Cardiovascular Research

Cardiovascular Research 2000 48(2):244-253; doi:10.1016/S0008-6363(00)00166-8
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Activity of the Na⁺/H⁺ exchanger is critical to reperfusion damage and preconditioning in the isolated rat heart

X-H. Xiao and D.G. Allen^*

Institute of Biomedical Research and Department of Physiology, University of Sydney, F13, Sydney, NSW 2006, Australia

^* Corresponding author. Tel.: +61-2-9351-4602; fax: +61-2-9351-2058 david.allen{at}physiol.usyd.edu.au

Received 22 March 2000; accepted 26 June 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Objective: Removal of protons from the heart during ischemia and/or reperfusion by the cardiac Na⁺/H⁺ exchanger (NHE1) leads to Na⁺ entry; this causes Ca²⁺ influx and is thought to contribute to ischemic and/or reperfusion damage. The extent to which Na⁺ enters during ischemia as opposed to reperfusion is disputed and has important implications for the therapeutic use of NHE1 inhibitors as protection against ischemic damage. Preconditioning has recently been proposed to inhibit NHE1 during reperfusion. The objective of the present study was to determine the activity of NHE1 during ischemia, reperfusion and following preconditioning. Methods: The experiments were on isolated perfused rat hearts in which left ventricular developed pressure (LVDP) and intracellular sodium and pH were measured. Results: LVDP following 30 min of ischemia recovered to 14±3% of pre-ischemic level. Application of the NHE1 inhibitor HOE 642 during ischemia and reperfusion improved recovery of LVDP to 77±9%. When HOE was applied at the moment of reperfusion the recovery of LVDP was reduced to 54±6%. To overcome possible delays in the delivery of HOE, the drug was applied at 28 min of ischemia; under these conditions recovery of LVDP (71±7%) was not significantly different to HOE throughout ischemia and reperfusion. HOE had no effect on the recovery of preconditioned hearts. NHE1 activity was assessed by the [Na⁺]_i and pH_i changes in response to brief exposure to Na⁺ lactate (NaL). In control hearts, activity of NHE1 caused a pH_i recovery of 0.034±0.007 pH units and was associated with a [Na⁺]_i rise of 7.5±0.5 mmol/l. After 5-min reperfusion following ischemia, NaL application caused a pH_i recovery (0.046±0.007) and a larger [Na⁺]_i rise (15.8±0.6 mmol/l). After 5-min reperfusion of preconditioned hearts, NaL application caused a smaller recovery pH recovery (0.013±0.002) and a smaller [Na⁺]_i rise (4.2±0.5 mmol/l). Conclusions: These findings suggest that NHE1 is activated in early reperfusion after ischemia but inhibited during early reperfusion in preconditioned hearts. Overall our results point to a critical period of activity of NHE1 in early reperfusion which is inhibited by preconditioning.

KEYWORDS Acidosis; Ischemia; Na/H exchanger; Preconditioning; Reperfusion

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This article is referred to in the Editorial by J.W.T. Fiolet (pages 185–187) in this issue.

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Protons accumulate in the heart during ischemia and are rapidly removed on reperfusion [1]. The cardiac Na⁺–H⁺ exchanger (NHE1) is one of the pathways which extrudes protons [2] and its activity will lead to a Na⁺ influx (for review see Refs. [3,4]). The resulting increase in [Na⁺]_i will increase Ca²⁺ entry on the Na⁺/Ca²⁺ exchanger and the resulting increase in [Ca²⁺]_i is thought to contribute to ischemic and/or reperfusion injury [5–8]. However there is controversy as to whether NHE1 is active during ischemia or whether its major activity is during early reperfusion [3,4]. This controversy is important because inhibitors of the NHE1 have a substantial protective effect on the heart recovering from ischemia [9–11] and, in developing protocols for their use, it is important to know the period of maximum efficacy.

