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(Circulation. 1997;96:3617-3625.)
© 1997 American Heart Association, Inc.

Articles

Na⁺/H⁺ Exchanger Activity Does Not Contribute to Protection by Ischemic Preconditioning in the Isolated Rat Heart

Alex R. Shipolini, FRCS; Hiroyuki Yokoyama, MD; Manuel Galiñanes, MD, PhD; Stephen J. Edmondson, FRCS; David J. Hearse, PhD, DSc; ; Metin Avkiran, PhD

From Cardiovascular Research, The Rayne Institute, St Thomas' Hospital, and the Cardiothoracic Department, St Bartholomew's Hospital (S.J.E.), London, UK.

Correspondence to Dr Metin Avkiran, Cardiovascular Research, The Rayne Institute, St Thomas' Hospital, Lambeth Palace Rd, London SE1 7EH, UK. E-mail m.avkiran{at}umds.ac.uk

	Abstract

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Background Despite evidence that pharmacological inhibition of the Na⁺/H⁺ exchanger (NHE) is cardioprotective, activation of NHE has been proposed as a protective mechanism of ischemic preconditioning (PC).

Methods and Results In isolated rat ventricular myocytes (n=8 to 11 per group) loaded with the fluorescent pH indicator C-SNARF-1, we showed that HOE-642 (HOE) was a potent inhibitor of the sarcolemmal NHE (80% inhibition at 1 µmol/L); such inhibition was readily reversible by washout of the drug. We confirmed that 1 µmol/L HOE produces significant and reversible inhibition of NHE activity in isolated rat hearts as well (n=4), and in this model, we tested (n=8 per group) whether the presence of the drug during (1) the prolonged period of ischemia (40 or 60 minutes) or (2) the preceding brief periods of PC ischemia (3 minutes plus 5 minutes) modulates the protective efficacy of PC. In protocol 1, HOE was infused for 5 minutes immediately before the prolonged ischemic period. With 40 minutes of prolonged ischemia, the postischemic recovery of left ventricular developed pressure (LVDP) was 15±2% in controls and was improved to 45±7% with HOE (P<.05), 55±5% with PC (P<.05), and 68±2% with PC+HOE (P<.05 versus all groups). When the prolonged ischemic period was extended to 60 minutes, an additive effect of PC and HOE was readily apparent and LVDP recovery with PC+HOE (66±2%) was almost double that observed with HOE (37±4%) or PC (34±5%) alone (P<.05). In protocol 2, HOE was infused for 3 minutes immediately before each episode of PC ischemia and was subsequently washed out before a 40-minute prolonged ischemic period (HOE+PC). LVDP recovery was 34±4% in controls and was improved to 57±2% with PC (P<.05) and 55±3% with HOE+PC (P<.05). Improved recovery of LVDP was matched by reduced creatine kinase leakage in all cases.

Conclusions Because coadministration of HOE (at a concentration sufficient to inhibit NHE activity) did not reduce the efficacy of PC in either protocol, we conclude that NHE activity does not contribute to the cardioprotective actions of PC. On the contrary, NHE inhibition during the prolonged ischemic period may enhance the protection afforded by PC.

Key Words: ischemia • sodium • myocytes

	Introduction

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The remarkable cardioprotective efficacy of ischemic preconditioning has stimulated an intense investigative effort aimed at delineating its cellular mechanisms (for a recent review, see Downey and Cohen¹). One noteworthy consequence of ischemic preconditioning, which has been reported by several independent investigators,^2–6 is a significant reduction in the severity of intracellular acidosis during the prolonged period of ischemia. This "antiacidotic" effect has received considerable attention as a potential mechanism mediating the cardioprotective actions of ischemic preconditioning.^7–10 However, despite the general agreement that ischemic preconditioning attenuates intracellular acidosis during subsequent ischemia, there is no consensus regarding the mechanism that underlies this effect. Recently, Ramasamy and colleagues⁸ proposed that the sarcolemmal Na⁺/H⁺ exchanger (NHE), which is a primary route for H⁺ efflux from cardiac myocytes,^11,12 may be stimulated after ischemic preconditioning and that this may contribute to both the attenuation of intracellular acidosis and the amelioration of ischemia/reperfusion–induced injury in preconditioned hearts.⁸

On the basis of available evidence, it is reasonable to suggest that ischemic preconditioning may result in a greater activity of the sarcolemmal NHE and that this may contribute to a reduced severity of intracellular acidosis during the prolonged ischemic period. However, the proposal⁸ that such stimulation of NHE may be a cardioprotective mechanism appears contrary to the substantial body of evidence suggesting that in the setting of myocardial ischemia and reperfusion, pharmacological inhibition of NHE is protective, whereas pharmacological activation of NHE is detrimental (for recent reviews, see References 12 through 15^{12 13 14 15} ). An alternative possibility is that activation of NHE is an epiphenomenon that accompanies ischemic preconditioning but is not causally involved in its cardioprotective actions. On the contrary, it is possible that any activation of NHE may limit the extent of the cardioprotection afforded by preconditioning.

