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(Circulation. 1999;100:2519.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Inhibition of the Na⁺/H⁺ Exchanger Confers Greater Cardioprotection Against 90 Minutes of Myocardial Ischemia Than Ischemic Preconditioning in Dogs

Richard J. Gumina, MD, PhD; Erich Buerger, PhD; Christian Eickmeier, PhD; Jeannine Moore, BS; Juergen Daemmgen, PhD; Garrett J. Gross, PhD

From the Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee (R.J.G., J.M., G.J.G.), and Boehringer Ingelheim Pharma KG, Germany (E.B., C.E., J.D.). Dr Gumina is now at the Mayo Clinic and Foundation, Rochester, Minn.

Correspondence to Garrett J. Gross, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.

	Abstract

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Background—This study compared the efficacy of ischemic preconditioning (IPC) and sodium-hydrogen exchanger (NHE)-1 inhibition to reduce infarct size (IS) induced by a 90-minute ischemic insult and examined the interaction between NHE-1 inhibition and IPC.

Methods and Results—In a canine infarct model, either IPC, produced by 1 or four 5-minute coronary artery occlusions, or the specific NHE-1 inhibitor BIIB 513, 0.75 or 3.0 mg/kg, was administered 15 minutes before either a 60- or 90-minute coronary artery occlusion followed by 3 hours of reperfusion. IS was determined by TTC staining and expressed as a percentage of the area at risk (IS/AAR). Although both IPC and BIIB 513 at 0.75 mg/kg produced comparable and significant reductions in IS/AAR in the 60-minute occlusion model, insignificant reductions in IS/AAR were observed in the 90-minute occlusion model. However, BIIB 513 at 3.0 mg/kg markedly reduced IS in both models (P<0.05). Next, to examine the interaction between NHE-1 blockade and IPC, BIIB 0.75 mg/kg was administered either before IPC or during the washout phase of IPC before 90 minutes of coronary artery occlusion. Both combinations resulted in a greater-than-additive reduction in IS/AAR (P<0.05).

Conclusions—These data demonstrate that although IPC and NHE-1 inhibition provide comparable protection against 60 minutes of myocardial ischemia, NHE-1 inhibition is more efficacious than IPC at protecting against a 90-minute ischemic insult. Furthermore, the combination of NHE-1 inhibition and IPC produces a greater-than-additive reduction in IS/AAR, suggesting either that NHE activity limits the efficacy of IPC or that different mechanisms are involved in the cardioprotective effect of IPC and NHE-1 inhibition.

Key Words: myocardial infarction • ions • ischemia

	Introduction

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Ischemic preconditioning (IPC) is a phenomenon in which brief periods of ischemia confer cardioprotection against prolonged ischemia.^{1 2} Our laboratory first demonstrated that IPC produced cardioprotection via activation of the ATP-sensitive potassium (K_ATP) channel.³ Pharmacological opening of the channel mimicked IPC, and closing the channel blocked cardioprotection. Interestingly, reexamination of our data indicated that IPC produced a greater and more prolonged cardioprotective effect than that produced by K_ATP channel openers, suggesting that other factors besides the K_ATP channel contribute to IPC-induced cardioprotection.

IPC reduces the severity of acidosis during prolonged ischemia,^{4 5} leading to the suggestion that IPC induces sodium-hydrogen exchanger (NHE) activity, which contributes to the attenuation of intracellular acidosis and the reduction of ischemia-reperfusion injury.⁶ However, this suggestion contradicts numerous reports demonstrating that NHE inhibition is cardioprotective.⁷ Furthermore, in isolated hearts, IPC and NHE inhibition are additive.^{8 9} Thus, enhanced NHE activity may in fact limit the cardioprotection conferred by IPC.

Although numerous studies have examined the cardioprotective effect produced by either IPC or NHE inhibition, only a few reports compare their efficacy or the interaction between these 2 modalities.^{6 8 9 10 11 12} Furthermore, although the length of the time interval during which the cardioprotective effects of IPC are maintained, ie, the "window of protection," has been elucidated,¹³ the efficacy of IPC against ischemic insults of increasing duration, ie, the "ceiling of protection," has received little attention. Similarly, the efficacy of NHE inhibition against ischemic insults of >60 minutes has not been examined.^{14 15} Because the NHE isoform 1 (NHE-1) is predominant in the myocardium,¹⁶ the purpose of this study was to compare the efficacy of IPC and NHE-1 inhibition against ischemic insults of either 60 or 90 minutes and to examine the relationship between IPC and NHE-1 inhibition.

