Cardiovascular Research

Cardiovascular Research 2003 57(4):934-941; doi:10.1016/S0008-6363(02)00836-2
© 2003 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Role of the cardiac Na⁺/H⁺ exchanger during ischemia and reperfusion

David G Allen^* and Xiao-Hui Xiao

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

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

Received 16 July 2002; accepted 2 December 2002

	Abstract

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 
The coupled exchanger theory describes one of the central mechanisms of damage in the ischemic heart. The theory proposes that anaerobic glycolysis produces lactate and protons and that the protons can leave the cardiac cell on the cardiac Na⁺/H⁺ exchanger (NHE1). The subsequent rise in [Na⁺]_i stimulates the cardiac Na⁺/Ca²⁺ exchanger (NCX) and results in an increase in [Ca²⁺]_i which promotes myocardial cell damage. Although the general features of this theory are widely accepted, there is dispute about some aspects, specifically whether the NHE1 remains active during ischemia or not. We review the evidence on this issue and conclude that NHE1 is substantially inhibited during ischemia. This issue is central to the design of a clinical trial of NHE1 inhibitors in the treatment of human cardiac ischemia and the existing clinical trials are considered in this light.

KEYWORDS Ischemia; Na/Ca-exchanger; Na/H-exchanger; Reperfusion

	1. Introduction

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 
Patients with early myocardial infarcts are currently treated with either thrombolytic therapy or primary angioplasty in order to reperfuse the ischemic myocardium. Both these treatments have been shown to reduce the size of the infarct and to improve patient recovery. A limitation of these treatments is that the reperfusion of the ischemic myocardium needs to occur early after the infarct to achieve optimal recovery [1]. Inevitably some patients do not reach hospital in time for these therapies to be most effective. Furthermore, hearts are intentionally made ischemic during cardiac surgery and during the preparation and transport of donor heart for transplantation and in both cases it would be advantageous if the period of safe ischemia could be extended. Consequently the intracellular mechanisms associated with ischemic and reperfusion damage are of great interest as they may lead to strategies for improving cardiac recovery after moderate periods of ischemia.

Studies on isolated animal hearts have clearly shown that the damage associated with ischemia/reperfusion is partly reversible. The discovery of preconditioning [2] in which several short periods of ischemia improve the recovery of the heart after a long period of ischemia, has triggered intense interest in the mechanisms of recovery. It is also known that inhibitors of the cardiac Na⁺/H⁺ exchanger (NHE1) exert substantial protection when present throughout ischemia and reperfusion [3]. In this short review, we focus on the possible role of NHE1 in ischemic and reperfusion damage.

	2. The coupled exchanger theory

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 
The interactions amongst H⁺, Na⁺ and Ca²⁺ during ischemia/reperfusion of the heart can be understood in the context of the coupled exchanger theory [4–6] (Fig. 1). During ischemia, oxidative phosphorylation rapidly ceases and anaerobic glycolysis is accelerated. The net products of anaerobic glycolysis are lactate and protons and result in an acidosis of around 1 pH unit within 10 min [7]. The intracellular acidosis stimulates acid extrusion mechanisms, including NHE1 and the Na⁺ HCO₃^– cotransporter, that lead to the entry of one Na⁺ ion for each H⁺ removed. The resulting rise in [Na⁺]_i will be minimised by activation of the Na⁺ pump though there is good evidence that the Na⁺ pump is inhibited late in ischemia [8]. Of course other sources of Na⁺ entry may also contribute to the rise in [Na⁺]_i during ischemia and of particular interest is the persistent Na⁺ current (I_Na,P). A rise in [Na⁺]_i, from whatever source, will affect the Na⁺/Ca²⁺ exchanger (NCX) decreasing the driving force for Ca²⁺ efflux and/or increasing the driving force for Ca²⁺ entry, both of which will tend to increase [Ca²⁺]_i. Accumulation of Ca²⁺ inside cells is a common feature of injury and promotes cell damage by a range of mechanisms including activating proteases and entering mitochondria and disrupting their function. Many previous reviews have summarised the evidence that NHE1 and NCX are involved in ischemic and/or reperfusion damage [9–12]. While there is general agreement that the coupled exchanger theory has a central role in many aspects of ischemic and reperfusion damage, there is dispute about certain aspects of this theory [13,14]. In this brief review, we focus on one area of disagreement which is whether or not NHE1 remains active during ischemia.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Schematic diagram illustrating some of the pathways involved in [Na+]i and pHi regulation in the heart during ischemia/reperfusion. During ischemia, anaerobic glycolysis is stimulated leading to the net breakdown of glycogen to lactate and protons. Protons which leave the myocardial cell on NHE1 contribute to Na+ loading. Increased [Na+]i causes Na+ to leave and Ca2+ to enter the myocardial cell on NCX. The persistent Na+ current (INa,P) represents an addition pathway for Na+ entry. The Na+ pump will normally remove excess Na+.