In light of the above, the primary objective of the present study was to determine whether NHE activity during either (1) the prolonged ischemic period or (2) the brief preconditioning ischemic periods contributes to the cardioprotective effect of ischemic preconditioning. To attain this objective, we first determined the effects of HOE-642, a novel benzoyl guanidine–based NHE inhibitor,¹⁶ on sarcolemmal NHE activity in rat ventricular myocytes. Subsequently, we subjected isolated rat hearts to an ischemic preconditioning protocol (which we have previously shown^6,17,18 to provide significant cardioprotection) in conjunction with the administration of HOE-642 at a concentration sufficient to inhibit sarcolemmal NHE activity.

	Methods

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Animals
Adult male Wistar rats (200 to 300 g body weight; B&K Universal Ltd, Hull, UK) were used in all studies. The animals were anesthetized with sodium pentobarbital (60 mg/kg IP) and systemically anticoagulated with heparin (300 IU IV). After a transverse thoracotomy, the heart was excised and immediately immersed in perfusion solution at 4°C for subsequent use in isolated myocyte or whole-heart studies. The investigation was conducted in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationery Office, London, UK.

Effects of HOE-642 on NHE Activity in Isolated Myocytes
Myocyte Isolation
Ventricular myocytes were isolated by a collagenase-based enzymatic digestion technique, as we have described previously.¹⁹ In brief, hearts were retrogradely perfused (37°C) in the Langendorff mode at a constant flow rate of 10 mL · min^-1 · g^-1 for four sequential periods, as follows: (1) with Tyrode's solution (containing, in mmol/L, NaCl 137, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.5, HEPES 10, and glucose 10, adjusted to pH 7.4 at 34°C with NaOH) for 5 minutes, (2) with nominally Ca²⁺-free Tyrode's solution (NaCl 135, KCl 5.4, NaH₂PO₄ 0.33, MgCl₂ 1.0, HEPES 10, and glucose 10, adjusted to pH 7.2 at 34°C with NaOH) for 5.5 minutes, (3) with nominally Ca²⁺-free Tyrode's solution containing collagenase (Worthington type 1, 100 U/mL) for 10 minutes, and (4) with storage buffer (KOH 78, KCl 30, KH₂PO₄ 30, MgSO₄ 3, EGTA 0.5, HEPES 10, glutamic acid 50, taurine 20, and glucose 10, adjusted to pH 7.2 at 34°C with KOH) for 5 minutes. All solutions were gassed with 100% O₂. After the perfusion procedure, the ventricles were removed and chopped into several pieces in storage buffer. The tissue fragments were then gently agitated to facilitate cell dispersion, and the cell suspension was maintained in storage buffer at 25°C for at least 1 hour before use in the microepifluorescence studies.

Measurement of pH_i and NHE Activity
pH_i was measured in single ventricular myocytes with the pH-sensitive fluorescent dye C-SNARF-1, as we have described previously.¹⁹ Cells loaded with C-SNARF-1 were allowed to settle on a glass coverslip at the bottom of a chamber mounted on the stage of an inverted microscope (Nikon Diaphot) and were superfused (3.5 mL/min, 34°C) with Tyrode's solution. Cells were excited with light at 540 nm, and the resulting fluorescence emission intensity from a selected area of a single myocyte was measured simultaneously at 580 nm (I₅₈₀) and 640 nm (I₆₄₀) with a dual-emission photometer system (model D104C, Photon Technology International). The emission intensity ratio (I₅₈₀/I₆₄₀) was calculated and converted to a pH_i scale by use of in situ calibration data obtained by exposing cells loaded with C-SNARF-1 to nigericin-containing calibration solutions.¹⁹

All experiments were carried out in the nominal absence of HCO₃^- (thereby precluding an involvement of HCO₃^--dependent pH_i-regulatory mechanisms), such that the rate of acid efflux (J_H) could be used as a direct index of sarcolemmal NHE activity.¹⁹ J_H was estimated during recovery from acute intracellular acidosis from the equation J_H=ß_i · dpH_i/dt, where ß_i is the intrinsic buffering power and dpH_i/dt is the rate of recovery of pH_i.

Experimental Protocol
The main objective of these studies was to determine the inhibitory efficacy of HOE-642 on sarcolemmal NHE activity. To this end, intracellular acidosis was induced (to activate sarcolemmal NHE) in the cells by the washout of NH₄Cl (20 mmol/L) after its transient (3 minutes) application. The initial (1 minute) washout of NH₄Cl was with Na⁺-free Tyrode's solution (NaCl replaced by choline chloride) to ensure NHE inactivity during H⁺ loading. Subsequently, NHE was reactivated by the reintroduction of Na⁺-containing Tyrode's solution in the absence or presence of various concentrations of HOE-642 (0.0001 to 1 µmol/L; n=8 to 11 cells per group). J_H was calculated from the initial dpH_i/dt value (obtained by linear regression analysis of pH_i data collected during the first 1 minute after the reintroduction of Na⁺) and the ß_i value corresponding to the appropriate pH_i (estimated from the equation ß_i=-34.9 · pH_i+273.5).¹⁹

In additional experiments, the reversibility of sarcolemmal NHE inhibition by HOE-642 was studied. Myocytes (n=3) were subjected to two consecutive acid pulses by the NH₄Cl washout method, separated by 15 minutes of normal superfusion. During both pulses, NH₄Cl washout was with normal Tyrode's solution; however, during the second pulse the initial (3 minutes) washout solution additionally contained 1 µmol/L HOE-642.

