The cellular injury observed during myocardial ischemia and reperfusion is a consequence of a complex mechanism in which alteration of the cation homeostasis and intracellular pH are believed to play a major role (1). The Na+/H+ exchanger (NHE) has been proposed as one of the major systems responsible for sodium entry and proton extrusion. Thereby, NHE is of primary importance in the regulation of electrolyte homeostasis and pH during myocardial ischemia and reperfusion (2,3). Besides the intracellular pH, several endogenous factors, including catecholamines (4), endothelin (5), and angiotensin (6), whose activity is modified during ischemia and reperfusion, have been shown to regulate the activity of the NHE.
During reperfusion of ischemic myocardium, the role of the NHE has been widely investigated by using protocols of acidic reperfusion (7,8) or by the addition of NHE blockers administered either before ischemia or after initiation of reperfusion (9-11). Because amiloride has been shown not to be a specific and selective inhibitor of the NHE, several derivatives, including 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and HOE-694, have been used to investigate the role of the Na+-H+ exchange. All the studies performed with NHE inhibitors showed a salutary effect on cardiac function (9,11), calcium and sodium overload (10), and ventricular arrhythmias (12) during reperfusion. However, little is known about the role of the NHE during ischemia.
The NHE is generally considered not to be operative during ischemia since the administration of NHE inhibitors has been reported to have no effect on cardiac function and intracellular pH measured during ischemia (13). However, for an NHE inhibitor to protect the heart during the reperfusion period, it is generally accepted that it must be administered before induction of ischemia (9). In a recent study, HOE-694 administered to anesthetized pigs before initiation of ischemia reduced infarct size and improved cardiac function (14).
Although in this study it was suggested that the inhibition of the NHE during ischemia was necessary for induction of cardioprotection, it was not possible to ascertain whether the beneficial effect was actually due to inhibition of NHE during the ischemic or reperfusion period.
Therefore, we investigated the direct effect of specific inhibitors of the NHE, i.e., EIPA and 3-methylsulphonyl-4-piperidinobenzoyl guanidine methanesulphonate (HOE-694), on ischemic dysfunction. For these experiments, we used isolated working rat heart, in which low-flow ischemia has been shown to mimic the cardiac dysfunction characteristic of stable angina (15). Using this model, Pijl and colleagues showed that calcium channel antagonists may improve recovery of cardiac function after ischemia (15,16), which suggests that a reduction in intracellular calcium, as a consequence of a reduced inward calcium current, may produce a beneficial effect. We hypothesized that with the use of potent inhibitors of the NHE, a reduction in intracellular calcium, secondary to a reduction in the intracellular sodium concentration, should lead to a beneficial effect on cardiac function.
Male Wistar rats weighing 280-320 g were maintained in accordance with National Institutes of Health (NIH) guidelines (publication no. 85-23) for animal care. Rats were anesthetized by intraperitoneal (i.p.) injections of sodium thiopental (50 mg/kg). Hearts were quickly removed and immersed in ice-cold buffer to produce immediate cessation of contractility. The aorta was dissected free and then mounted on a cannula attached to a perfusion apparatus. The heart was perfused retrogradely for 10 min by the Langendorff mode and then switched to perfusion according to the working-heart technique (17). The perfusion fluid was a Krebs-Henseleit buffer (pH 7.4) of the following composition (in mM): NaCl 118, NaHCO3 23, KCl 4.7, KH2PO4 1.2, MgCl2 1.2, CaCl2 1.8, glucose 11, and pyruvate 2. This buffer was continuously gassed with an O2/CO2 (95%/5%) mixture and maintained at 37°C throughout the experiment. The perfusate was not recirculated. Preload was maintained at 15 cm H2O, and afterload was maintained at 80 cm H2O.