	Methods

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Materials
To perform this study, benzamide-N-(aminoiminomethyl)-4-[4-(2-furanylcarbonyl)-1-piperazinyl]-3-(methylsulfonyl)methanesulfonate (BIIB 513) was used (Figure 1). All reagents were obtained from Sigma Chemical Co or Gibco BRL unless otherwise indicated.

View larger version (14K): [in this window] [in a new window]   Figure 1. Chemical structure of NHE-1 inhibitor BIIB 513.

Binding Assays
The experimental conditions for analysis of binding to each specific channel have been published previously.^{17 18 19 20 21 22 23 24 25} BIIB 513 was tested in triplicate in each assay at 10 µmol/L. The specific radioligand binding was defined as the difference between total binding and nonspecific binding determined in the presence of an excess of unlabeled ligand. Results are expressed as a percentage of inhibition of specific binding obtained in the presence of BIIB 513. IC₅₀ values were determined for the reference compounds by nonlinear regression analysis of their competition curves.

NHE Assays
An NHE-deficient cell line or cells expressing either NHE isoform 1 or 3 (PS120 hNHE-1 and PS120 rNHE-3, respectively) were obtained from Professor J. Pouyssegur (Nice, France) and cultured as previously described.²⁶ HT-29 cells, a human colon carcinoma cell line that expresses wild-type hNHE-1, was purchased from American Type Culture Collection.

The inhibitory effects of the various inhibitors on wild-type NHE-1 were examined via intracellular pH measurements. HT-29 cells, grown to confluence at 37°C with 5% CO₂ in DMEM, were incubated with 2.5 µmol/L BCECF-AM at 37°C without CO₂. Next, the cells were incubated in acid-loading medium, pH 7.5 (in mmol/L: NH₄Cl 50, choline chloride 70, KCl 5, MgCl₂ 1, CaCl₂ 1.8, glucose 5, and HEPES 15) for 30 minutes at 37°C without CO₂. Cells were rinsed and incubated at 37°C for 4 minutes without CO₂ in recovery medium, pH 7.0 (in mmol/L: NaCl 120, KCl 5, MgCl₂ 1, CaCl₂ 1.8, glucose 5, and MOPS 15), with or without specific NHE inhibitors. BCECF fluorescence was monitored with a CytoFluor 2350. The cytoplasmic pH was calculated from the ratio of fluorescence at excitation wavelengths of 485 and 440 nm. Dose-response curves were generated from single-cell intracellular pH measurements taken every 10 to 20 seconds for 20 minutes (data not shown).

The selectivity of the various NHE inhibitors for NHE-1 versus NHE-3 was compared by the acute acid load recovery test.²⁶ Cells (PS120 hNHE-1 and PS120 rNHE-3) were incubated for 30 minutes at 37°C without CO₂ or Na⁺ in acid-loading medium. Cells were washed and incubated for 24 hours at 37°C with CO₂ in recovery medium containing the specific NHE inhibitors. After the 24-hour incubation, cell viability was determined by fluorescein diacetate staining (excitation 485 nm; emission 530 nm).

Concentrated stock solutions (10^-2 mol/L) of NHE-1 inhibitors tested were prepared in DMSO, and aliquots were added to the recovery medium. The final concentration of DMSO was <1%. For generation of concentration-response curves, concentrations of inhibitors ranged from 10^-10 to 10^-4 mol/L.