The evidence that NHE1 is active during ischemia comes partly from measurements of [Na⁺]_i during ischemia. Many studies have show substantial rises in [Na⁺]_i during ischemia which can be reduced by NHE inhibitors [10,12–14]. In addition many studies of NHE1 inhibitors suggest that they are most effective when present throughout ischemia and reperfusion (for review see Refs. [3,4]). However there is also substantial evidence that NHE1 is inhibited during ischemia [5,11,15,16]. For instance pH_i shows no recovery during ischemia and NHE1 inhibitors do not change the pH_i during ischemia [10,15,16]. Our own studies show a small rise in [Na⁺]_i during ischemia which is unaffected by NHE1 inhibitors but a substantial rise in [Na⁺]_i on reperfusion which is blocked by NHE1 inhibitors [16,17]. An alternative explanation for the reduction of [Na⁺]_i during ischemia caused by NHE1 inhibitors is based on the fact that most of these inhibitors also block Na⁺ channels [18] and it has been suggested that reduction in [Na⁺]_i during ischemia arises through this action on Na⁺ channels [19]. Thus we believe the weight of evidence is that NHE1 is inhibited during ischemia and rapidly reactivates on reperfusion. An important prediction from this hypothesis is that NHE1 inhibitors should exert their main protective effect against myocardial damage during the early reperfusion period which would represent a critical window of opportunity for these drugs.

Recently we showed that preconditioning appeared to inhibit the reactivation of NHE1 which occurs on reperfusion after a long period of ischemia and suggested that this is the basis of the protective effect of preconditioning [17]. This finding was based on the interpretation of [Na⁺]_i and pH_i measurements during ischemia and reperfusion. In the present study we sought further evidence for this novel hypothesis by use of the NHE1 inhibitor HOE 642 and by direct measurement of the activity of NHE1. We chose HOE 642 as the NHE1 inhibitor because it is relatively specific for the cardiac isoform of NHE (NHE1) [20] and because it is currently in a clinical trial for protection against ischemic/reperfusion damage [4]. Activity of NHE1 was assessed by measuring the changes in [Na⁺]_i and pH_i after imposition of an acid load.

Overall our results show that NHE1 is inhibited during ischemia but rapidly reactivates causing Na⁺ influx during early reperfusion. This interpretation is strongly supported by our finding that HOE 642 is mainly active on reperfusion and that its presence during ischemia produces only a moderate improvement in the recovery of the heart during reperfusion. Our measurements of NHE1 activity strongly support our hypothesis that the activity of NHE1 during early reperfusion from a long ischemia is inhibited by preconditioning. Our results confirm that NHE1 has a key role in reperfusion damage and in the protection produced by preconditioning.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
The experiments used Langendorff-perfused rat hearts and were approved by the Animal Ethical Committee of the University of Sydney (for details see Refs. [16,17]). Female Sprague–Dawley rats were anaesthetised with pentobarbitone, the hearts were excised, and perfused with a modified Tyrode solution at 10 ml/min (12–15 ml/min/g wet weight) at 37°C. The perfusate had the following composition (mmol/l): NaCl 119, KCl 4, NaH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 25, CaCl₂ 1, glucose 11. The solutions were equilibrated with 95% O₂/5% CO₂ to give a pH 7.4. Hearts were continuously stimulated at 2 Hz after the sinoatrial node was excised and the atrio-ventricular node was crushed. The low rate of stimulation was chosen to minimise the consequences of the low O₂ content of the perfusate which lacks haemoglobin (for discussion see Ref. [16]). Isovolumic left ventricular developed pressure (LVDP) was monitored with a balloon in the left ventricle.

In some experiments [Na⁺]_i and pH_i were measured in different hearts using fluorescent indicators loaded in their membrane-permeable acetoxymethyl (AM) ester form. [Na⁺]_i was measured with SBFI and pH_i with SNARF; the resulting fluorescent signals were calibrated by standard methods. In the [Na⁺]_i experiments correction was made for the changes in autofluorescence which occur during ischemia (for details see Ref. [16]). We have previously established that the fluorescent methods measure ionic concentrations in the epicardium and myocardium to a depth of about 0.1–0.2 mm [21]. In experiments in which pH_i was measured the extracellular bath solution was replaced during ischemia with Tyrode equilibrated with 70% N₂/30% CO₂; this maintains the acidosis which develops during ischemia [17].