Soon after the initiation of the perfusion through the right atria, the hearts began to pump and to perform pressure-volume work. Hearts were then perfused in the working mode for an initial 30-min control perfusion period. We induced low-flow ischemia by reducing the cardiac afterload from 60 to 11 mm Hg by adjusting the Starling resistance, thus causing a reduction in coronary flow (CF) by ≈90% (16). Global low-flow ischemia was maintained for either 30 or 60 min, during which time temperature was maintained at 37°C, followed by 30-min reperfusion in the working-heart mode. Global mechanical function [i.e., left ventricular pressure (LVP) and its first derivatives dP/dtmax and dP/dtmin] was continuously recorded by a high-fidelity catheter-tipped manometer (Millar Instruments, Houston, TX, U.S.A.) placed in the left ventricle. Aortic output (AO) and CF were determined volumetrically, and heart rate (HR) was measured from the ECG.
When used, EIPA and HOE-694 were added to the perfusate 15 min before induction of ischemia and were maintained throughout the ischemia and reperfusion. Additional experiments were performed in which EIPA and HOE-694 were added to the perfusate either 15 or 30 min after initiation of the low-flow ischemia. In these additional experiments, in which the low-flow ischemia was maintained for 60 min, each drug was also present during the reperfusion period.
For the determination of tissue metabolites after reperfusion, the heart was clamped with Wollenberger tongs precooled in liquid nitrogen, removed from the cannula, and stored in liquid nitrogen until the assays were performed. Assays for ATP, creatine phosphate (CP), and lactate were performed using neutralized perchloric acid extracts. Energy metabolite content was determined enzymatically with a Beckman spectrophotometer as previously described (18,19). The same enzymatic procedure was used to determine lactate in myocardial effluent samples collected before ischemia, after ischemia, and during the first minute of reperfusion.
EIPA (Research Biochemicals International, Natick, MA, U.S.A.) and HOE-694 (a gift from Hoechst, Frankfurt, Germany) were dissolved to the highest concentration possible in deionized water before their addition to the perfusate, to yield a final concentration of 1 μM. This concentration, identical for both compounds, was chosen on the basis of similar potency for EIPA and HOE-694 in inhibiting NHE (20,21). In addition, preliminary studies showed a very limited effect of 0.1 μM and dramatic reduction of heart function induced by 10 μM EIPA and HOE-694 (data not shown). All other reagents were of analytical grade.
The data are mean ± SEM. Statistical significance of differences was determined by analysis of variance (ANOVA) followed by Student-Newman-Keuls test. Differences of p < 0.05 were considered statistically significant.
Initial experiments were performed in the absence or in the presence of NHE inhibitor (n = 3 per group) in nonischemic hearts to evaluate the stability of the preparation for a 2-h period, a time that represented the maximum duration of perfusion of the ischemia/reperfusion experiments. During this period, the parameters used to assess the function of hearts perfused with or without addition of HOE-694 (1 μM) remained stable. Less than 5% change from predrug values in any parameter of contractile function was observed. On the other hand, EIPA (1 μM) induced a gradual decrease in the parameters of mechanical function (≈30% of reduction at the end of 2-h perfusion).
Table 1 shows the values of the functional parameters measured in isolated working rat hearts after 30 min of normal perfusion (i.e., before the induction of ischemia) with or without addition of the NHE inhibitors. During the preischemic period, EIPA and HOE-694 generally showed no direct effect on the parameters of cardiac function. The only change observed was a slight reduction in HR when EIPA was added (Table 1). This effect may explain the gradual reduction in cardiac function observed within the 2 h of normal perfusion.