Ischemia-Reperfusion Protocol
A standard canine myocardial ischemia-reperfusion protocol was used (Figure 2).¹⁵ The in vivo doses of BIIB 513 were based on the t_1/2 and plasma levels. The t_1/2 for BIIB 513 is 60 minutes with a maximum plasma level of 10 µmol/L after dosing with 7.5 mg/kg. Thus, doses to provide 0.3 µmol/L and 1 µmol/L concentrations were used. Dogs were randomly assigned to 1 of 11 groups. All dogs were subjected to either 60 or 90 minutes of left anterior descending coronary artery (LAD) occlusion and 3 hours of reperfusion. In groups 1 to 3, either saline (control group) or 1 of 2 doses of BIIB 513 (0.75 or 3.0 mg/kg) was infused intravenously for 15 minutes before 60 minutes of LAD occlusion. In group 4, IPC was achieved by 5 minutes of LAD occlusion followed by 10 minutes of reperfusion before 60 minutes of LAD occlusion (1x5' IPC). In groups 5 to 7, either saline or 1 of 2 doses of BIIB 513 (0.75 or 3.0 mg/kg) was infused intravenously for 15 minutes before 90 minutes of LAD occlusion. In group 8, IPC, 5 minutes of LAD occlusion followed by 10 minutes of reperfusion, was conducted before 90 minutes of LAD occlusion (1x5' IPC). In group 9, IPC, four 5-minute periods of LAD occlusion followed by 5 minutes of reperfusion, was conducted before 90 minutes of LAD occlusion (4x5' IPC). In group 10, 0.75 mg/kg of BIIB 513 was infused 10 minutes before 1x5' IPC. In group 11, 1x5' IPC was followed by 10 minutes of reperfusion, during which time 0.75 mg/kg of BIIB 513 was infused before 90 minutes of LAD occlusion. In all groups, hemodynamics and blood gas analyses were measured before LAD occlusion, at 30 minutes during LAD occlusion, and every hour after reperfusion. Regional myocardial blood flows were determined at 30 minutes during the LAD occlusion period and at the end of the experiment.

View larger version (44K): [in this window] [in a new window]   Figure 2. Experimental protocols used to examine effects of NHE-1 inhibition on myocardial IS. Dogs were assigned to 1 of 11 groups. All animals were subjected to either 60 or 90 minutes of LAD occlusion and 3 hours of reperfusion. Hatched bars indicate ischemia; clear, reperfusion; stippled, 0.75 mg/kg BIIB 513; solid, 3.0 mg/kg BIIB 513; and alternating hatched/clear, ischemic preconditioning.

At the end of the 3-hour reperfusion period, the anatomic area at risk (AAR) and the nonischemic area were differentiated, and the hearts were processed and stained with 2,3,5-triphenyl tetrazolium chloride (TTC) to differentiate the infarcted myocardium.¹⁵ Infarct size (IS) was expressed as a percentage of the AAR. Regional myocardial blood flow was measured by the radioactive microsphere technique.²⁷

Dogs were excluded if (1) heartworms were found, (2) transmural collateral blood flow was >0.20 mL · min^-1 · g^-1, (3) heart rate was >180 bpm at the beginning of the experiment, or (4) >3 consecutive attempts were needed to convert ventricular fibrillation.

Statistical Analysis
All values are expressed as mean±SEM. Differences between groups in hemodynamics and blood gases were compared by use of a 2-way ANOVA with repeated measures. Differences between groups in tissue blood flows, AAR, and IS were compared by 1-way ANOVA, and comparisons between individual groups were made with a 2-tailed t test. ANCOVA was used to determine whether the relationship between transmural collateral blood flow and IS differed between the control and treated groups. For all experiments, differences between groups were considered significant if the probability value was P<0.05.

	Results

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Selectivity of BIIB 513
To determine the selectivity of BIIB 513 for NHE isoforms, the recovery from an acute acid load in cells expressing either NHE-1 or NHE-3 was compared. The IC₅₀ for inhibition of recovery from acid load in cells expressing wild-type NHE-1 was 27 nmol/L (Table 1). This is 8 times more potent than that obtained for HOE 642 (210 nmol/L).²⁸ The IC₅₀ determined from cells expressing recombinant human NHE-1 is 10-fold greater than that obtained from cells expressing wild-type NHE-1; however, this is due to an 15-fold overexpression of recombinant human NHE-1 in the transfected PS120 cells due to gene amplification.²⁹ Finally, the IC₅₀ for BIIB 513 in cells expressing recombinant rat NHE-3 was >1000 µmol/L. On the basis of these data, BIIB 513 is 37 000 times more selective toward NHE-1.

View this table: [in this window] [in a new window]   Table 1. Inhibition of NHE Subtypes

Exclusions to Canine Ischemia-Reperfusion Studies
One hundred ten dogs were used in this study. Four were excluded because transmural collateral blood flow was >0.2 mL · min^-1 · g^-1 (1 in the 60-minute control group, 1 in the 60-minute low-dose BIIB 513–treated group, 1 in the 60-minute high-dose BIIB 513–treated group, and 1 in the 90-minute high-dose BIIB 513–treated group). One dog in the 60-minute control group and 1 in the 60-minute group treated with 3.0 mg/kg of BIIB 513 were excluded because of intractable ventricular fibrillation. Thus, 104 dogs successfully completed the protocol.