Activity of the NHE1 was assessed by measuring the increase in [Na⁺]_i and the rate of recovery of pH_i during an acid load caused by 5 min exposure to 20 mmol/l Na⁺ lactate (pH_o 7.4) [16,22]. We have previously shown that this manoeuvre produces a rapid acidosis of about 0.15 pH units followed by a slow recovery which is prevented by inhibitors of NHE1 [16,23]. Because both [Na⁺]_i and pH_i change during ischemia and reperfusion the timing of the acid load is critical. Under our conditions, ischemia causes a small rise in [Na⁺]_i and reperfusion causes a larger rise which reaches a peak within 3–5 min and thereafter shows a slow and variable decrease. In contrast in preconditioned hearts [Na⁺]_i decreased on reperfusion and reached a steady level after about 5 min [17]. Reperfusion of both the ischemia-only and the preconditioned heart caused a rapid recovery of pH_i which was largely complete by 5 min [2,17]. Thus in the present experiments the activity of NHE1 was assessed in control conditions and 5 min after reperfusion in both ischemia-only and reperfused hearts.

Ischemia was produced by stopping perfusion inflow to the heart while the heart was maintained at 35°C. The standard period of ischemia was 30 min; preconditioning consisted of three periods of 5 min ischemia each followed by 5 min reperfusion and then followed by the standard 30 min of ischemia. The NHE1 inhibitor, HOE 642 (cariporide; 4-isopropyl-3-methlsulphonylbenzoyl-guanidine methanesulphonate), was kindly donated by Hoechst AG (65926 Frankfurt/Main, Germany).

Recovery from ischemia was assessed by measuring LVDP, averaged over 1 min, after 30 min of reperfusion and was expressed as % of control LVDP. The ischemic contracture was the peak pressure developed during ischemia; the reperfusion contracture (RC) was the additional increase in diastolic pressure observed from the end of ischemia to the peak during reperfusion. Reduction of LVDP and increases in RC have been shown to correlate with other markers of cell damage such as histological changes, protein release, frequency of arrhythmias [24].

In some experiments HOE was applied to the heart 2 min before the end of ischemia. This was done by commencing perfusion of the HOE containing Tyrode at 28 min for 15 s. This provides 2.5 ml of solution which is more than enough to completely perfuse the vasculature of the heart. The heart then remained ischemic until 30 min when reperfusion with HOE present occurred in the normal way.

All data are expressed as mean±S.E.M. Comparison of paired results was made with Student's t-test. Comparison within or between groups was made by one way analysis of variance (ANOVA) using the Student–Newman–Keuls correction for multiple comparisons. Statistical significance was taken as P<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
3.1 Effects of HOE 642 on NHE1 activity
Preliminary experiments were undertaken to determine the appropriate concentration of HOE 642 and its timecourse of action. Application of extracellular Na⁺ lactate (NaL; 20 mmol/l) causes an acidosis which develops in 1–2 min as uncharged lactic acid enters the cell and releases a proton [16,23]; this acidosis causes the rapid reduction of developed pressure (Fig. 1A). Subsequently the NHE1 extrudes some of the protons leading to a slower pH_i recovery (Fig. 5), a rise in [Na⁺]_i (Fig. 1A) and a recovery of developed pressure (Fig. 1A). In the presence of inhibitors of NHE1 all three parameters are reduced. For instance in 16 hearts the rise in [Na⁺]_i on NaL application was 7.5±0.5 mmol/l but in the presence of 10 µmol/l HOE the rise in [Na⁺]_i was reduced to 0.9±0.1 mmol/l. The recovery of developed pressure is the simplest parameter to measure and Fig. 1B shows a plot of the inhibition of this recovery of developed pressure as a function of HOE concentration. The fitted line shows a least-squares fit of a single site binding model and gives an apparent binding constant of 0.9±0.2 µmol/l; 10 µmol/l HOE gives a near maximal effect and was used subsequently. In most experiments HOE 642 was applied simultaneously with the NaL () as shown in Fig. 1A; thus the effects of HOE will only be optimal if it binds rapidly to NHE1. In a smaller number of experiments HOE was applied 5 min before NaL () but these results were not significantly different. Thus it appears that HOE 642 binds and inhibits NHE1 relatively rapidly.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Inhibition by HOE 642 of the effects of Na+ lactate on the [Na+]i and developed pressure of the rat heart. (A) [Na+]i (above) and left ventricular developed pressure (below) from an isolated rat heart. At the times indicated 20 mmol/l Na+ lactate (NaL) was added to the perfusate for 5 min. In the central application 10 µmol/l HOE 642 was added simultaneously. (B) Collected results from nine hearts showing the degree of inhibition of the recovery of LVDP during a 5-min application of Na+ lactate in the presence of various concentrations of HOE 642. () HOE 642 applied simultaneously with the NaL (n = 5); () HOE applied 5 min before NaL application (n = 4). There were no differences between the inhibition of LVDP recovery as a function of the time of HOE 642 (comparison of () with () points, unpaired t-test).

