Introduction

Top Abstract Introduction Materials & Methods Results Discussion Conclusions References

Activation of the NHE is an important regulator of intracellular pH during ischemia (Piper et al., 1996) and is known to extrude H⁺ in exchange for Na⁺ in cardiac myocytes immediately after the onset of ischemia (Lazdunski et al., 1985). The increase in intracellular Na⁺ has been shown to lead to cytosolic Ca⁺⁺ overload (Tani and Neely, 1989) due to effects on the Na⁺/Ca⁺⁺ exchanger. Calcium overload is thought to result in cardiac arrhythmias, myocardial stunning and irreversible cell injury (Scholz and Albus, 1993). Furthermore, there is evidence that NHE is reactivated at the onset of reperfusion when a rapid washout of extracellular H⁺ provides a large concentration gradient for the extrusion of intracellular H⁺ via NHE activation accompanied by a rapid influx of Na⁺. Increase in intracellular Na⁺ correlates with an accumulation of Ca⁺⁺ presumably via effects on the Na^+/Ca⁺⁺ exchanger (Tani and Neely, 1990).

Based on the pathophysiology that occurs as a result of NHE activation in the myocardium, there has been considerable interest in developing inhibitors of this antiport for therapeutic use in the treatment of ischemia-reperfusion injury. A number of studies have shown that inhibition of NHE produces a marked cardioprotective effect against cardiac arrhythmias and myocardial stunning and infarction (Duff, 1995; Scholz et al., 1995). Although there is firm evidence to suggest that administration of NHE inhibitors before an ischemic insult results in a marked reduction in infarct size in rabbits and pigs (Klein et al., 1995; Rohmann et al., 1995; Bugge et al., 1996; Miura et al., 1997), controversy still exists as to the efficacy of these agents to reduce infarct size when administered only before the onset of reperfusion. Rohmann et al. (1995) found that the selective NHE-1 isoform inhibitor, HOE 694 (Scholz and Albus, 1993) significantly reduced infarct size in pigs when administered 15 min before occlusion and 15 min before the onset of reperfusion although the cardioprotective effect was greater in the pretreated group. In contrast, Klein et al. (1995) and Miura et al. (1997) found that treatment with HOE 694 or HOE 642 just before reperfusion produced small but insignificant reductions in infarct size in pigs or rabbits, respectively. The reasons for these conflicting results are not clear but may be related to differences in models, the dose of drug used, or the precise time of administration before reperfusion. Based on this preceding work, we report the characterization of EMD 85131 as a new selective inhibitor of NHE-1 that reduces myocardial infarct size when administered either before ischemia or before reperfusion in a canine model.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion Conclusions References

Materials. To perform our studies, we used the new selective NHE-1 isoform inhibitor, EMD 85131 (2-methyl-5-methylsulfonyl-1-(1-pyrrollyl)-benzoylguanidine) (fig. 1). EMD 85131 is the hydrochloride salt of the developmental compound EMD 96785, a methane sulfonate salt (Baumgarth et al., 1997)_.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Chemical structure of the Na+/H+ exchange-isoform 1 (NHE-1) inhibitor, EMD 85131.

Characterization of EMD 85131 as a NHE-1 isoform selective inhibitor using stable transfected mouse fibroblast cell lines. The characterization of NHE inhibitors has been previously published (Baumgarth et al., 1997). Three mouse fibroblast cell lines expressing the different NHE isoforms NHE-1, NHE-2 and NHE-3 as well as the original LAP-1 cell line (NHE deficient) were obtained from Professor J. Pouyssegur (Nice, France). The expression of the three different isoforms as well as the cell culture were carried out as previously described for the Chinese hamster fibroblast cells (CCL-39 cell line) (Counillon et al., 1993)_. The cDNAs for the NHE-1, NHE-2, and NHE-3 isoforms as well as their sources have been previously described (Orlowski et al., 1992; Tse et al., 1993).

