with a peak value occurring 15 min after reperfusion. Thereafter, pH stabilized at a lower value ( 6 . 8 6 _ 0 . 0 4 p H units) than before ischaemia (7.14+_0.05pH units). This overshoot was explained by Na+-H + exchanger sensitivity to the proton and sodium transmembrane concentrations and to their rate of change, which can alter surface pH (pHs). Hotokebuchi et al. (1991) reported that amiloride induced alkalinization of the surface solution in guinea-pig ventricular papillary muscle, providing indirect support for the idea that the lower pHs value, compared to the pH of the perfusion solution, was due to the extrusion of H + via the Na+-H + exchange. In our experiments, the wide pH oscillations could be responsible for pHs alterations which could be modified by the rate of proton extrusion and washout of the cell membrane surface. But we cannot exclude the possibility that H +lactate co-ettlux contributed to modifications in pH

recovery and pH oscillations (Vandenberg et al., 1993).

Na+-HCO; symport The contribution of a Na+-HCO3-dependent mechanism to pH~ regulation in mammalian myocardial tissue has recently been demonstrated by LagadicGossmann et al. (1992) in the guinea-pig ventricular myocyte. A bicarbonate-dependent alkalinizing mechanism has also been demonstrated in the rat ventricular myocyte (Terzic et al., 1992). Its effect can be observed in our preparation in bicarbonate-buffered solutions after addition of 10 -6 M EIPA to selectively block Na+-H + exchange. The time course of the pH~ decline during ischaemia was similar to that observed in HEPES buffer, with an identical pH~ at the end of ischaemia. The Na+-HCO; - symport is therefore an important controller of pHi in the heart during intracellular acidosis. During reperfusion, pH oscillations were slightly weaker than in HEPES buffer and in bicarbonate buffer, and there was no overshoot, but rather, slow stabilization to an identical equilibrium pH value. Its sensitivity is similar to the sensitivity of the Na+-H + exchanger, but these two pH control systems can be differentiated by their respective dynamic responses.

pH Regulation of Rat Hearts Comparison of pH recovery via Na+-H + antiport and Na+-HCO~- symport showed that the two mechanisms are almost equally important (60% and 40% respectively, in guinea-pig papillary muscle, LagadicGossmann et al., 1992). In group I, both mechanisms contribute to the regulation of pH~ during ischaemia and reperfusion. During ischaemia the drop in pH was slower and the end ischaemia pH~ value slightly higher than in HEPES buffer or bicarbonate buffer plus EIPA. The pH~ oscillations were strong at the onset of reperfusion ( 0 . 2 0 6 + 0 . 0 3 0 pH oscill.) and prior to stabilization of pH~ values at the same level as with the other two buffers. These oscillations were clearly observed on individual data curves, but were slightly smoothed on the mean curves, since oscillations were not in phase. For this reason we used the sum of the squared pH~ derivatives during recovery to quantify oscillations. Such oscillations might be explained by two independent systems contributing to pH regulation, or by the fact that these mechanisms are sensitive to the rate of change of transmembrane Na +, H + or HCO~- gradients. The high pH~ rate may exert deleterious effects on membrane and regulatory proteins by affecting surface pH, as discussed above. A "pH paradox" has been described by Currin et al. (1991) in peffused rat liver, where a fast return of pH from an acidic to a physiologically normal range causes the ceils to deteriorate rather than to recover. Better functional recovery during reperfusion was associated with slow pH~ oscillations in group KI compared with group I. This improvement in function suggests that the increase in intracellular Na ÷ load at .the time of reperfusion was a determinant in cellular damage. The degree of activation of the Na÷-H ÷ antiport and of the Na+-HCO~- symport will determine the increase in intracellular Na + and the amplitude of cytosolic Ca-' + increase secondarily mediated by activation of the Na+-Ca 2÷ exchanger. The latter can be correlated with depressed ventricular function, as described by Tani and Neely (1989). The N a ÷ - H ÷ exchange inhibitors could interfere with the development of reperfusion injury, and recent studies have demonstrated the protective effects of EIPA and other N a + - H + exchange inhibitors (Meng and Pierce, 1990). This effect was demonstrated in our experiments in bicarbonate buffer plus EIPA (group III). In this group, EIPA induced marked increase in the rate pressure product compared to the other two groups (I and II).