 

View larger version (7K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 pHi in response to Na+ lactate application in various conditions. (A) Control heart. Dashed lines indicate the resting pHi, the peak of the acidosis caused by Na+ lactate application and the pHi at the end of the exposure. These pHi values were used to calculate the initial acidosis (I) and the recovery caused by activity of NHE1 (R). (B) Application of NaL after 5 min of reperfusion of an ischemia-only heart. (C) Application of NaL after 5 min of reperfusion of a preconditioned heart. Note the pHi recovery during lactate application was nearly abolished by preconditioning.

 
3.2 Developed pressure and contracture following ischemia
Fig. 2 shows representative examples of left ventricular developed pressure (LVDP) in a heart subjected to 30 min ischemia (A) and the hearts treated with HOE 642 (B–D). The relevant data are summarised in Table 1. Note that following ischemia-only (Fig. 2A) the reperfusion contracture (RC) was large and there was little recovery of LVDP. In eight hearts the RC was 62±7 mmHg while the recovery of LVDP was 14±3%. Fig. 2B illustrates the effect of HOE 642 applied only during reperfusion. The RC is smaller than in the ischemia-only record and LVDP shows a moderate recovery. In 11 hearts the RC was smaller (26±6 mmHg) and the recovery of LVDP was greater (54±6%) than in the ischemia-only hearts. Fig. 2D illustrates the effect of HOE applied 5 min before onset of ischemia and throughout reperfusion. Note that the RC was smaller than in the ischemia-only record and that DP shows a better recovery. In 11 hearts the RC was much smaller (13±3 mmHg) and the recovery of LVDP was much greater (77±9%) than in the control ischemic hearts. There was also a significant difference between the results when HOE applied throughout ischemia and reperfusion was compared to HOE applied just during reperfusion (ANOVA; P<0.05).

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effect of HOE 642 on mechanical performance of isolated rat hearts during and after ischemia. Records show left ventricular developed pressure (LVDP). (A) 30 min of ischemia. Note large reperfusion contracture and absence of recovery of developed pressure. (B) Na+–H+ exchanger inhibitor, HOE 642 (10 µmol/l), applied at the onset of the reperfusion. Note smaller reperfusion contracture and substantial recovery of developed pressure. (C) HOE 642 (10 µmol/l) perfused for 15 s 2 min before the start of reperfusion. The vertical dashed line shows the start of the main period of reperfusion. (D) HOE 642 (10 µmol/l) applied 5 min before the start of ischemia and present throughout the rest of the experiment. Note smaller reperfusion contracture and substantial recovery of developed pressure.

 

View this table: [in this window] [in a new window]   Table 1 Mechanical performance during ischemia and reperfusion under various conditionsa

 
One possible explanations for the greater protection provided by HOE when applied throughout ischemia and reperfusion is that when applied at the moment of reperfusion it takes some time for the drug to perfuse the heart, cross the capillary wall and bind and inhibit the exchanger [11]. To test this possibility we reperfused the heart for 15 s starting at 28 min so that the heart had an additional 2-min exposure to HOE before normal reperfusion commenced at 30 min. As shown in Fig. 2C and Table 1, this protocol produced a marginally but not significantly better protection (LVDP 71±7%; RC 20±4 mmHg) than HOE applied at the moment of reperfusion. Furthermore the LDVP and RC in this situation were not significantly different to HOE applied throughout ischaemia and reperfusion. We also performed three control experiments in which control hearts were reperfused with standard perfusate for 15 s at 28 min followed by reperfusion in the normal way at 30 min (not shown). The recovery of LVDP and RC in these hearts was not significantly different to that shown in Fig. 2A. Thus these data suggest that the main value of HOE is in the early reperfusion period and that the presence of HOE during ischaemia provides no additional protection.