Effect of low-flow ischemia in isolated working rat heart
When the afterload was reduced from 60 to 11 mm Hg, CF decreased from 17.5 ± 0.7 to 2.16 ± 0.4 ml/min in the control group; this reduction remained stable for 60 min of ischemia (1.3 ± 0.4 ml/min after 60 min of low-flow ischemia). The reduction in CF to ≈10% of the preischemic value induced a rapid decrease in LVP and HR. After 10 min of ischemia, LVP and HR were reduced from 125.7 ± 3.5 to 15.4 ± 1.4 mm Hg and from 300.5 ± 9.2 to 246.3 ± 20.7 beats/min, respectively. However, no diastolic contracture was observed since glucose was provided throughout the low-flow ischemia, as previously described (22). Because of the occurrence of episodes of ventricular tachycardia and fibrillation during ischemia in 62.5% of the hearts, the parameters of contractility (LVP and dP/dt) were no longer analyzed in those hearts. In the remainder, AO was considered to reflect ventricular contractility, which is a valid assumption with the perfusion system used (17) in which the afterload is maintained at a fixed value. As shown in Fig. 1, AO rapidly decreased after the initiation of ischemia and reached 10.1 ± 1.3 ml/min after 30 min of low-flow ischemia (≈18% of the preischemic value). In the second set of experiments in which low-flow ischemia was maintained for 60 min (n = 10), the cardiac function was decreased by no greater amount than that in hearts in which the ischemia was maintained for 30 min. AO was 9.1 ml/min after 60 min of ischemia (Fig. 2). The recovery of cardiac function during the reperfusion period, though incomplete, was better when ischemia was maintained for 30 min (Fig. 1) than when maintained for 60 min (Fig. 2), reflecting a reduced amount of irreversible damage of contractile function.
Effect of pretreatment with EIPA and HOE-694 on cardiac function during ischemia and reperfusion
The effects of EIPA (1 μM) and HOE-694 (1 μM) on the different parameters of the cardiac function were assessed both during ischemia, when the low-flow ischemia was maintained for 30 or 60 min, and during the ensuing reperfusion. EIPA and HOE-694 did not significantly change CF during the preischemic period (16.7 ± 0.7 and 16.8 ± 0.6 ml/min for EIPA and HOE-694, respectively; p > 0.05 vs. time-matched control value 17.5 ± 0.7 ml/min). Similarly, CF measured at the end of the 30-min ischemic period was not significantly different (p > 0.05) in the presence of EIPA and HOE-694 (1.8 ± 0.3 and 2.08 ± 0.2 ml/min, respectively) from that of the control group (2.16 ± 0.4 ml/min). CF remained ≈10% of the preischemic values after 60-min ischemia.
During the preischemic period, HR was slightly reduced by EIPA (261 ± 14 beats/min) but was unchanged by HOE-694 (301 ± 8 beats/min) as compared with that of the control group (300 ± 9 beats/min), which was similar to that observed in the initial experiments on basal function. After the first 10 min of ischemia, HR was reduced by 15-20% for all groups (246 ± 21, 228 ± 22, and 245 ± 13 beats/min for control, EIPA, and HOE-694, respectively). This slight bradycardia was associated with a reduction in the occurrence of arrhythmias in the groups treated with the NHE inhibitors, as previously demonstrated in experimental models of ischemia involving coronary artery occlusion (23,24) and was maintained throughout the ischemic period. Because of the occurrence of arrhythmias in some hearts, HR was not measured during the reperfusion period.
Neither EIPA nor HOE-694 changed LVP during the preischemic period (125.7 ± 3.5, 123.3 ± 10.8, and 123.5 ± 6.7 mm Hg for control, EIPA-, and HOE-694-treated groups, respectively). LVP was dramatically reduced during ischemia but to a similar degree after 10-min ischemia in all groups (15.4 ± 1.4, 13.1 ± 0.8, and 16.3 ± 1.5 mm Hg for control, EIPA-, and HOE-694-treated groups, respectively). The AO, which represents a better index of cardiac function than LVP in this model, was partially preserved with NHE inhibitors after 30-min low-flow ischemia (Fig. 1), although only the effect of EIPA was significantly different (p < 0.05) from the control. During reperfusion, both compounds induced a dramatic improvement in the cardiac function; HOE-694 was apparently slightly more effective than EIPA (Fig. 1).
To investigate whether the protection observed in the presence of NHE inhibitors can be maintained during longer ischemic periods, we measured the effects of EIPA and HOE-694 on the AO after 60-min low-flow ischemia. The effects of EIPA and HOE-694 before, during, and after a 60-min ischemic period on CF, HR, and LVP were similar to those observed for 30-min ischemia (data not shown). The results summarized in Fig. 2 show that the NHE inhibitors protected the AO during a long-lasting ischemia, although again the effect was statistically significant (p < 0.05) only for EIPA. The protection was maintained for the 60-min ischemic period. During the reperfusion, both NHE inhibitors induced a significant improvement in the AO during reperfusion (Fig. 2).