Effects of HOE-642 on NHE Activity in Whole Hearts
Isolated Heart Perfusion
Hearts were retrogradely perfused in the Langendorff mode at a constant coronary flow rate of 12 mL/min via a roller pump (Gilson Minipuls 3). The nominally HCO₃^--free perfusion solution was of the following composition (in mmol/L): NaCl 143.5, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.2, HEPES 20.0, CaCl₂ 1.4, and glucose 11.0 (adjusted to pH 7.4 at 37°C with NaOH, maintained at 37°C, and gassed continuously with 100% O₂). The solution contained, in addition, 20 mmol/L NH₄Cl when required. The pulmonary artery was incised to facilitate coronary effluent drainage. Left ventricular pressure was monitored by means of an intraventricular, isovolumic balloon²⁰ connected to a pressure transducer and was recorded with an ink-jet recorder. The right atrium was excised, and hearts were paced at 360 bpm throughout via an electrode attached to the right ventricle to avoid potential complications in data interpretation arising from pH-induced changes in sinus rate and/or atrioventricular conduction. HOE-642 (a gift from Hoechst-Marion-Roussel, Frankfurt, Germany) was dissolved in perfusion solution immediately before use to obtain a drug concentration of 14.3 µmol/L. When required, this solution was infused into the perfusion line at 7% of the total flow rate to give a final drug concentration of 1 µmol/L (chosen on the basis of the isolated myocyte studies; see below).

Experimental Protocol
The main objective here was to confirm that 1 µmol/L HOE-642 was sufficient to produce significant inhibition of sarcolemmal NHE activity in the whole heart. In the absence of a facility for the continuous measurement of pH_i in the whole heart (eg, nuclear magnetic resonance spectroscopy), we used LVDP as a surrogate for pH_i during the infusion and washout of NH₄Cl (20 mmol/L). The selection of this surrogate index was based on the work of Grace et al,²¹ who used hearts perfused with HCO₃^--free solution under conditions of constant coronary flow and heart rate (as in the present study), which has shown that (1) changes in LVDP during NH₄Cl pulses mirror closely the changes that occur in pH_i and (2) NHE inhibition suppresses the recovery of LVDP after NH₄Cl washout, which reflects a delay in the recovery of pH_i from acidosis. After 15 minutes of perfusion with the standard perfusion solution, hearts (n=4) were perfused with solution containing NH₄Cl for 5 minutes and with standard solution for a further 20 minutes (first acid pulse). Subsequently, this cycle was repeated (second acid pulse), but this time with HOE-642 (1 µmol/L) also present during the infusion of NH₄Cl and the first 10 minutes of its washout.

During initial perfusion with standard solution, the intraventricular balloon was inflated to obtain a LVEDP of 4 mm Hg, and the balloon volume was kept constant thereafter. LVDP was calculated as the difference between LVEDP and LVSP and was noted at 1- to 2-minute intervals throughout each experiment.

Effects of HOE-642 on Cardioprotective Efficacy of Ischemic Preconditioning
Isolated Heart Perfusion
Hearts were perfused in the Langendorff mode as described above, but this time at a constant perfusion pressure of 75 mm Hg. Furthermore, the perfusion solution contained the physiological buffer HCO₃^- (25.0 mmol/L NaHCO₃) rather than HEPES and was gassed continuously with a mixture of 95% O₂/5% CO₂ (pH 7.4 at 37°C). During preischemic perfusion, hearts were paced at 360 bpm via an electrode attached to the right atrium; pacing was discontinued from 2 minutes into the prolonged ischemic period and recommenced on reperfusion. HOE-642 was dissolved in deionized water to make a 1 mmol/L stock solution, which was stored at 4°C for a maximum of 5 days. The stock solution was diluted in perfusion solution to obtain a final drug concentration of 1 µmol/L immediately before use.

Experimental Protocols
As summarized schematically in Fig 1, there were two main protocols in this part of the project (n=8 hearts per group), which were designed to determine the effects of NHE inhibition during either (1) the prolonged ischemic period (protocol 1) or (2) the preceding brief preconditioning ischemic periods (protocol 2). After the interventions illustrated in Fig 1, hearts were subjected to normothermic global zero-flow ischemia for 40 or 60 minutes in protocol 1 and for 40 minutes in protocol 2, followed in each case by 40 minutes of reperfusion. Associated with protocol 2, an additional experiment was performed to confirm the adequate washout of HOE-642 before the prolonged ischemic period, as described in "Results."

View larger version (31K): [in this window] [in a new window]   Figure 1. Schematic of main experimental protocols for PC studies. Only periods before induction of prolonged ischemia are shown; open bars indicate aerobic perfusion with standard perfusion solution, shaded bars aerobic perfusion with perfusion solution containing HOE-642 (1 µmol/L), and solid bars global zero-flow ischemia. All hearts (n=8/group) were subsequently subjected to 40 minutes (protocols 1 and 2) or 60 minutes (protocol 1 only) of global zero-flow ischemia, followed by 40 minutes of reperfusion. Functional indices were measured at end of reperfusion period and expressed as percentage of their respective values obtained at end of initial 15 minutes of aerobic perfusion.