Hemodynamic and Blood Gas Data
Table 2 summarizes the hemodynamic data from all groups. There were no significant differences within or between groups.

View this table: [in this window] [in a new window]   Table 2. Hemodynamics for 60-Minute and 90-Minute Ischemia-Reperfusion Protocols

IS Measurements
Figure 3 and Table 3 demonstrate the effect of IPC, NHE-1 inhibition, or the combination on the AAR and IS expressed as a percentage of the AAR (IS/AAR) and left ventricle (IS/LV). In the 60-minute model, both IPC and administration of 0.75 or 3.0 mg/kg of BIIB 513 produced comparable reductions in IS/AAR (Figure 3). However, in the 90-minute occlusion model, no statistically significant reduction in IS/AAR was observed with either IPC (1x5') or 0.75 mg/kg of BIIB 513 (Figure 3, Table 3). In contrast, 3.0 mg/kg of BIIB 513 significantly reduced IS/AAR (P<0.05) against 90 minutes of ischemia. Furthermore, comparison of the efficacy of IPC and NHE-1 inhibition against 60 and 90 minutes of occlusion revealed that NHE-1 inhibition with 3.0 mg/kg of BIIB 513 produced marked cardioprotection in both models, whereas the cardioprotection from IPC or 0.75 mg/kg of BIIB 513 was limited to the 60-minute model (Figure 3).

View larger version (32K): [in this window] [in a new window]   Figure 3. Effect of NHE-1 inhibition and ischemic preconditioning on IS from either 60 or 90 minutes of myocardial ischemia. Before occlusion of LAD bed for either 60 or 90 minutes, either BIIB 513 (0.75 mg/kg or 3.0 mg/kg) was administered or ischemic preconditioning [(1x5') or (4x5')] was conducted. AAR was visualized by staining with patent blue dye, and IS was visualized by TTC staining. AAR and IS were determined gravimetrically. IS/AAR is expressed as percentage. All values are mean±SEM (n=6 to 16). *P<0.05 vs control group.

View this table: [in this window] [in a new window]   Table 3. Infarct Size Data and Transmural Blood Flow Data

Increasing the number of brief occlusions before the sustained coronary artery occlusion has been reported to significantly reduce IS/AAR against 90 minutes of ischemia.³⁰ However, as shown in Figure 3, no greater cardioprotection was conferred with multiple periods of IPC (4x5') compared with the single period of IPC (1x5').

Next, we used IPC and 0.75 mg/kg of BIIB 513 to determine whether the combination of these modalities was antagonistic, additive, or synergistic. Administration of 0.75 mg/kg of BIIB 513 during the interval surrounding IPC should be sufficient to inhibit NHE-1 activity while providing minimal cardioprotection against 90 minutes of ischemia. As shown in Figure 3, administration of BIIB 513 either before IPC or during the washout phase of IPC resulted in a greater-than-additive reduction in IS.

There were no significant differences in LV weight, AAR, AAR/LV, or transmural collateral blood flow between groups (Table 3). Against 60 minutes of ischemia, the regression lines describing the relationship between transmural collateral blood flows and IS/AAR were shifted down in BIIB 513– and IPC-treated animals compared with the control group (Figure 4A). In contrast, the regression lines describing this relationship between groups treated with 0.75 mg/kg of BIIB 513, (1x5') IPC, and (4x5') IPC were not different from that of the control group after 90 minutes of ischemia (Figure 4B). However, in animals treated with 3.0 mg/kg BIIB 513 or the combinations of IPC and 0.75 mg/kg, the regression lines describing this relationship were shifted down compared with the 90-minute control group (Figure 4C).