Effects of EIPA and HOE-694 on ischemic function when administered during ischemia
To evaluate whether the reduction in cardiac function induced by 60-min low-flow ischemia can be reversed by the administration of the NHE inhibitors after the initiation of ischemia, EIPA and HOE-694 were administered after either 15 or 30 min of reduced flow. EIPA administered either 15 min (Fig. 3) or 30 min (Fig. 4) after the beginning of the low-flow ischemia allowed the cardiac function to be maintained near predrug value. The AO that represented 15.7 ± 1.8% (n = 8) of the preischemic value at the end of the 60-min ischemia in the control group was increased to 38.9 ± 4.9% (n = 6) and 28.6 ± 6.2% (n = 6) of the preischemic values when EIPA was added to the perfusion solution 15 min (Fig. 3) and 30 min (Fig. 4) after the flow was reduced, respectively. HOE-694 had a slightly weaker protective effect that was statistically significant (p < 0.05) only when the compound was administered 15 min after initiation of the low-flow ischemia.
When EIPA was added to the perfusion solution 15 min after the initiation of ischemia, AO recovered to 51.5 ± 5.6% of the preischemic value at the end of reperfusion (p < 0.05 versus control group, 19.9 ± 7.1%). When administered after 30-min ischemia, EIPA did not significantly improve the recovery of AO during the reperfusion. HOE-694 administered either 15 min (Fig. 3) or 30 min (Fig. 4) after the initiation of ischemia did not significantly improve AO during reperfusion.
Effect of EIPA and HOE-694 on energy metabolite content
To measure a possible biochemical effect of EIPA or HOE-694, we assessed the energy metabolites at the end of the reperfusion period. Table 2 shows that the energy metabolite content measured after 30- or 60-min ischemia followed by 30-min reperfusion was not markedly altered by EIPA or HOE-694. Finally, the release of lactate into coronary effluents was increased during the low-flow ischemia by the NHE inhibitors (Fig. 5). However, although the effect of HOE-694 was significantly different from the pre- and postischemic values, it did not reach significance over the control value during ischemia.
The results of the present study confirm the previous observations that inhibitors of the NHE, such as EIPA and HOE-694, can improve cardiac function during reperfusion. The present results obtained after low-flow global ischemia reinforce the hypothesis that myocardial stunning can be related to an enhanced activity of the NHE after a reversible ischemic period (25). They also show that EIPA can enhance cardiac function during the ischemic period when administered either before or after the induction of ischemia. Indeed, administered during ischemia, EIPA attenuated the dysfunction related to the reduced flow, maintaining AO at the predrug level. However, this administration was less able to reverse the reduced AO on reperfusion than was administration before ischemia. These and other data indicated that the effects of EIPA become less pronounced the later relative to onset of ischemia it is administered. Therefore, NHE appears to function during ischemia but to decrease in activity as the ischemia continues.
A finding of the present study is that the protection of the ischemic function was more pronounced with EIPA than with HOE-694. Because EIPA and HOE-694 exhibit similar potency in blocking NHE (20,21), other mechanisms, e.g., an inhibition of the Na+/HCO3- co-transport, may be involved in the cardioprotective action of EIPA.
The NHE may play a key role in the pathophysiology of cardiac ischemia and reperfusion. At the time of reperfusion, a large outwardly directed gradient of protons forms across the sarcolemma, which activates the NHE and leads to an increase in intracellular sodium (2,26). Because cellular sodium and calcium transports are linked together by the Na+/Ca2+ exchange, an increase in intracellular sodium leads to a significant entry of calcium, resulting in myocardial dysfunction and arrhythmias (12). These effects can be reversed by NHE inhibitors, which have been shown to cause an improvement in cardiac function during reperfusion after regional ischemia and global zero-flow ischemia (11,12,27). The present results, obtained in isolated working rat hearts with HOE-694 and EIPA, reinforce the results of previous studies in demonstrating a protective effect of NHE inhibitors against reperfusion-induced dysfunction after low-flow ischemia (9-11,21,24,28). However, in our experimental conditions of low-flow ischemia, during which the residual CF of ≈2 ml/min allows the heart to produce a certain though reduced level of work, the cardioprotection provided by EIPA was not restricted to the reperfusion episode. The presence of EIPA in the perfusion solution induced an improvement in cardiac function during ischemia, as evidenced by the higher AO. In contrast, in the presence of HOE-694, only marginal protection was evident during ischemia.