Our previous study suggested that in preconditioned hearts NHE1 was inhibited during early reperfusion following the long ischemia and contributed to the protection afforded by preconditioning [17]. We therefore investigated whether the application of an NHE1 inhibitor had any effect on preconditioned hearts. We first confirmed that preconditioning produced a substantial improvement in recovery when compared to ischemia-only (compare Fig. 2A and Fig. 3A). Note that in Fig. 3A the RC is much smaller than in the ischemia-only record and that LVDP shows a good recovery. In 7 hearts the RC was much smaller (24±7 mmHg) and the recovery was much greater (72±8%) when compared to ischemia-only.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of HOE 642 on LVDP of isolated rat hearts during and after preconditioned ischemia. Records show left ventricular developed pressure (LVDP). (A) 30 min of ischemia preceded by 3x5 min periods of preconditioning ischemia. Note reduction in reperfusion contracture and substantial recovery of developed pressure. (B) Na+–H+ exchanger inhibitor, HOE 642 (10 µmol/l) applied 5 min before the start of ischemia and throughout the rest of the experiment. Note a similar reduction in reperfusion contracture and substantial recovery of developed pressure.

 
Fig. 3B shows that the effect of HOE 642 applied 5 min before onset of 30 min ischemia in preconditioned hearts. In seven hearts, the RC was much smaller (16±4 mmHg) and the recovery of LVDP was much greater (73±9%) when compared to ischemia-only. However, there were no significant difference between either the recovery of DP or the size of RC when preconditioned hearts were compared with and without HOE 642.

We also measured the magnitude of the ischemic contracture. The magnitude of the ischaemic contracture is quite variable in these experiments. Some ischaemia only experiments show no ischaemic contracture in contrast to the examples in Fig. 2. Some preconditioned ischaemias show a substantial ischaemic contracture in contrast to the examples in Fig. 3. Overall the magnitude of the ischaemic contracture was not significantly affected by any of the above interventions.

3.3 Activity of NHE1 after ischemia and preconditioning; [Na⁺]_i measurements
In our previous study [17] we proposed that the NHE1 rapidly reactivated on reperfusion following ischemia-only but remained inhibited on reperfusion of the preconditioned heart. This hypothesis was based on the interpretation of [Na⁺]_i and pH_i measurements in the presence and absence of NHE1 inhibitors. A more direct approach would be to measure the activity of the NHE1 by imposing acid loads on the heart and measuring the resulting consequences of NHE1 activity.

Fig. 4A shows the effect of application of NaL (20 mmol/l Na⁺ lactate) for 5 min on [Na⁺]_i under control conditions. Note that the increases in [Na⁺]_i are about 10 mmol/l and are repeatable on multiple exposures. In subsequent panels ischemia-only and preconditioned ischemia are shown and the data are collated in Table 2.

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Records of [Na+]i during exposure to Na+ lactate at various stages of ischemia-only and preconditioned hearts. (A) Repeated application of NaL under control conditions. Note reproducibility. (B) NaL applied during control and 5 min after reperfusion after 30 min of ischemia. Note rise of [Na+]i on reperfusion and subsequent rise when NaL was applied. (C) NaL applied during control and 5 min after reperfusion of a preconditioned heart. Note absence of [Na+]i rise on reperfusion and reduced [Na+]i rise when NaL was applied after 5 min of reperfusion.

 

View this table: [in this window] [in a new window]   Table 2 Effect of 5 min exposure to 20 mmol/l sodium lactate on [Na+]i and pHi under control conditions and after 5 min of reperfusion on ischemia-only hearts and in preconditioned hearts

 
Fig. 4B shows a control NaL application before ischemia caused a [Na⁺]_i rise of about 10 mmol/l. 30 min of ischemia induced a relatively small [Na⁺]_i rise of about 3 mmol/l but reperfusion caused a substantial additional increase of [Na⁺]_i of about 15 mmol/l similar to our previous study [17]. After 5 min of reperfusion [Na⁺]_i was relatively stable and NaL was applied. Note that this caused a further increase in [Na⁺]_i of about 20 mmol/l which is strikingly larger than that observed in response to the the control NaL application. In four hearts the [Na⁺]_i rise caused by NaL exposure under control conditions was 8.2±0.6 mmol/l but the [Na⁺]_i rise was much bigger (15.8±0.6 mmol/l) when NaL was applied during reperfusion of ischemia-only hearts.