View larger version (17K): [in this window] [in a new window]   Figure 4. Regression of transmural collateral blood flow (TCCF) vs IS/AAR for control and IPC- and NHE-1 inhibitor–treated animals. TCCF to ischemic area at 30 minutes of occlusion vs IS expressed as a percentage of AAR (IS/AAR) was plotted. A, 60 minutes of ischemia. IPC- and BIIB 513–treated groups had regression lines that were significantly shifted (P<0.05) downward by ANCOVA. indicates control, y=-72.5x+29.8; , 0.75 mg/kg BIIB 513, y=-73.4x+15.4; •, 3.0 mg/kg BIIB 513, y=-22.2x+5.1; , IPC (1x5'), y=-11.6x+6.8. B, 90 minutes of ischemia. IPC (1x5' or 4x5') and 0.75 mg/mg BIIB 513–treated groups did not have regression lines that were significantly shifted downward by ANCOVA. =control, y=-245.9x+47.6; , 0.75 mg/kg BIIB 513, y=-146.2x+34.9; , IPC (1x5'), y=-213.7x+41.7; , IPC (4x5'), y=-146.5x+37.8. C, 90 minutes of ischemia. Groups treated with 3.0 mg/kg BIIB 513, IPC/0.75 mg/kg BIIB 513, or 0.75 mg/kg BIIB 513/IPC had regression lines that were significantly shifted (P<0.05) downward by ANCOVA. indicates control, y=-245.9x+47.6; •, 3.0 mg/kg BIIB 513, y=-124.6x+20.4; , IPC (1x5')/0.75 mg/kg BIIB 513, y=-60.2x+14.0; , 0.75 mg/kg BIIB 513/IPC (1x5'), y=-78.9x+12.9.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study demonstrates that IPC and NHE-1 inhibition do not antagonize one another but rather produce greater-than-additive myocardial protection against prolonged ischemia-reperfusion injury in vivo. Furthermore, this study demonstrates that although IPC and NHE-1 inhibition provide comparable cardioprotection against 60 minutes of myocardial ischemia, NHE-1 inhibition provides significantly greater protection against 90 minutes of ischemia in vivo.

Efficacy of IPC Versus NHE-1 Inhibition Against Prolonged Myocardial Ischemia
Because the interval from onset of symptoms of myocardial infarction to treatment is often delayed, any clinically efficacious treatment must be effective against prolonged ischemic injury. Although the cardioprotective effect of IPC and NHE inhibition has been demonstrated consistently in a number of animal species,⁷ until now, no studies have compared the efficacy of these treatments in an in vivo model of prolonged ischemia-reperfusion injury. This study demonstrates that although IPC and NHE-1 inhibition conferred comparable cardioprotection against 60 minutes of ischemia, the efficacy of IPC and 0.75 mg/kg of BIIB 513 was greatly diminished against 90 minutes of ischemia. Interestingly, no greater cardioprotection was conferred by a 4x5' IPC protocol, confirming that the cardioprotection induced by the 1x5' IPC protocol was optimal. This contrasts with previous work showing that against a 90-minute ischemic insult, 4x5' IPC reduces IS to a degree comparable to that generally observed in a 60-minute occlusion model in dogs.³⁰ However, NHE-1 inhibition with 3.0 mg/kg of BIIB 513 still afforded significant cardioprotection against 90 minutes of ischemia. Interestingly, in a model of canine myocardial ischemia-reperfusion injury that used 90 minutes of ischemia, a significant proportion of myocytes within the infarct region were shown to be viable at the time of reperfusion and to subsequently lose viability during reperfusion, suggesting cell death due to reperfusion injury.³¹ NHE-1 activity increases in cardiomyocytes not only during ischemia³² but also during the first few minutes of reperfusion,³³ resulting in an accumulation of Ca²⁺, which contributes to cellular damage. Thus, in contrast to IPC, NHE-1 inhibition may prevent both ischemia-induced and reperfusion-induced myocardial injury against prolonged ischemia.

The fact that the efficacy of IPC is significantly diminished as the duration of ischemia is increased is intriguing. Although much attention has been focused on defining the window of protection induced by IPC, ie, the time interval between episodes of brief coronary artery occlusions and the final sustained ischemia during which cardioprotection is still observed, no studies have examined the efficacy of IPC against myocardial ischemia of increasing duration. Similarly, no studies have examined the efficacy of NHE inhibition against ischemic insults >60 minutes.^{14 15} Thus, the results of this study demonstrate that just as a window of protection for IPC exists, a ceiling of cardioprotection also exists, ie, IPC is cardioprotective only against ischemia of a defined duration. However, the same ceiling does not appear to be present for NHE-1 inhibition.