²²Na⁺ uptake in the transfected mouse fibroblast cells. The cells expressing the three different NHE-isoforms were seeded in 24-well plates and grown to confluence. The culture medium was removed and the cells were incubated for 60 min at 37°C in 50 mM NH₄Cl, 15 mM 4-morpholinopropanesulfonic acid, 70 mM choline chloride, pH 7.0. Thereafter, the cells were washed twice rapidly with the wash buffer (120 mM choline chloride, 15 mM 4-(2-hydroxyethyl-)1-piperazineethanesulfonic acid/Tris, pH 7.4) and then incubated in the uptake buffer containing 9.3 mEq carrier-free ²²Na⁺/ml, 120 mM choline chloride, 15 mM 4-(2-hydroxyethyl-)1-piperazineethanesulfonic acid/Tris, 0.1 mM ouabain, 1 mM MgCl₂, 2 mM CaCl₂, pH 7.4 in the absence or presence of increasing concentrations of EMD 85131. The incubation was carried out for 6 min. At the end of the incubation time, the supernatants of the cell monolayers were aspirated from four wells at a time and washed with ice cold phosphate buffered saline. The cells were solubilized in a total of 0.9 ml (3 × 0.3 ml) of 0.1 N NaOH; the NaOH washes of one cavity were collected into a scintillation vial to which 3 ml of scintillation cocktail was added. The radioactivity was determined by liquid scintillation counting in a -counter. The Na⁺/H⁺-dependent ²²Na⁺-uptake was defined as the difference between the uptake of ²²Na⁺ in the absence and presence of 1 µM ethylispropylamiloride (EIPA). It was shown for the ²²Na⁺-uptake in NHE-1- and NHE-2-expressing cells that in the presence of 1 µM EIPA the uptake was the same as that seen in the presence of the highest concentrations of EMD 85131; in case of the NHE-3-expressing cell line, high enough concentrations of EMD 85131 could not be obtained due to the low solubility of the compound at the high (millimolar) concentrations needed.

General surgical preparation in dogs. Adult mongrel dogs of either sex, weighing 19.5 to 29.3 kg, were fasted overnight, anesthetized with a combination of sodium barbital (200 mg/kg) and sodium pentobarbital (15 mg/kg) and ventilated by a respirator with room air supplemented with 100% oxygen. Atelectasis was prevented by maintaining an end-expiratory pressure of 5 to 7 cm H₂O with a trap. Arterial blood pH, P_CO2 and P_O2 were monitored at selected intervals by an automatic blood gas system (AVL 995, AVL Scientific Corp., Roswell, GA) and maintained within normal physiological limits (pH 7.35 to 7.45; P_CO2, 30 to 35 mm Hg and P_O2, 85 to 100 mm Hg) by adjustment of the respiration rate and oxygen flow or by i.v. administration of 1.5% sodium bicarbonate if necessary. Body temperature was maintained at 38 ± 1°C with a heating pad. Aortic blood pressure and LV pressure were monitored by insertion of a double-pressure transducer-tipped catheter (PC 771, Millar Instruments, Houston, TX) inserted into the aorta and LV through the left carotid artery. LV dP/dt was recorded by electronic differentiation of the LV pressure pulse, and heart rate was determined by a tachometer. The right femoral vein and artery were cannulated for drug administration and for blood gas analysis and measurement of the reference blood flow used to determine myocardial tissue blood flow, respectively. A left thoracotomy was performed at the fifth intercostal space, the lung was carefully retracted, the pericardium was incised, and the heart was suspended in a cradle. A proximal portion of the LAD distal to the first diagonal branch was isolated from surrounding tissue, and a calibrated electromagnetic flow probe (Statham SP 7515, Gould-Statham) was placed around the vessel. A flow meter (Statham 2202) was used to measure LAD blood flow. A mechanical occluder was placed distal to the flow probe so that there were no branches between the flow probe and the occluder. The occluder was used to set the flow probe to zero (20 min before coronary occlusion, the LAD was occluded for 10 sec), to occlude the LAD and to reperfuse the myocardium. If the basal heart rate was <150 bpm, the heart was paced at that rate with rectangular pulses of 4 msec duration and with a voltage twice the threshold through bipolar electrodes clipped to the left atrial appendage. Pacing was not used in the few animals with initial rates > 150 bpm. Hemodynamics, heart rate and LAD blood flow were monitored and recorded by a polygraph (model 7, Grass Instrument) throughout the experiment. The left atrium was cannulated through the appendage for radioactive microsphere injection.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Experimental protocols used to examine the effects of EMD 85131 on myocardial infarct size. Dogs were assigned to one of eight groups. All animals were subjected to 60 min of LAD occlusion and 3 hr of reperfusion.