 

	3. Properties of NHE1

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 
The Na⁺/H⁺ exchangers are important pathways for the regulation of intracellular pH in all tissues. NHE1 is the predominant isoform in cardiac tissue and is one of the most important pathways by which the cardiac cell extrudes protons following ischemia [15,16]. NHE1 uses the inward chemical gradient of Na⁺ ions to drive the electroneutral extrusion of protons. The exchange rate is partly dictated by pH_i which exerts its effects through an allosteric intracellular binding site and results in a steep activation curve with essentially zero activity at around pH_i 7.2 or more alkaline and maximal activation at pH_i of 6.5 or more acid [17]. NHEs are also regulated by a broad range of growth hormones and other circulating factors [18]. The intracellular pathways involved modify the cytoplasmic tail of the exchanger which has a number of potential phosphorylation sites and a Ca/calmodulin binding site [15].

The earliest blocker of NHE1 was amiloride which has a half maximal inhibitory concentration (IC₅₀) of around 80 µM though dependent on the measurement conditions [19]. The most potent effect of amiloride is blockade of the epithelial Na⁺ channels (IC₅₀ 0.3 µM) and this effect underlies its use as a diuretic but amiloride has a wide range of additional actions including blocking sodium channels (IC₅₀ 600 µM) and Ca²⁺ channels (IC₅₀ 90 µM) and inhibiting NCX (IC₅₀ 1000 µM) and the Na⁺ pump (IC₅₀3000 µM). Extensive structure–function studies of amiloride led to a second generation of ‘high-affinity amiloride analogues’ including ethylisopropyl amiloride (EIPA) and methylisobutyl amiloride (MIA) with IC₅₀ for NHEs of around 0.5 µM and substantially reduced side-effects. The most recent group of NHE1 inhibitors (third generation) include cariporide (HOE 642), eniporide and zoniporide which are substantially more potent with IC₅₀s of, respectively, 5, 0.7 and 0.25 nM [20]. These compounds also show strong selectivity between the various NHE isoforms; for instance cariporide has an IC₅₀ of 3 µM for NHE2 and 1 mM for NHE3 [21]. However information about their specificity is limited. Cariporide, which is the most widely studied in animals and man, was originally reported as having no detectable effect on NCX and having a small, ‘biologically-irrelevant’ effect on the veratridine-induced depolarization of ventricular cells (IC₅₀100 µM) [21]. This is a significant issue because veratridine inhibits the inactivation of the Na⁺ current and induces a persistent (or long-lasting or non-inactivating) Na⁺ current. There is evidence that this is an important route for Na⁺ influx into the heart during ischemia (discussed below). Subsequently this issue was reinvestigated using voltage clamp protocols and it was found that each of the compounds tested (HOE 694, EIPA and cariporide) reduced the persistent Na⁺ current induced by either veratridine or lysophosphatidyl choline but had no effect on the peak conventional Na⁺ current [22]. Cariporide had the smallest effect of these three compounds but, nevertheless, at 1 µM, cariporide reduced Na⁺ entry by I_Na,P by around 20–25%.

	4. Activity of NHE1 during ischemia

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 
The fundamental role of the NHE1 is to remove protons from the myocardial cells when these are present in excess. It is natural to expect, therefore, that the NHE1 will operate during ischemia when the heart exhibits a very large intracellular acidosis. Nevertheless, a range of evidence suggests that NHE1 is inhibited during ischemia while other experimental data point to activity of NHE1 during ischemia. We first review the evidence for both points of view and then try to suggest possible interpretations of this apparently contradictory situation.

Studies of the accumulation of Na⁺ and Ca²⁺ in the heart during and after ischemia were initiated by the classic work of Shen and Jennings [23]. They measured the total Na⁺ and Ca²⁺ in hearts during ischemia and reperfusion and the key result was that neither Na⁺ nor Ca²⁺ increased during ischemia but both increased dramatically on reperfusion. Lazdunski et al. proposed the coupled exchanger theory to explain the increases in total Na⁺ and Ca²⁺ on reperfusion [4]. They suggested that NHE1 was initially active during ischemia but as protons were extruded from the myocardial cells they accumulated in the extracellular space and eventually inhibited further activity of NHE1. This could happen either through the extracellular inhibitory site on NHE1 [4,24] or by the development of a sufficiently large extracellular acidosis to prevent operation of the exchanger (if the chemical gradient of protons becomes equal to that of Na⁺ then exchange activity will cease). If the exchanger continued to operate during ischemia and protons simply accumulated in the extracellular space, then NHE1 activity must eventually cease but the question is whether some other inhibitory mechanisms acts to reduce NHE1 activity before this mechanism operates. Studies of extracellular pH during ischemia show pH_o of about 6.5 [25] and it is generally agreed that this degree of extracellular acidosis will substantially inhibit the flux on the exchanger at any given pH_i (5-fold reduction from pH_o 7.4 to 6.4 [25]). However the situation in ischemia is more complex with a pH_i of around 6.0, which markedly stimulates the exchanger, and a pH_o of around 6.5 which is inhibitory. Experimentally, the combination of these two changes causes only a moderate (<2-fold) inhibition of NHE1 [24,26,27]. These studies lead to the conclusion that other pathways may contribute to NHE1 inhibition during ischemia.