During ischemia, the role played by the NHE is controversial. At the beginning of a myocardial ischemic period, the decrease in intracellular pH stimulates the NHE (2). At later stages of ischemia, when extracellular pH is also decreased, the NHE is proposed to become inactive (2,13,26). The results of the present study on myocardial function corroborate data obtained from measurements of the intracellular pH (11,29). Indeed, when NHE inhibitors such as EIPA were administered before or early after the initiation of the low-flow ischemia, the ischemic dysfunction was attenuated. However, when NHE inhibitors were administered after >30-min of ischemia, only minor effects were observed. Therefore, we suggest that the inhibition of the NHE needs to occur early enough during ischemia to provide a protection on myocardial function is to be afforded. Less cardiac structural alteration occurred when HOE-694 was administered before rather than after ischemia in anesthetized pigs (24). Similarly, NHE inhibitors administered before ischemia were capable of preventing the occurrence of ventricular arrhythmias after coronary artery occlusion (20). Taken together, all these results provide evidence for a cardioprotective effect of NHE inhibition during ischemia. However, the observation that HOE-694, which unlike most other NHE inhibitors is potent and highly selective (20), did not show as great a beneficial effect as EIPA suggests that other mechanisms may play a role in the effect of EIPA.
The regulation of the cellular pH during ischemia and reperfusion is not under the control of the NHE only, but instead represents a multifactorial mechanism. Recent studies in single cardiac myocytes (30) and in perfused hearts (31,32) demonstrated the existence of both NHE- and HCO3--dependent mechanisms of cellular proton extrusion. Because HOE-694 and EIPA exhibit similar potency in inhibiting NHE (20,21) and because the concentration used in the present study was shown to be sufficient to block the NHE completely (31,33), we can hypothesize that the protons generated during ischemia cannot be extruded by NHE alone. Therefore, we suggest that an additional effect could explain the beneficial action of EIPA. During ischemia and reperfusion, both activation of the Na+/HCO3- coinflux carrier and NHE could favour sodium overload. In the presence of NHE inhibitors, intracellular sodium overload is reduced, resulting in less cellular injury (10). However, some sodium influx may still occur as a consequence of the Na+/HCO3- symport, suggesting that the inhibition of the NHE, together with that of the Na+/HCO3- symport and probably the H+-lactate coefflux (29,32) as well, would be necessary to provide optimal protection of the heart from ischemic injury. Although HOE-694 is fairly specific for the isoform 1 of the NHE (34), EIPA was recently shown to inhibit the Na+/HCO3- cotransporter in vascular smooth muscle (35). Therefore, the greater beneficial effect of EIPA as compared with HOE-694 may be related to an effect on both NHE and Na+/HCO3- cotransporter.
Under conditions of zero-flow ischemia or during severe underperfusion, cardiac cells rapidly proceed to an anaerobic metabolism, causing an accumulation of harmful metabolic products such as protons, NADH, inorganic phosphate, and lactate. A role of lactate in development of ischemic damage has been acknowledged for several years (36,37). In the present study, EIPA caused a slightly greater release of lactate than HOE-694 during ischemia which may be another explanation of the better cardiac protection afforded by EIPA as compared with that afforded by HOE-694.
In conclusion, our results reinforce previous data indicating that the inhibition of the NHE may provide an effective mechanism to reduce reperfusion injury. Furthermore, our results obtained in an experimental condition of low-flow global ischemia that may correspond to a clinical form of stable angina suggest that although inhibition of NHE alone may provide some beneficial effect, additional mechanisms, such as an inhibition of the Na+/HCO3- cotransporter, may be necessary to provide better cardioprotection.
Acknowledgment: We thank Sonia Métayer-Saïdi for assistance in manuscript preparation.
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