Regional myocardial blood flow. Regional myocardial blood flow was measured by the radioactive microsphere technique as described previously in this laboratory (Gross et al., 1982). Microspheres were administered 30 min into the prolonged 60-min occlusion period and at the end of reperfusion. Carbonized plastic microspheres (15 µm diameter, New England Nuclear, Boston, MA) labeled with ¹⁴¹Ce or ⁹⁵Nb were suspended in isotonic saline with 0.01% Tween 80 added to prevent aggregation. The microspheres were ultrasonicated for 5 min and vortexed for another 5 min before injection. One milliliter of the microsphere suspension (2 to 4 × 10⁶ spheres) was given through the left atrial catheter and flushed by 5 ml of saline. A reference blood flow sample was drawn from the right femoral artery at a constant rate of 9.4 ml/min starting 30 sec before microsphere injection and continuing for 3 min. The next day, the tissue slices were sectioned into subepicardium, midmyocardium and subendocardium of nonischemic (three pieces) and ischemic (five pieces) regions. Transmural pieces were obtained from the center of several transverse sections used to determine the AAR and were at least 1 cm from the perfusion boundaries as indicated by Patent blue dye. All samples were counted in a gamma-counter (Tracor Analytic 1195) to determine the activity of each isotope in each sample. The activity of each isotope was also determined in the reference blood flow samples. Myocardial blood flow was calculated by use of a preprogrammed computer to obtain the true activity of each isotope in individual samples and tissue blood flow was calculated from the equation Q_m = Q_rxC_m/C_r, where Q_m is myocardial blood flow (in milliliters per minute per gram of tissue), Q_r is the rate of withdrawal of the reference blood flow (9.4 ml/min), C_r is the activity of the blood flow sample (cpm) and C_m is the activity of the tissue sample (cpm per gram). Transmural blood flow was calculated as the weighted average of the three layers in each region.

Exclusion criteria. Dogs were excluded if: 1) heartworms were found after the dogs were killed, 2) transmural collateral blood flow was >0.20 ml/min/g, 3) heart rate was >180 bpm at the beginning of the experiment or 4) more than three consecutive attempts were needed to convert ventricular fibrillation with low-energy DC pulses applied directly to the heart.

Statistical analysis. All values are expressed as mean ± S.E.M. Differences between groups in hemodynamics and blood gases were compared by use of a two-way (for time and treatment) analysis of variance with repeated measures and Fisher's least significant difference test if significant F ratios were obtained. Differences between groups in tissue blood flows, AAR and infarct size were compared by one-way analysis of variance and comparisons between groups were made with Fisher's least significant difference test. Analysis of covariance was used to determine whether the relation between transmural collateral blood flow and infarct size differed between the control and drug-treated groups. Differences between groups were considered significant if the probability value was *P < .05.

	Results

Top Abstract Introduction Materials & Methods Results Discussion Conclusions References

Inhibition of the ²²Na⁺-uptake in NHE-1, -2 or -3 expressing mouse fibroblast cells. The concentration-related effects of EMD 85131 to inhibit the uptake of ²²Na⁺ into NHE-1, -2 or -3 expressing mouse fibroblast cells (n = 3, each group) are shown in figure 3. The IC₅₀ for inhibition of Na⁺-uptake are 10.4 ± 1.0 nM, 306.8 ± 27.0 nM and 454.0 ± 47.0 µM, respectively. Based on these data, EMD 85131 is approximately 30-fold more selective toward NHE-1 compared to NHE-2 and 45,000-fold more selective towards NHE-1 compared to the NHE-3 isoform. Furthermore, no effects were observed on the L-type Ca⁺⁺ channel, the Na⁺ channel, the Na⁺/K⁺ ATPase or the Na⁺/K⁺/Cl cotransporter, indicating the specificity of EMD 85131 for the NHE-1 (Beier N, personnel communication)

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Inhibition of 22Na+-uptake in NHE-1, -2, -3 expressing mouse fibroblast cells by the Na+/H+-exchange inhibitor EMD 85131. Three cell lines expressing the different NHE isoforms. 22Na+-uptake was measured as described in "Methods" using either NHE-1, NHE-2 or NHE-3 stably transfected mouse fibroblast cells (NHE) as well as the original LAP-1 cell line (NHE deficient) which were obtained from Professor J. Pouyssegur (Nice, France). The Na+/H+-dependent 22Na+-uptake was defined as the difference between the uptake of 22Na+ in the absence and presence of 1 µM EIPA. The percent 22Na+-uptake data were plotted in a semilogarithmic plot against the concentration of EMD 85131. A sigmoid curve was fitted to the data according to the equation 100/(1 + IC50/x) by a nonlinear regression analysis, assuming a steepness of 1 for the curve; n = 3 for each group.