Fig. 4C shows the equivalent results from a preconditioned heart. The control NaL application produced the usual rise in [Na⁺]_i. The short preconditioning ischemias did not change [Na⁺]_i concentration very much and the 30 min of ischemia caused a small [Na⁺]_i rise comparable to the ischemia-only heart. However, reperfusion caused a decline in [Na⁺]_i as we have previously reported [17]. After 5 min of reperfusion NaL was applied and it is clear that the rise of [Na⁺]_i is smaller than the control and much smaller than that observed when NaL was applied after ischemia-only. In four hearts the [Na⁺]_i rise caused by NaL application was 8.4±0.6 mmol/l under control conditions but [Na⁺]_i rise was much smaller (4.2±0.5 mmol/l) when the NaL was applied after 5 min reperfusion of the preconditioned heart.

Taken at face value, these results suggest that the activity of NHE1 is enhanced after ischemia but reduced following a preconditioned ischemia. However interpretation of these changes in [Na⁺]_i is dependent on the amount of lactic acid entering the myocytes and the size of the rise of [Na⁺]_i will be dependent on both the activity of the NHE1 and on the activity of the Na⁺ pump and the Na⁺/Ca²⁺ exchanger. These issues can be resolved by examining the pH_i changes since the initial acidosis is proportional to the lactic acid entry while the rate of pH_i recovery is a function of the activity of NHE1 which is insensitive to the activity of the Na⁺ pump or the Na⁺/Ca²⁺ exchanger.

3.4 Activity of NHE1 after ischemia and preconditioning; pH_i measurements
Fig. 5 illustrates experiments designed to determine the magnitude of lactic acid entry into the myocytes and the activity of NHE1 by measurement of pH_i following NaL application. Previous studies have shown that pH_i is largely recovered within 5 min of reperfusion following ischemia-only and preconditioned ischemia [2,17]. Thus application of NaL after 5 min of reperfusion should be largely uncomplicated by residual lactic acid and/or protons remaining in the cells. Fig. 5 shows examples of the pH_i records obtained from control, post-ischemia-only and post-preconditioned ischemia. The resulting data is summarised in Table 2.

Fig. 5A illustrates the pH_i result obtained under control conditions; there is rapid, initial fall of pH_i complete in 2 min (marked I in Fig. 5A), subsequently pH_i shows a slower recovery (marked R in Fig. 5A) which is caused by activity of NHE1 [16,17]. In ten experiments, the resting pH_i was 7.45±0.03; the initial decline in pH_i was 0.110±0.018 pH units over 2 min and the slow recovery was 0.034±0.007 pH units at the end of 5 min. Fig. 5B shows NaL application after 5 min reperfusion following ischemia. In five hearts pH_i after 5 min reperfusion was 7.31±0.02 indicating that pH_i recovery after the preceding ischemia was almost complete. The initial decline of pH_i was significantly smaller than control at 0.070±0.008 pH units and the slow recovery was 0.046±0.007 pH units was larger than control though this difference was not significant. Fig. 5C illustrates the pH_i changes when NaL was applied 5 min after reperfusion from a preconditioned ischemia. In five hearts pH_i after 5 min recovery was 7.26±0.04. The initial pH_i decline was not significantly different to control at 0.092±0.007 pH units decline while the slow recovery was 0.013±0.002 pH units. This recovery was significantly smaller than control or the ischemia-only. Both the activity of NHE1 and the pH buffering are sensitive to pH_i and in this context it is important to note that the pH_i values were not significantly different after 5 min recovery in the ischemia-only compared to the preconditioned hearts. Thus the pH_i changes following NaL application were performed at comparable values of pH_i.