Left ventricular pressure was monitored via an intraventricular balloon, as described above, and coronary flow rate was measured by timed collection of the coronary effluent. Basal values of LVDP and coronary flow rate were measured at the end of the initial 15-minute period of aerobic perfusion. Left ventricular pressure was also monitored during the period of ischemic arrest and throughout reperfusion to allow assessment of the temporal profiles of the development of ischemic contracture and the postischemic recovery of contractile function. The final postischemic recoveries of LVDP and coronary flow were assessed by expressing the values obtained at the end of the reperfusion period as a percentage of their respective basal values. Total creatine kinase leakage (expressed as IU/g heart dry wt) was assessed by spectrophotometric analysis of enzyme activity in the coronary effluent collected during reperfusion with a commercially available kit (Sigma Diagnostics).

Statistical Analysis
Experiments within each protocol were carried out in a prospectively randomized manner. Gaussian-distributed variables were expressed as mean±SEM and were subjected to one-way ANOVA. If a difference among mean values was established, intergroup comparisons were performed with the Student-Newman-Keuls test. A value of P<.05 was considered significant.

	Results

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Effects of HOE-642 on NHE Activity in Isolated Myocytes
Basal and Minimal pH_i
Values for basal pH_i (measured immediately before exposure to NH₄Cl) and minimal pH_i (measured at the end of NH₄Cl washout with Na⁺-free Tyrode's solution) did not differ significantly between the various study groups. The ranges observed were from 7.16±0.04 to 7.24±0.04 for basal pH_i and from 6.68±0.04 to 6.77±0.05 for minimal pH_i.

Sarcolemmal NHE Activity
Fig 2A shows representative recordings of pH_i during NH₄Cl pulses in a control cell and in a cell in which extracellular Na⁺ was reintroduced in the presence of 1 µmol/L HOE-642. There was a rapid recovery of pH_i from acidosis under control conditions, whereas pH_i recovery was slowed significantly by HOE-642. The quantitative effects of HOE-642 on sarcolemmal NHE activity are illustrated in Fig 2B, which shows J_H as a function of drug concentration. J_H was 7.51± 1.49 mmol/L per minute in control cells and was reduced by HOE-642 in a concentration-dependent manner, by 10%, 27%, 50% (P<.05), 68% (P<.05), and 77% (P<.05) at 0.0001, 0.001, 0.01, 0.1, and 1 µmol/L, respectively.

View larger version (22K): [in this window] [in a new window]   Figure 2. A, Intracellular pH recordings during acid pulses (induced by transient exposure to 20 mmol/L NH4Cl in HCO3--free medium) in control myocyte (open symbols) and one that received 1 µmol/L HOE-642 during reintroduction of extracellular Na+ (solid symbols). B, Initial H+ efflux rate (JH) in control myocytes (open bar) and those that were exposed to various concentrations of HOE-642 during reintroduction of extracellular Na+ (solid bars) (n=8 to 11/group). *P<.05 vs control.

Fig 3 shows representative pH_i recordings from a myocyte subjected to two consecutive acid loads, the first in the absence of HOE-642 and the second in the transient presence of 1 µmol/L HOE-642. As expected from the above observations, pH_i recovery was markedly suppressed in the presence of HOE-642; however, pH_i recovery (at a rate similar to that under control conditions) was rapidly restored on removal of HOE-642 from the superfusion solution, indicating that the inhibition of exchanger activity was readily reversible.

View larger version (16K): [in this window] [in a new window]   Figure 3. Intracellular pH recordings in an isolated rat ventricular myocyte during two consecutive acid pulses (induced by transient exposure to 20 mmol/L NH4Cl in HCO3--free medium). During first pulse (open symbols), NH4Cl washout was under control conditions. During second pulse (solid symbols), initial (3 minutes) washout of NH4Cl was in presence of 1 µmol/L HOE-642, which was subsequently removed from superfusion solution. Recording shown is representative of values obtained from three such experiments.

Effects of HOE-642 on NHE Activity in Whole Hearts
Basal Cardiac Function
The basal value of LVDP, measured immediately before the first exposure to NH₄Cl, was 117±7 mm Hg. LVDP declined slightly after recovery from the first acid pulse, and the basal value measured immediately before the second exposure to NH₄Cl was 104±5 mm Hg.

Cardiac Function During Acid Pulses
Fig 4 illustrates LVDP, expressed as a percentage of the basal value, at various time points during the two consecutive acid pulses. As can be seen, during both pulses, the 5-minute infusion of NH₄Cl produced a positive inotropic effect (probably because of a rise in pH_i²¹), and the washout of NH₄Cl depressed LVDP within 1 minute (probably because of a rapid drop in pH_i²¹). In the first pulse, which occurred in the absence of HOE-642, there was a rapid biphasic recovery of LVDP. In the second pulse, which occurred in the presence of 1 µmol/L HOE-642, LVDP was further depressed by 2 minutes of washout, and recovery was markedly delayed, with a significant difference in LVDP values between the pulses during the first 2 to 10 minutes of NH₄Cl washout. This most likely reflected a delayed recovery of pH_i from acidosis due to drug-induced inhibition of NHE activity,²¹ the major H⁺ extrusion pathway under these experimental conditions. After the removal of HOE-642 from the perfusion solution, there was a rapid secondary recovery of LVDP, such that there was no significant difference between the pulses in LVDP values by 16 minutes of NH₄Cl washout.