Exclusionsdog studies. Fifty-two dogs were initially used in this study. Four were excluded because transmural collateral blood flow was >0.20 ml/min/g [1 each in the control and EMD-pretreated groups and 1 in the EMD posttreatment (3.0 mg/kg) group]. One dog in the control and one in the 0.75 mg/kg posttreatment group was excluded due to intractable ventricular fibrillation. Thus 46 dogs successfully completed the protocol and were used in data analysis.

Hemodynamic data. Table 1 summarizes the hemodynamic data. There were no significant differences between groups throughout the experiment with the exception of the RPP in the high dose (3.0 mg/kg) EMD posttreatment group where the baseline value and that at 30 min of occlusion were significantly lower than the corresponding values in the control group. Infusion of the high dose (3.0 mg/kg) of EMD 85131 resulted in a transient increase in mean blood pressure (approximately 14 mmHg), LV dP/dt and the RPP (data not shown). The effect on hemodynamics was transient and all values returned to baseline within 10 min after drug infusion. There were also no significant differences in pH and blood gas values for P_O2 and P_CO2 between groups at the times studied (data not shown).

IS data. Figures 4 and 5 and table 2 summarize the effect of pre- and posttreatment with different doses of EMD 85131 on the AAR and IS expressed as a percent of the AAR (IS/AAR) and left ventricle (IS/LV). Both pre- and posttreatment with the two higher doses of EMD 85131 (0.75 and 3.0 mg/kg) resulted in significant (*P < .05) and comparable reductions in IS, IS/LV (table 2) and IS/AAR (figs. 4 and 5). Although the effect of EMD 85131 to reduce infarct size had a tendency to be greater in the two pretreatment groups, these values were not significantly different from those obtained in the posttreatment groups at comparable doses. There were no significant differences in LV weight, AAR, or AAR/LV between groups (table 2). The threshold dose for the cardioprotective activity of EMD 85131 appeared to be between 0.52 and 0.75 mg/kg when administered just before reperfusion (fig. 5). There were no differences in transmural collateral blood flow at 30 min into the 60-min occlusion period between groups (table 2). These data indicate that all groups were subjected to equivalent degrees of ischemia. However, when transmural collateral blood flow in each experiment was plotted vs. IS/AAR, the four regression lines describing this relationship in EMD 85131-treated animals were shifted down as compared to the control group by analysis of covariance (fig. 6). These data indicate that for any level of collateral blood flow IS/AAR would be predicted to be smaller in EMD 85131-treated animals.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of EMD 85131 on IS/AAR when administered before left anterior descending artery occlusion. Effects of two doses of EMD 85131 on myocardial infarct size (IS/AAR) in dogs when administered 15 min before occlusion of the LAD for 60 min. All values are the mean ± S.E.M. (n = 7-8). *P < .05 vs. the control group (CON).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of EMD 85131 on IS/AAR when administered before reperfusion of the left anterior descending artery bed. Effects of five doses of EMD 85131 on myocardial infarct size (IS/AAR) in dogs when administered 15 min before reperfusion of the LAD after a period of 60 min of occlusion. All values are the mean ± S.E.M. (n = 7-8). *P < .05 vs. the control group (CON).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Regression of TCCF vs. IS/AAR for EMD 85131 and control animals. Plot of transmural collateral blood flow (TCCF) to the ischemic area at 30 min of occlusion versus infarct size as expressed as a percent of the area-at-risk (IS/AAR). All four EMD 85131-treated groups had a regression line that was significantly shifted (P < .05) downward by analysis of covariance (ANCOVA). A = control vs. pretreatment with 0.75 mg/kg EMD 85131, B = control vs. posttreatment with 0.75 mg/kg EMD 85131, C = Control vs. pretreatment with 3.0 mg/kg EMD 85131, D = control vs. posttreatment with 3.0 mg/kg EMD 85131