	5. Measurements of [Na+]i and pHi

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 
One approach to assessing the activity of NHE1 during ischemia comes from measurements of pH_i and [Na⁺]_i in the heart during and after ischemia. Work from our laboratory using standard acid loading techniques, which cause an intracellular acidosis of about 0.15 pH units, has shown that under control conditions, this acidosis recovers through activity of NHE1 over 5–10 min. The operation of NHE1 also causes a rise of [Na⁺]_i of around 2–4 mM over 10 min [25] though no doubt this would eventually be reversed by increased activity of the Na⁺ pump. However in ischemia, an acidosis of 1 pH unit causes no significant change in [Na⁺]_i over a 10-min period. The simplest explanation is that NHE1 is inhibited during ischemia so that the large acidosis fails to activate NHE1 or to cause the expected rise in [Na⁺]_i. However work from many other laboratories shows much larger rises of [Na⁺]_i which would be more consistent with continuous activity of NHE1 during ischemia [5,28,29].

An alternative approach is to measure [Na⁺]_i and pH_i during ischemia before and after application of NHE1 inhibitors. The results with pH_i measurements show an acidosis of approaching 1 pH unit after 10 or so minutes of ischemia and the magnitude of this acidosis is generally unaffected by inhibitors of NHE1 [25,30,31]. However the rate of recovery of pH_i after ischemia is slowed in the presence of NHE1 inhibitors and this is consistent with NHE1 having a significant role in recovery from acidosis during reperfusion [16]. The simplest interpretation of these data is that NHE1 is not contributing to proton extrusion during ischemia and, since the intracellular acidosis would be expected to maximally activate NHE1, it seems that NHE1 is inhibited. Conversely, it is widely accepted that during reperfusion the activity of NHE1 rapidly reactivates and it contributes to the recovery of pH_i during early reperfusion [16,31].

Measurements of [Na⁺]_i during ischemia have given quite variable results. Our own work shows only a small (5 mM) rise in [Na⁺]_i during 30 min of ischemia and this rise is unaffected by NHE1 inhibition [32]. Again this result suggests inhibition of NHE1 during ischemia. However there are many reports in the literature that [Na⁺]_i rises very substantially during ischemia (sodium NMR signal rise between 200 and 400% [12] suggesting increases of [Na⁺]_i of about 15–30 mM) and that, in the presence of Na⁺/H⁺ exchange inhibitors, the rise of [Na⁺]_i is substantially reduced [28,29,33]. Generally this observation is interpreted as evidence that NHE1 is active during ischemia. In the original report [28] amiloride was used as the NHE1 inhibitor; however, as noted above, amiloride is a much more potent inhibitor of Na⁺ channels than NHE1; thus if Na⁺ entry is via a Na⁺ channel which is blocked by amiloride then the conclusion that NHE1 is active during ischemia could be incorrect. Subsequent studies generally used second or third generation inhibitors, but as noted above, all those tested show significant inhibition of the persistent Na⁺ current. Thus the question of the mechanism of the reduction of [Na⁺]_i caused by NHE1 inhibitors during ischemia remains unresolved and will not be finally resolved until there is a NHE1 inhibitor whose Na⁺ channel blocking effects have been comprehensively explored and shown to be negligible.

A substantial body of evidence indicates that some of the Na⁺ entry during ischemia and metabolic inhibition occurs via channels. A number of studies have found that Na⁺ channel blockers are very effective at reducing [Na⁺]_i during ischemia or metabolic inhibition [6,32,34,35]. During ischemia and metabolic inhibition there is depolarization of the membrane potential so that conventional Na⁺ channels are inactivated. In contrast, a persistent form of the Na⁺ channel is activated by metabolic blockade, either directly by hypoxia or by some consequence of hypoxia such as lysophosphatidyl choline accumulation [36,37]. The following points support the idea that Na⁺ influx during ischemia occurs by persistent Na⁺ channels. (i) R 56865, which has little effect on I_Na but is an effective blocker of I_Na,P, is effective at reducing [Na⁺]_i during metabolic blockade [6]. (ii) I_Na,P is more sensitive than I_Na to tetrodotoxin [38] and a low concentration of tetrodotoxin which did not inhibit I_Na nevertheless reduced the rise of [Na⁺]_i during ischemia [32]. Given that there is good evidence that I_Na,P is one source of Na⁺ entry during ischemia, it is of particular significance that this channel is blocked by many NHE1 inhibitors and means that the interpretation of the decline of [Na⁺]_i with NHE1 inhibitors is equivocal.