View larger version (23K): [in this window] [in a new window]   Figure 4. LVDP at various time points during two consecutive NH4Cl pulses (comprising the 5-minute infusion and 20-minute washout of 20 mmol/L NH4Cl) in isolated hearts perfused with nominally HCO3--free solution. LVDP is expressed as percentage of basal value obtained immediately before start of NH4Cl infusion during each pulse. Open bars indicate first pulse; solid bars, second pulse. During second pulse, 1 µmol/L HOE-642 was present during NH4Cl infusion and first 10 minutes of its washout. *P<.05 vs first pulse.

Effects of HOE-642 on Cardioprotective Efficacy of Ischemic Preconditioning
Basal Cardiac Function
Basal values of LVDP and coronary flow did not differ significantly between groups within each study protocol and ranged from 136±4 to 148±5 mm Hg and from 11.4±0.5 to 12.2±0.5 mL/min, respectively.

Postischemic Cardiac Function
Protocol 1: Effects of NHE inhibition during prolonged ischemic period. In this protocol, the objective was to determine whether NHE inhibition during the prolonged ischemic period influences the cardioprotection afforded by ischemic preconditioning. Fig 5 shows the left ventricular pressure profiles during 40 minutes of ischemia and subsequent reperfusion in the four study groups. As can be seen, the time to onset of ischemic contracture (defined as the time at which left ventricular pressure rose 4 mm Hg above baseline) was significantly shorter in PC (5.0±0.2 minutes) relative to control (9.0±0.6 minutes). HOE-642 did not alter the time to onset of ischemic contracture when given alone (HOE; 9.0±0.6 minutes) and did not inhibit the acceleration of the onset of ischemic contracture by PC when given in combination (PC+HOE; 5.0±0.2 minutes). It is also apparent from this figure that in all three treatment groups, the postischemic recovery of contractile function was markedly improved relative to control, with end-reperfusion LVSP values of 96±2, 111±4 (P<.05), 115±4 (P<.05), and 121±3 (P<.05) mm Hg and LVEDP values of 75±2, 53±6 (P<.05), 40±4 (P<.05), and 28±2 (P<.05) mm Hg in control, HOE, PC, and PC+HOE, respectively. At this time, LVDP recovery was 15±2% in controls. This was significantly increased, to 45±7% by HOE-642 alone (HOE) and to 55±5% by ischemic preconditioning alone (PC). With the combination of both interventions (PC+HOE), LVDP recovery was further improved to 68±2%, a value that was significantly greater than those obtained in HOE and in PC. Creatine kinase leakage during reperfusion measured 494±49 IU/g in controls and was reduced significantly to 350±35, 291±36, and 272±33 IU/g in HOE, PC, and PC+HOE, respectively (with no significant difference between the values obtained in the three treatment groups). Postischemic recovery of coronary flow was 56±3% in the untreated control group; this was significantly increased in all treatment groups, to 78±3%, 72±3%, and 80±4% in HOE, PC, and PC+HOE, respectively.

View larger version (22K): [in this window] [in a new window]   Figure 5. Left ventricular pressure profiles during the 40-minute period of prolonged ischemia and subsequent reperfusion in various study groups in protocol 1 (n=8/group). HOE indicates hearts that received HOE-642 (1 µmol/L) immediately before prolonged ischemic period; PC, preconditioned hearts; and PC+HOE, preconditioned hearts that also received HOE-642 (1 µmol/L) immediately before prolonged ischemic period (see Fig 1 for details). Horizontal black bar above time axis illustrates period of ischemia, and vertical dashed lines indicate times to onset of ischemic contracture in control and PC groups. At each time point during reperfusion, higher symbol indicates LVSP and lower symbol LVEDP; thus, difference (shaded area) represents LVDP.

As shown above, although the recovery of contractile function after 40 minutes of prolonged ischemia was significantly enhanced in PC+HOE relative to PC or HOE alone, the improvement in final LVDP recovery was small and was not matched by a significant reduction in creatine kinase leakage during reperfusion. Therefore, we performed an additional study with the objective of testing whether any additive protection afforded by the combination of ischemic preconditioning and HOE-642 would be more readily revealed under more severe conditions. To this end, the duration of prolonged ischemia was extended from 40 minutes to 60 minutes. Fig 6 shows the postischemic recovery of LVDP (Fig 6A) and creatine kinase leakage during reperfusion (Fig 6B) in the control, HOE, PC, and PC+HOE groups. Under these conditions also, LVDP recovery was significantly improved and creatine kinase leakage significantly reduced in all three treatment groups relative to control. However, with this extended duration of prolonged ischemia, LVDP recovery in PC+HOE (66±2%) was almost double that in HOE (37±4%) or PC (34±5%); furthermore, this time the improved contractile recovery was accompanied by a significant reduction in creatine kinase leakage, supporting an additive effect.