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion Conclusions References

Summary. We have characterized EMD 85131, (2-methyl-5-methylsulfonyl-1-(1-pyrrollyl)-benzoylguanidine), as a selective inhibitor of the Na⁺/H⁺ exchanger, isoform 1 (NHE-1) and demonstrated that in vivo administration either 15 min before 60 min of ischemia or 15 min before 180 min of reperfusion resulted in marked cardioprotection as measured by infarct size in the dog.

Selectivity of EMD 85131 for NHE-1. To date, five isoforms of the Na⁺/H⁺ exchanger have been characterized (Orlowski and Grinstein, 1997). In myocardium, mRNA for NHE 1-3 has been demonstrated, however, it appears that NHE-1, which is ubiquitously expressed, is the predominant isoform (Fliegel and Dyck, 1995). To determine the specificity of EMD 85131 for the NHE isoforms, the compound was examined for its ability to block ²²Na⁺ flux in fibroblasts expressing a specific NHE isoform. EMD 85131 was shown to be a selective inhibitor of NHE-1, with an IC₅₀ of 10.4 nM, approximately five times more potent than that published for HOE 642 (50 nM), a structural analog previously shown to have cardioprotective activity (Scholz et al., 1995). Furthermore, the difference in inhibition of NHE-1 vs. NHE-2 was approximately 30-fold and NHE-1 vs. NHE-3 was approximately 4500-fold. Thus, EMD 85131 appears to be a potent and selective inhibitor of NHE-1.

The Na⁺/H⁺ exchanger in myocardial ischemia-reperfusion. The role of the Na⁺/H⁺ exchanger in normal and ischemic myocardium has been extensively studied (Piper et al., 1996)). In the normal myocardium, intracellular Na⁺ and Ca⁺⁺ are actively regulated by Na⁺/K⁺-ATPase and Ca⁺⁺-ATPase, respectively. Furthermore, the NHE and the Na⁺/Ca⁺⁺-exchanger also link intracellular sodium to hydrogen and calcium concentrations, respectively. With myocardial ischemia, mitochondrial ATP production ceases and glycolysis ensues resulting in a net breakdown of ATP and an accumulation of lactate and intracellular H⁺ (Dennis et al., 1991). The increase in intracellular H⁺ activates the pH regulatory systems which include the lactate transport (Vandenberg et al., 1993)_, the Na⁺-HCO₃-cotransport (Tonnessen et al., 1990) and the Na⁺/H⁺-exchanger (Frelin et al., 1984; Lazdunski et al., 1985). Adrenergic stimulation, which occurs during ischemia, also activates the Na⁺/H⁺ exchanger (Lagadic-Gossmann et al., 1992). The net effect of the activation of NHE is the extrusion of H⁺ and the influx of Na⁺ in an attempt to restore intracellular pH. The influx of Na⁺ is thought to accelerate ATP depletion in the early phase of ischemia via stimulation of the Na⁺/K⁺-ATPase (Frelin et al., 1984; Rasmussen et al., 1989). The net result is an increase in intracellular Na⁺ and the establishment of a Na⁺ gradient. An increase in intracellular Na⁺ has been shown to correlate with an increase in intracellular Ca⁺⁺ (Tani and Neely, 1989). Calcium levels in the myocyte are regulated by the Na⁺/Ca⁺⁺ exchanger which under physiological conditions extrudes calcium, however, the Na⁺/Ca⁺⁺ exchanger can transport Ca⁺⁺ in either direction (Kohmoto et al., 1994), and therefore may promote both Ca⁺⁺ extrusion as well as entry. Increased intracellular sodium has been reported to both diminish the normal transport rate of calcium extrusion (Frelin et al., 1985; Tani and Neely, 1989) and to possibly "reverse" the transport direction (Frelin et al., 1984; Tani and Neely, 1990; Doering and Lederer, 1993). Thus, regardless of the exact mechanism, the net effect of increasing intracellular sodium is an accumulation of Ca⁺⁺ in the ischemic myocardium that contributes to cellular damage resulting in arrhythmias and contraction band necrosis. Interestingly, the Na⁺/H⁺ exchanger has been reported to be inhibited by extracellular acidosis (Vaughan-Jones and Wu, 1990) which may exceed intracellular acidosis within 10 to 20 min of ischemia (Yan and Kleber, 1992)_. Furthermore, the Na⁺/Ca⁺⁺ exchanger is also inhibited by intracellular acidosis (Doering and Lederer, 1993). Thus, the establishment of a Na⁺ gradient and the compensatory Ca⁺⁺ overload that ensues occurs relatively early in ischemia. With reperfusion, extracellular H⁺ rapidly decreases again establishing a large intracellular to extracellular H⁺ gradient. This gradient reactivates the Na⁺/H⁺ exchanger resulting in an increase in intracellular Na⁺ which via effects on the Na⁺/Ca⁺⁺ exchanger contributes to Ca⁺⁺ overload. Cardiomyocytes accumulate an abnormally large amount of Ca⁺⁺ during reperfusion (Tani and Neely, 1989)., contributing to reperfusion arrhythmias, myocardial contracture and necrosis (Steenbergen et al., 1990). Thus, during both ischemia and reperfusion, the Na⁺/H⁺ exchanger plays a critical role in contributing to cellular damage in an attempt to maintain intracellular pH.