BDM-treated hearts When cardiac contraction was inhibited by BDM, we observed, in all experiments, better recovery of

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contractile function, a slighter decrease in pH~ at the end of ischaemia and an increase in pH~ at the end of the reperfusion period. In groups [ and II, the decrease in the oscillations of pH in BDM-treated hearts during early reperfusion can be explained by the less severe drop in pill at the end of ischaemia and the resulting slowdown in the activity of the Na+-H + exchanger. BDM is known to inhibit Ca -'+ release from sarcoplasmic reticulum and Ca 2+ channel current. This decrease in intracellular Ca 2+ could explain the lower drop in pH~ at the end of ischaemia, since an increase in intracellular Ca -'+ would lead to an increase in intracellular H + via steps involving Ca2+-H + (Kaila et al., 1987). Indeed the same effect is observed with verapamil, but to a greater extent, owing to its potent selective Ca 2+ channel blocker properties (Nayler, 1992). In the HEPES group, we observe a significant decrease in the pH recovery rate. This decrease could be related to the higher pH value at the end of ischaemia (Fig. 2c), but such a decrease is not observed with BDM in the other two groups. Furthermore, in the HEPES group, verapamil did not decrease the pH recovery rate, while pH at the end of ischaemia remained higher. BDM is known to have a "phosphatase-like effect" and might act to dephosphorylate the Na+-H + exchanger itself or an associated regulatory protein whose phosphorylation state could influence Na+-H + exchange activity (Guizouarn et al., 1993). The improvement in post-ischaemic recovery of cardiac function observed with BDM cannot be explained simply by a decrease in intracellular calcium during ischaemia. Indeed, the recovery of RPP is not increased by verapamil. An alternative explanation of the protective effect of BDM m a y lie in its metabolic effect. In our experiments, inhibition of cardiac contraction by BDM did not induce any significant change in high energy phosphate levels such as described by Hajjar et al. (1994:). This can be interpreted as a decrease in both ATP consumption and ATP production, reflected by the absence of a decrease (or even a slight increase with verapamil) in PCr. Nevertheless, the ischaemic period was too brief, and there were no significant differences in ATP and PCr ratios in the groups with v those without BDM. However, the accumulation of glycolytic products, particularly lactate, during ischaemia can significantly contribute to diminished recovery of ventricular function after reperfusion. Lactate accumulation could enhance the N a + - H ÷ exchange at the time of reflow (Karmazyn, 1993) and might therefore increase intracellular Na + and subsequently cellular Ca2+ concentrations

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via the Na+--Ca 2+ exchanger. Recently, Vandenberg et al. (1993) have demonstrated that, during ischaemia, accumulation of CO, and lactate may, w h e n washed out during reperfusion, contribute to pH~ recovery via H+-lactate coeffiux. By directly inhibiting the contractile apparatus without an induced energy supply deficit before ischaemia, BDM could decrease lactate accumulation at the end of ischaemia and thus reduce N a * - H + exchange activation during early reperfusion.

Conclusion The N a + - H + exchanger is sensitive to relative proton and sodium t r a n s m e m b r a n e concentrations. Na+-HCO~ - symport, like N a + - H + exchange, is an important controller of pH, in the heart during intracellular acidosis. The improvement in ventricular function after ischaemia is dependent on the dynamics of pH recovery at the onset of reperfusion. The protective effects of BDM can be explained by (i) a metabolic effect induced by inhibition of contractile activity via direct interaction with myosin ATPase, without a deficit in energy supply and (ii) a possible consequence of the phosphataselike effect of BDM upon the phosphorylation State of the exchanger and symport, or an associated regulatory protein, which would decrease pH oscillations and the recovery rate at the onset of reperfusion.

Acknowledgements This work was supported by grants from the "Minist~re de la Recherche et de l'Espace" MRE 92.C.0746 and Universit6 Paris 7.

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