View larger version (17K): [in this window] [in a new window]   Figure 6. A, Postischemic recovery of LVDP and B, creatine kinase leakage during reperfusion in various study groups in protocol 1 (n=8/group), which were subjected to 60 minutes of prolonged ischemia. HOE indicates hearts that received HOE-642 (1 µmol/L) immediately before prolonged ischemic period; PC, preconditioned hearts; and PC+HOE, preconditioned hearts that additionally received HOE-642 (1 µmol/L) immediately before prolonged ischemic period (see Fig 1 for details). *P<.05 vs control; **P<.05 vs control, HOE, and PC.

Protocol 2: Effects of NHE inhibition during preconditioning ischemic periods. In this protocol, the objective was to determine whether NHE inhibition during the short periods of preconditioning ischemia influences the cardioprotection afforded by ischemic preconditioning. Relative to control, the postischemic recovery of LVDP was once again significantly increased in PC (57±2% versus 34±4%), and this effect was accompanied by a significant reduction in creatine kinase leakage during reperfusion (410±25 versus 523±31 IU/g) (Fig 7). However, in contrast to our observations with the infusion of HOE-642 immediately before the prolonged ischemic period, when the drug was infused before each of the preconditioning ischemic periods and subsequently washed out (HOE+PC), there was no significant difference in LVDP recovery or creatine kinase leakage relative to PC (Fig 7). A similar pattern was seen with respect to the postischemic recovery of coronary flow: 57±4% in control, 70±3% in PC (P<.05), and 72±2% in HOE+PC (P<.05). The time to onset of ischemic contracture was once again significantly shortened in PC (from 10.9±0.4 minutes in control to 6.4±0.4 minutes), and this effect was unaffected by the coadministration of HOE-642 (7.3±0.6 minutes in HOE+PC, P<.05 versus control).

View larger version (15K): [in this window] [in a new window]   Figure 7. A, Postischemic recovery of LVDP and B, creatine kinase leakage during reperfusion in the various study groups in protocol 2 (n=8/group), which were subjected to 40 minutes of prolonged ischemia. PC indicates preconditioned hearts; HOE+PC, preconditioned hearts that also received HOE-642 (1 µmol/L) for 3 minutes immediately before each of two preconditioning ischemic periods (drug was washed out before prolonged ischemic period; see Fig 1 for details). *P<.05 vs control.

A potential complication in the interpretation of the above study is the possibility that the washout of HOE-642 in the HOE+PC group might have been inadequate. Thus, even if NHE inhibition during the brief preconditioning ischemic periods did abolish the cardioprotective actions of preconditioning, such an effect might have been obscured by any cardioprotection arising from residual drug presence during the prolonged ischemic period. To test whether the washout period used was sufficient, an additional study was performed in which 1 µmol/L HOE-642 was infused for 6 minutes (equivalent to the total duration of drug infusion in the above protocol) and hearts were subjected to ischemia either (1) immediately after drug infusion or (2) after 15 minutes of washout (equivalent to the duration of drug washout in the above protocol). The control group once again received no intervention. Postischemic recovery of LVDP was significantly improved, from 32±2% in control to 46±4% by the infusion of HOE-642 immediately before ischemia. In contrast, there was no significant change in LVDP recovery (31±4%) when the drug was washed out for 15 minutes before the induction of ischemia, indicating that a 15-minute washout period was sufficient to reduce the tissue drug content to a level that does not affect postischemic cardiac function.

	Discussion

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The present study has demonstrated that, in isolated rat hearts, administration of HOE-642 (at a concentration sufficient to inhibit sarcolemmal NHE activity in isolated myocytes and delay recovery of contractile function from acidosis-induced depression in whole hearts) before either the prolonged ischemic period or the preceding brief preconditioning ischemic periods fails to impair the cardioprotective efficacy of ischemic preconditioning. This finding questions the suggested role⁸ of NHE activity as a determinant of the cardioprotective effect of ischemic preconditioning.

Is NHE Activity Necessary for Cardioprotection by Preconditioning?
Ramasamy et al⁸ recently proposed a role for stimulation of NHE activity in the protective action of ischemic preconditioning in the isolated rat heart. This proposal was based on the observation that the intracoronary infusion of ethylisopropylamiloride (EIPA), an inhibitor of NHE, immediately before the prolonged ischemic period could attenuate the improved LVDP recovery and reduced creatine kinase leakage afforded by ischemic preconditioning. This finding contrasts with a large body of evidence (for recent reviews, see References 12 through 15^{12 13 14 15} ), obtained with a variety of pharmacological NHE inhibitors (including EIPA) and species, that inhibition of NHE is cardioprotective. Indeed, in accordance with our earlier work,²² the present study has confirmed that infusion of the NHE inhibitor HOE-642 immediately before the prolonged ischemic period affords significant cardioprotection. Furthermore, the present study has shown that the cardioprotective effects of ischemic preconditioning and NHE inhibition, as assessed by increased LVDP recovery and reduced creatine kinase leakage (as in the study by Ramasamy et al⁸), are additive rather than counteractive.