The cardioprotective efficacy of EMD 85131. The cardioprotective efficacy of blockade of the Na⁺/H⁺ exchanger during myocardial ischemia-reperfusion injury has been clearly demonstrated using amiloride derivatives and the more specific NHE-1 inhibitors HOE 694 and HOE 642. Thus, with the characterization of EMD 85131 as a potent and specific inhibitor of NHE-1, we sought to determine if this compound demonstrated similar cardioprotective effects in a canine model of myocardial ischemia-reperfusion injury. Specifically, we wished to examine if administration of EMD 85131 either before ischemia or before reperfusion could confer myocardial protection in vivo. As demonstrated, administration of EMD 85131 before ischemia resulted in a significant reduction in myocardial infarct size. The cardioprotection observed with administration of EMD 85131 before ischemia confirms numerous reports using other less selective inhibitors of NHE-1 such as amiloride and its derivatives. These reductions in infarct size occurred independent of differences in hemodynamics, area at risk and coronary collateral blood flow, the three major determinants of myocardial infarct size. Furthermore, the magnitude of the reduction in infarct size produced by EMD 85131 was equivalent to that previously observed after ischemic preconditioning in the canine heart (Gross and Auchampach, 1992). Thus, our study implies that the action of EMD 85131 in the first several minutes of ischemia is sufficient to protect the myocardium. However, while mechanistically interesting, pretreatment with NHE-1 inhibitors before myocardial infarction is a less likely clinical scenario in the acute setting. Thus, examination of the effect of EMD 85131 when administered before reperfusion was also conducted. Although the cardioprotection afforded by EMD 85131 was somewhat greater when it was administered before ischemia, particularly with the high dose, there were no statistically significant differences between pre- and posttreatment at either dose. The timing of administration of Na⁺/H⁺ exchange inhibitors (i.e., preischemia, prereperfusion, postreperfusion) has been extensively studied using isolated heart preparations. Several groups have reported that ex vivo, Na⁺/H⁺ exchange inhibitors must be present before and during ischemia to exert a cardioprotective effect (Karmazyn, 1993; Bugge et al., 1996; Myers et al., 1995), although other groups have demonstrated that addition of Na⁺/H⁺ exchange inhibitors just before or at the initiation of reperfusion conferred significant myocardial protection (Tani and Neely, 1989; Meng and Pierce, 1990; Maddaford and Pierce, 1997). It is unclear if the discrepancies observed with regard to timing of Na+/H+ exchange inhibitor administration are due to differences in the drugs used, the various models or the parameters examined. Thus, in the isolated heart system, the efficacy of Na⁺/H⁺ exchange inhibitors administered just before or at reperfusion remains controversial. Similarly, several groups have examined the effect of administration of Na⁺/H⁺ exchange inhibitors in vivo before reperfusion with conflicting results (Klein et al., 1995; Rohmann et al., 1995; Bugge et al., 1996; Miura et al., 1997). Most notably, Klein et al. (1995) failed to demonstrate a cardioprotective effect with HOE 694 when administered at 3 mg/kg i.v., 10 min before reperfusion in a porcine model of myocardial ischemia-reperfusion injury. However, using a porcine model, Rohmann et al. (1995) demonstrated marked cardioprotection with HOE 694 when administered at 7 mg/kg i.v., 15 min before reperfusion. The authors commented in their discussion that preliminary studies demonstrated that an increased dose of HOE 694 was required to observe cardioprotection when administered before reperfusion. The marked cardioprotection observed with administration of EMD 85131 before reperfusion is quite intriguing. That EMD 85131 reduced infarct size when administered 15 min before reperfusion also suggests that this compound reduces reperfusion injury which is in agreement with results obtained by Rohmann et al. (1995) with HOE 694 in anesthetized pigs. Because pigs and rabbits both have a sparse native collateral circulation (Sjoquist et al., 1984; Miura et al., 1989), the smaller effect of the NHE inhibitors when administered before reperfusion in these two species may have been the result of a lack of sufficient drug being delivered to the ischemic tissue before reperfusion (Maddaford and Pierce 1997). Therefore, in the present study, performed in anesthetized dogs which are known to have collateral blood flows that are normally 10 to 15% of the flow that is present before ischemia (Gross et al., 1982), it is more likely that a significant quantity of drug reached the ischemic area when it was administered 15 min before reperfusion. Alternatively, it is possible that EMD 85131 exerts its protective effect by reducing a late component of ischemic injury. Nevertheless, the marked cardioprotection observed is most likely the result of differences in the models, with more drug reaching the affected myocytes within the area-at-risk before reperfusion in our canine model. We also tested the importance of dose in determining the efficacy of NHE inhibition to reduce infarct size since Rohmann et al. (1995) previously demonstrated that this was an important factor in determining the efficacy of HOE 694 to reduce infarct size in pigs when administered just before reperfusion. As shown in figure 5, a dose between 0.5 to 0.75 mg/kg was required to observe myocardial protection when EMD 85131 was administered 15 min before reperfusion. Thus, a difference in dose may account for the cardioprotection observed in our study and that of Rohmann et al. (1995) compared to that of Klein et al. (1995) regardless of the model.