3.5 Activity of the NHE1 following preconditioning ischemias
To test whether the three short preconditioning ischemias affected the activity of NHE1 we measured the recovery of LVDP as a percentage of the initial fall in LVDP during a 5-min exposure to Na⁺ lactate (see Fig. 1). In a control NaL exposure this recovery was 73±8% (n = 5); following three short ischemias (see Fig. 3) a test exposure to NaL produced a recovery of 78±11% (n = 5). Thus there was no evidence that the preconditioning ischemias alone affected the activity of NHE1.

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
This study seeks to understand the mechanism of myocardial damage after moderate periods of ischemia and the improvement in recovery produced by preconditioning. In our previous paper we proposed that preconditioning prevents the reactivation of NHE1 which normally occurs after a long ischemia and that this was the mechanism of the improved recovery [17]. In the present paper we provide further evidence for this novel hypothesis by showing that the NHE1 inhibitor HOE 642 has no effect in the preconditioned heart and that the activity of the NHE1 exchanger appears to be inhibited on reperfusion of the preconditioned heart. Secondly we provide further evidence that NHE1 is inactive during ischemia but rapidly reactivates on reperfusion. Although this hypothesis has considerably support [5,11,15,16] recent reviews conclude that the NHE is active during ischemia [3,4] so that the issue remains controversial.

4.1 Mechanism of damage during reperfusion
Shen and Jennings [25] first noted the large increase in total calcium in ischemic hearts which occurred on reperfusion and showed that the increase in Ca²⁺ uptake on reperfusion was related to the duration of ischemia and the degree of damage. Lazdunski et al. [5] provided a theoretical framework for these observations suggesting that NHE1 was inhibited by extracellular acidosis during ischemia but that the exchanger reactivated during reperfusion as the extracellular protons were flushed away. Then the coupled activity of the NHE1 and the Na⁺/Ca²⁺ exchanger led to Ca²⁺ entry which triggered the damage.

While there is broad agreement on the general features of the coupled ion-exchanger theory outlined above, there is dispute about the extent to which Na⁺ entry occurs during ischemia as opposed to reperfusion. Recent reviews by Murphy [26] and Karmazyn [4] discuss this evidence in detail and support the view that Na⁺ entry during ischemia via NHE1 is an important component. In contrast a number of studies have led to the view that NHE1 is inhibited during ischemia and cannot therefore be a source of Na⁺ influx during ischemia [5,11,15,16]. (i) Most studies agree that pH_i during ischemia is unaffected by NHE1 inhibitors which is difficult to explain if the NHE1 is active (for review see Refs. [3,4]). (ii). Under the conditions of our experiments there was only a small increase in [Na⁺]_i during ischemia and this was unaffected by an NHE inhibitor. In contrast there was a pronounced increase in [Na⁺]_i during reperfusion which was largely inhibited by NHE inhibitors [16,17]. Thus there are two important experimental difference between our results and those of others. The first is that we find a relatively small increase in [Na⁺]_i during ischemia whereas many other observe large rises [10,14,24,27]. We believe that the fact that we stimulate our hearts at 2 Hz whereas most others use the intrinsic heart rate (5 Hz) is the cause of this difference (for more detailed discussion see Refs. [16,17]). The second experimental difference is that we find that inhibitors of NHE1 have no effect on the rise of [Na⁺]_i during ischemia. As noted in the Introduction the resolution to this discrepancy may be that many NHE inhibitors are relatively non-specific and block Na⁺ channels as well as NHE [18,19].

The results in the present study provide further support for our interpretation that NHE1 is inhibited during ischemia but reactivates on reperfusion.

1. Although the NHE1 inhibitor HOE 642 was less potent when applied at the moment of reperfusion as compared to being present throughout ischemia and reperfusion, we believe that this is because [Na⁺]_i rises rapidly on reperfusion (peak [Na⁺]_i occurs within 5 min [16,17]) so it is necessary to have the inhibitor present for 1–2 min before reperfusion in order to completely inhibit Na⁺ influx. Our results are similar to earlier studies by Maddaford and Pierce [11] in which inhibitor was applied before the end of ischemia and dramatically improved the effectiveness of myocardial protection. However our results extend their study several ways. First, we observe effective protection in the Langendorff perfused heart which they did not. Second, we found protection in a HCO₃/CO₂-buffered solutions, which is obviously closer to the clinical situation, while they did not.