The observations of the present study do not support a role for NHE activity in determining the cardioprotective consequences of ischemic preconditioning. On the contrary, in light of the present study, it may be speculated that any increase in NHE activity in preconditioned hearts (the evidence for which is critically assessed below) may represent an undesirable side effect of ischemic preconditioning, which detracts from its cardioprotective efficacy. Thus, the true protective potential of ischemic preconditioning may be revealed only by concomitant inhibition of NHE activity during the prolonged ischemic period. The observation in the present study that the combination of ischemic preconditioning and infusion of HOE-642 immediately before the prolonged ischemic period afforded significantly greater protection than either intervention alone is consistent with this hypothesis.

Cardiac Actions of NHE Inhibitors
The factors that may potentially account for the divergent findings of the present study compared with that by Ramasamy and colleagues⁸ must be considered. Both studies used identical species, models, and functional end points, although the mode of perfusion (constant pressure versus constant flow) differed and may have contributed to the divergence in findings. However, the most significant factor is likely to have been the difference between the studies in the characteristics and concentration of the pharmacological NHE inhibitor used. Although 5-amino-substituted derivatives of amiloride (such as EIPA) are potent inhibitors of NHE, they are relatively nonspecific²³ and have been shown to produce electrophysiological abnormalities^24,25 and cardiodepressant effects,^25,26 particularly at concentrations that exceed 1 µmol/L.²⁵ For this reason, we chose not to use an amiloride derivative and selected HOE-642, which is a novel, benzoylguanidine-based NHE inhibitor that exhibits marked selectivity for the cardiac isoform of the exchanger.¹⁶ Indeed, in our myocyte studies, we confirmed that HOE-642 is a potent inhibitor of the sarcolemmal NHE in rat ventricular myocytes, with the 1 µmol/L concentration (as used in our preconditioning studies) resulting in 80% inhibition of exchanger activity at a pH_i of 6.75. This finding is consistent with recent work with HOE-694 (a structural congener of HOE-642) in guinea pig ventricular myocytes.²⁷ It is important to note that these benzoylguanidine derivatives do not affect the activity of other pH_i-regulating carriers²⁷ or Na⁺ transport mechanisms.¹⁶ Furthermore, unlike EIPA, they do not appear to exhibit cardiodepressant effects at NHE-inhibitory concentrations,^16,26 a property that enhances their value as pharmacological tools in the delineation of the physiological/pathophysiological role(s) of NHE. In light of the above arguments, it may be speculated that the results of the study by Ramasamy and colleagues⁸ were complicated by the use of a relatively high concentration of a less selective NHE inhibitor, whose nonspecific actions might have contributed to the apparent abolition of the protective effect of ischemic preconditioning. In this regard, it is important to note that Bugge and Ytrehus²⁸ showed that coadministration of EIPA at a lower concentration (1 µmol/L versus 3 µmol/L in the study by Ramasamy et al⁸) provides additional protection to preconditioned rat hearts, which is in keeping with our findings with HOE-642.

Role of NHE Activity During Preconditioning Ischemic Periods
In both previous studies in which EIPA was used in combination with ischemic preconditioning,^8,28 the NHE inhibitor was present during the prolonged ischemic period. In the present study, we additionally addressed, for the first time, the question of whether NHE activity during the preconditioning ischemic periods might be involved in the signaling mechanism(s) mediating the protective response. Our observation that the infusion of HOE-642 before each of the preconditioning ischemic periods (followed by its washout) does not diminish the cardioprotective action of preconditioning suggests that NHE activity during these periods also is not involved in the underlying protective mechanisms. It may be argued that residual drug presence during the prolonged ischemic period might have complicated the interpretation of these studies. However, our demonstration of the ready reversibility by drug washout of (1) HOE-642–induced depression of pH_i recovery in acid-loaded myocytes (Fig 3), (2) HOE-642–induced depression of LVDP recovery in acid-loaded hearts (Fig 4), and (3) the cardioprotective effect of HOE-642 in hearts subjected to ischemia/reperfusion would argue against significant residual drug presence.

NHE Activity and Ischemic Contracture
In the present study, the infusion of HOE-642 immediately before the prolonged ischemic period did not alter the time to onset of ischemic contracture. However, consistent with recent observations from our laboratory,^6,18 ischemic preconditioning significantly accelerated the onset of ischemic contracture. The combination of the two interventions resulted in an accelerated contracture profile similar to that observed with ischemic preconditioning alone. On the basis of these observations, it can be concluded that, in the isolated rat heart, NHE activity is not a determinant of the rate of development of ischemic contracture (although different observations have been made in the rabbit heart²⁹). Previous studies by Hearse et al³⁰ in the isolated rat heart have shown that the onset of ischemic contracture is closely linked to the rate at which the tissue ATP content declines, a relationship that appears to hold true in preconditioned hearts as well.^6,18 Thus, the inability of HOE-642 to modify the profile of ischemic contracture may be due to the inability of NHE inhibition to significantly alter the rate of ATP depletion during global zero-flow ischemia, as revealed by studies that used NMR spectroscopy for continuous analysis of tissue ATP content.^29,31,32

Is NHE Activity Increased by Preconditioning?
Within the context of the present study and the arguments presented above, a key issue to consider is whether ischemic preconditioning actually increases NHE activity. Before the evidence for this can be critically assessed, it should be stressed that the primary activator of NHE is intracellular acidosis.³³ Activation by other stimuli, such as ₁-adrenoceptor agonists^34,35 and thrombin,¹⁹ arises from a change in the pH_i sensitivity of the exchanger, so that at a given pH_i the exchanger has greater activity after stimulation. Therefore, comparisons of NHE activity between two or more groups are informative only if activity is determined at a similar pH_i in all cases.