	Conclusions and future studies

Top Abstract Introduction Materials & Methods Results Discussion Conclusions References

We have demonstrated that administration of the Na⁺/H⁺ exchanger inhibitor EMD 85131 either before ischemia or before reperfusion provides significant cardioprotection. Furthermore, the magnitude of the reduction in infarct size produced by the highest dose of EMD 85131 was equivalent to that previously observed after ischemic preconditioning in the canine heart (Gross and Auchampach, 1992) which suggests that NHE-1 inhibition may be a viable alternative to ischemic preconditioning as a cardioprotective mechanism in the clinical arena. Although the exact mechanisms by which EMD 85131 provides cardioprotection were not elucidated in our study, numerous studies have examined Na⁺/H⁺ exchange during myocardial ischemia and reperfusion. Although the presumption based on this work is that inhibition of NHE-1 ultimately prevents Ca⁺⁺ overload, we have no direct proof at this juncture that this is the mechanism that confers cardioprotection. Future experiments will focus upon the mechanisms by which blockade of the NHE-1 confers myocardial protection. Furthermore, NHE-1 is ubiquitously expressed and the Na⁺/H⁺ exchanger has been shown to be involved in the activation of neutrophils (Fukushima et al., 1996), platelets (Siffert, 1995) and endothelial cells (Ghigo et al., 1988), all of which have been shown to be intimately involved in reperfusion injury. The effect of EMD 85131 on these elements has not been adequately addressed and will be the focus of future experiments.

In conclusion, our experiments have demonstrated that EMD 85131, (2-methyl-5-methylsulfonyl-1-(1-pyrrollyl)-benzoylguanidine), is a potent, selective inhibitor of NHE-1 with marked cardioprotective effects when administered either 15 min before ischemia or 15 min before reperfusion. The clinical applicability of such a compound is exciting and might include scenarios such as adjunctive therapy with percutaneous transluminal coronary angioplasty or thrombolysis during an acute myocardial infarction, or as a pretreatment administered before or during coronary artery bypass grafting or heart transplantation.