Interaction Between NHE Activity and IPC
As stated earlier, there is contradictory evidence on the interaction between IPC and NHE activity.^{6 8 9 10 11 12} During the brief periods of coronary artery occlusion of IPC, intracellular pH decreases and intracellular Na⁺ increases.^{5 6} Because the primary activator of NHE is acidosis,³³ it would be expected that NHE activity would increase with IPC. The observation that ischemic preconditioned rat hearts recover more rapidly from ammonium chloride pulse–induced acidosis has been used as evidence for increased NHE activity during IPC.⁶ Studies that used isolated heart preparations demonstrate an additive effect between IPC and NHE-1 inhibition and suggest either that IPC and NHE inhibition confer cardioprotection by different mechanisms or that NHE activity is a detrimental consequence of IPC that limits its cardioprotective effect.^{8 9} In isolated rat hearts, NHE inhibition with 1 µmol/L HOE 642 provided additive cardioprotection to IPC, as measured by creatine kinase leakage and LV developed pressure recovery.⁹ In a separate study, the combination of EIPA and IPC produced a greater-than-additive reduction in IS in isolated rat hearts.⁸ However, other studies suggest that NHE activity is necessary for IPC-induced cardioprotection and that NHE inhibition reduces the IPC-induced cardioprotection.^{6 12} In isolated rat hearts, 3 µmol/L of ethylisopropylamiloride (EIPA) inhibited IPC-induced cardioprotection as measured by creatine kinase leakage and LV developed pressure recovery.⁶ However, in the rabbit heart, administration of 1 µmol/L of EIPA provided no additive cardioprotection to IPC, although no antagonism was observed.¹¹ Finally, in an in vivo canine model of ischemia-reperfusion injury, intramyocardial injections of dimethylamiloride (DMA) antagonized cardioprotection induced by IPC.¹² Although the in vivo concentration of drug (30 injectionsx0.15 mL of 400 µmol/L DMA) is difficult to ascertain, estimating myocardial interstitial volume to be 0.5 L results in a concentration of 3.6 µmol/L, a concentration comparable to that used in isolated rat hearts in which antagonism with IPC was observed.⁶ Because amiloride and its analogues have numerous nonspecific effects at higher doses,^{34 35} the discrepancies observed at high doses of EIPA and DMA in the experiments reviewed above may not be due to specific inhibition of NHE-1 but rather to nonspecific effects. Such an interpretation is consistent with the overwhelming evidence that NHE inhibition is cardioprotective.^{7 8 9 14 15 36}

This study demonstrates that in vivo, NHE-1 inhibition either before IPC or during the washout phase of IPC results in a greater-than-additive reduction in myocardial IS. Several possible explanations might account for the greater-than-additive cardioprotection observed. An increase in NHE activity actually may represent a detrimental side effect of IPC, and the true cardioprotective potential of IPC may be unmasked only by concomitant inhibition of NHE. Alternatively, IPC and NHE-1 inhibition may act via separate complementary mechanisms to confer cardioprotection. In fact, preliminary results from our laboratory have previously demonstrated that glibenclamide and PD 115199, inhibitors of IPC, do not attenuate NHE inhibitor–mediated cardioprotection. Furthermore, although this was not examined in the present study, if NHE activity is a detrimental factor induced by IPC, then one might speculate that the combination of BIIB and IPC might also increase the window of protection as well as the ceiling of protection.

Conclusions
The present study demonstrates that NHE-1 inhibition is significantly more efficacious against prolonged myocardial ischemia than IPC. Furthermore, IPC and NHE-1 inhibition do not antagonize one another but rather produce greater-than-additive cardioprotection, suggesting either that NHE activity limits the efficacy of IPC or that different mechanisms may be involved in the cardioprotective effect of IPC and NHE-1 inhibition. On the basis of the data reported, NHE-1 inhibition would be predicted to have beneficial effects in patients with unstable angina, which is purported to be the clinical correlate to IPC. Thus, NHE-1 inhibitors may have wide clinical applicability in scenarios such as preventive therapy in patients at high risk for myocardial infarction.

	Acknowledgments

 
This study was supported by NIH grant HL-08311 and a grant from Boehringer Ingelheim Pharma KG. The authors thank Anna Hsu for her excellent technical assistance.

Received April 12, 1999; revision received July 9, 1999; accepted July 21, 1999.

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