The primary evidence for an increased NHE activity after ischemic preconditioning is the observation by Ramasamy et al⁸ that preconditioned rat hearts exhibit an enhanced ability to recover from acute intracellular acidosis induced in the absence of ischemia. However, because that study was carried out in hearts perfused with HCO₃^--containing medium, it is impossible to ascribe the accelerated recovery from acidosis to an increase in NHE activity.³⁶ Furthermore, it should be noted that the method used to induce acute intracellular acidosis (transient exposure to NH₄Cl) resulted in greater acidosis in preconditioned (pH_i=6.54±0.02) than in control (pH_i=6.72±0.02) hearts.⁸ Because the rate of acid-equivalent extrusion via not only NHE but also Na⁺/HCO₃^- symport is inversely related to pH_i,³⁶ it is likely that the faster initial recovery from acidosis in preconditioned hearts may have arisen as a consequence of the lower starting pH_i in this group. Indeed, de Albuquerque and colleagues³⁷ recently showed that, in the presence of a similar acid load, the rate of pH_i recovery is similar in control and preconditioned rat hearts.

Ramasamy et al⁸ provided additional evidence that ischemic preconditioning increases intracellular Na⁺ accumulation during the prolonged ischemic period and that this effect is attenuated by EIPA. When taken together with the earlier reports of reduced acidosis,^2–6 an enhanced Na⁺ accumulation that is sensitive to inhibition by EIPA is supportive of an increased NHE activity in preconditioned hearts. However, the reported⁸ enhancement of Na⁺ accumulation in preconditioned hearts contrasts with earlier observations by Steenbergen et al⁵ in a similar model. Thus, it would appear that the question of whether ischemic preconditioning results in increased NHE activity cannot be resolved on the basis of the evidence currently available.

Potential Limitations of the Study
In the present study, sarcolemmal NHE activity in control versus preconditioned hearts, with and without coadministration of HOE-642, was not determined. Nevertheless, on the basis of our work with acid-loaded isolated ventricular myocytes (Fig 2) and whole hearts (Fig 4), it is highly likely that the 1 µmol/L concentration of HOE-642 used in our preconditioning studies was sufficient to inhibit sarcolemmal NHE activity. This is supported by the ability of this concentration of the drug to afford significant protection in hearts subjected to ischemia/reperfusion.

The present interpretation of the data from our preconditioning studies is contingent on NHE inhibition being the primary pharmacological action of HOE-642 and the sole mechanism of its cardioprotective effect at the 1 µmol/L concentration used. If the cardioprotective effect arose from a hitherto unidentified secondary action of the drug (that is distinct from NHE inhibition), then any diminution of the cardioprotective efficacy of ischemic preconditioning by HOE-642–induced NHE inhibition might be masked by such a secondary action. Although this possibility cannot be discounted, because HOE-642 is a new drug whose actions may not yet be comprehensively characterized, it should also be noted that there is currently no evidence to support it.

Finally, caution should be exercised in extrapolating the findings of the present study to other species or models, particularly when a different index of injury (eg, infarct size, arrhythmias) might be used to quantify the cardioprotective efficacy of HOE-642 or ischemic preconditioning.

Concluding Comments
The present study has shown that the application of a potent NHE inhibitor in combination with ischemic preconditioning does not attenuate the cardioprotective efficacy of ischemic preconditioning; on the contrary, the NHE inhibitor provides additional protection when present during the prolonged ischemic period. Assuming that NHE inhibition is the principal action of the drug at the concentration used, these observations indicate that NHE activity during either the prolonged ischemic period or the preceding brief preconditioning ischemic periods does not contribute to the cardioprotection afforded by ischemic preconditioning. Furthermore, they dispute the proposal⁸ that increased NHE activity may represent a protective mechanism of ischemic preconditioning.

	Selected Abbreviations and Acronyms

 

C-SNARF-1 = carboxy-seminaphthorhodafluor-1 LVDP = left ventricular developed pressure LVEDP = left ventricular end-diastolic pressure LVSP = left ventricular systolic pressure NHE = Na+/H+ exchanger PC = preconditioning pHi = intracellular pH

	Acknowledgments

 
This project was funded in part by grants from the British Heart Foundation, the Special Trustees for St Thomas' Hospital, and The Wellcome Trust (048021/Z/96/Z). Metin Avkiran is the holder of a British Heart Foundation (Basic Science) Senior Lectureship Award (BS/93002). Dr Hiroyuki Yokoyama is a visiting research fellow from the Nippon Medical School, Tokyo, Japan.

Received June 9, 1997; revision received July 14, 1997; accepted August 1, 1997.

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