2. The measurements of NHE1 activity by application of Na⁺ lactate strongly suggest that the NHE1 shows enhanced activity during early reperfusion from ischemia. The [Na⁺]_i measurement show a significantly enhanced Na⁺ influx associated with NaL application. This occurred in spite of a moderate reduction in the initial acidosis caused by NaL application. This reduction could indicate that the heart still contained some lactate and/or protons remaining from the ischemic period which would tend to reduce the inward diffusion of lactic acid; alternatively it might imply that capillary flow was inhomogenous and that some underperfused myocytes were not able to take up lactic acid rapidly. Either way, the reduced lactic acid uptake implies that the increased rise in [Na⁺]_i is even more significant since it appears to occur despite a reduced lactic acid entry. An alternative interpretation of the increase [Na⁺]_i is that it reflects an impaired Na⁺ pump activity. Two arguments against this interpretation are that the fall of [Na⁺]_i seems to be rapid implying that the Na⁺ pump is fully active and, more important, the recovery of pH_i following lactate application is not significantly different to control and appears, if anything, to be somewhat larger. These arguments all point to the conclusion that the activity of NHE1 5 min after reperfusion is equivalent or larger than the activity under control conditions.

4.2 Mechanism of preconditioning
Murry et al. [28] discovered the phenomenon of preconditioning and it has attracted intense interest as a possible endogenous mechanism of protection against ischemic damage. A large number of studies suggest that the preconditioning ischemias release a trigger substance which binds to a receptor and leads to PKC activation (for review see Ref. [29]). Presumably this leads to phosphorylation of a key protein whose changed properties lead to improved recovery from a subsequent long ischemia [30]. We recently proposed that one end result of this sequence was inhibition of NHE1 during reperfusion of a preconditioned ischemia [17]. Our present experiments support this novel hypothesis in two ways. (i) Preconditioning leads to a pronounced improvement in recovery from ischemia. However, the presence of a NHE1 inhibitor throughout ischemia and reperfusion led to no further improvement. This finding is qualitatively similar to an earlier study [31] though by manipulating the conditions they could observe additive effects of preconditioning and NHE blockade. Both our study and the earlier study are consistent with the concept that preconditioning and NHE blockade share a common mechanism. (ii) Our measurements of NHE1 activity show convincingly that after 5 min reperfusion of the preconditioned heart the NHE1 is significantly inhibited. This shown by both the reduced [Na⁺]_i rise which is accompanied by a reduction in the rate of pH_i recovery from the acid load. Thus the evidence that NHE1 is inhibited in early reperfusion of the preconditioned heart is now very strong. However, as we discussed recently there is no clear cellular mechanism which explains this reduction in activity [17].

4.3 Implications for the use of NHE1 inhibitors to reduce ischemic damage
The experimental support for the protective effect of NHE1 inhibitors on ischemic damage is now very strong and several clinical trials are currently underway [4]. It remains controversial whether the main effect of these drugs is during ischemia or during reperfusion or both (for review see Refs. [3,4]). Our results contribute to this debate in two ways. First, our results suggest that there is a critical window of opportunity with these drugs which is the early minutes of reperfusion when the major Na⁺ influx occurs. Second, NHE1 inhibitors are ineffective when applied to a preconditioned heart. This is consistent with our hypothesis that NHE is already inhibited during ischemia and early reperfusion of the preconditioned heart so that the addition of a NHE1 inhibitor brings no further benefit.

	5 Conclusions

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Our results lead to the following conclusions about the NHE1 which differ in important respects from the opinions expressed in recent reviews [3,4]. (i) The NHE1 is inhibited during ischemia by a mechanism which is not related to extracellular acidosis. (ii) The NHE1 is rapidly activated, possibly to above control levels, in the first 5 min of reperfusion. (iii) Paradoxically, in the preconditioned heart the activity of NHE1 appears to be inhibited during the first 5 min of ischemia and this is the mechanism of protection. The cellular mechanisms which underlie these important observations remain unknown.

Time for primary review 27 days.

	Acknowledgements

 
Supported by a grant from the Australian National Heart Foundation.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 

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