The role of Na⁺ HCO₃^– cotransporter in Na⁺ loading and pH_i regulation during ischemia remains controversial. Experiments on isolated rat myocytes in which ischemia was simulated by anoxia and a pH_o 6.4 show a moderate intracellular acidosis under conditions in which either or both of NHE1 and the Na/HCO₃^– cotransporter could operate [39]. Only if both proton extruders were blocked did a much larger intracellular acidosis develop. These experiments suggest that there must be some residual NHE1 activity under these conditions. Surprisingly, inhibiting NHE1 under these conditions did not lead to a reduction in the raised [Na⁺]_i seen under these conditions suggesting that the residual NHE1 activity may be rather small. In an earlier study from the same laboratory [40] it was found that cariporide exerted a protective effect at reoxygenation but only when the Na⁺ HCO₃^– cotransporter was inactivated by excluding HCO₃^– from the perfusate. Although cariporide produced a small increase in acidosis during metabolic inhibition this was not accompanied by the expected fall in Na⁺. These authors conclude that the protective effect of cariporide may act through inhibiting rigor development rather than ionic consequences. This provocative hypothesis is weakened by the absence of a detailed explanation of how cariporide may act and by the many differences between metabolic inhibition in isolated cells and ischemia in the intact heart.

	6. Timing of the action of NHE1 inhibitors

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 
If NHE1 is active during ischemia, one would expect NHE1 inhibitors to exert their protective effects during ischemia; conversely, if NHE1 is inhibited during ischemia but active during reperfusion, NHE1 inhibitors should be most effective during reperfusion. This philosophy has inspired a number of experimental studies but unfortunately the results are very variable. Many studies have compared the application of an NHE1 inhibitor applied before the start of ischemia and continued during reperfusion with application simply during reperfusion. Most studies of this sort find that application throughout ischemia and reperfusion is more effective than just during reperfusion [3,41–43] but some studies have found the reperfusion period is more effective [44–46]. One difficulty in this type of study is that the [Na⁺]_i rises to a peak within 5 min of the start of reperfusion [32] so that the inhibitor has to reach its target and inhibit it effectively in a small fraction of this period to ensure maximum effectiveness. This consideration has stimulated studies in which the drug was briefly perfused into the ischemic region a few minutes before the end of ischemia (preperfusion) to ensure that the drug was present at the moment of reperfusion [44,46]. It is clear from Fig. 2 that under the circumstances of this experiment the recovery from ischemia was enhanced equally by preperfusion (2 min prior to reperfusion; Fig. 2C) compared to the presence of the drug throughout ischemia and reperfusion (Fig. 2D). However there are other studies where this type of concern has been eliminated. For instance Klein et al. [43] compared an NHE1 inhibitor applied to the early part of ischemia with application in the later period of ischemia including reperfusion and found a strong protective effect in the early period but not in the late. While this study gave unequivocal results it is noteworthy that it used a low flow model of ischemia in which inhibition of the NHE1 may be less pronounced than in full ischemia.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Protection against ischemic damage by an NHE1 inhibitor (10 µM cariporide). Records of left ventricular developed pressure (LVDP) from four different isolated rat hearts all subjected to 30-min global ischemia (37 °C, 2 Hz stimulation rate). Note failure of LVDP to recover in Panel A and pronounced reperfusion contracture. In Panel B, cariporide was only present during reperfusion. Note only moderate recovery of LVDP. In Panel C, the period of ischemia was interrupted at 28 min and the heart was perfused for 15 s with cariporide. Ischemia then continued to the end of the 30-min period and the heart was reperfused with cariporide present. Note improvement of recovery of LVDP compared to Panel B and equivalence to recovery in Panel D. In Panel D, cariporide was present throughout ischemia and reperfusion. Note the substantial recovery of LVDP. Data from Ref. [46].

 
Given the number of experimental studies which support the two alternative interpretations, our view is that both results are part of the biological spectrum and the challenge is to identify possible differences in conditions which contribute to the different results. We consider this issue below.

	7. Possible mechanisms of inhibition of the NHE1 during ischemia

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 
Given the strength of the evidence suggesting that NHE1 may be inactive during ischemia, it is of interest to consider possible mechanisms involved. As noted above, Lazdunski et al. [4] proposed that developing extracellular acidosis would inhibit the exchanger but experimental studies of this proposal have failed to support this pathway as the main mechanism of NHE1 inhibition during ischemia.

A wide variety of studies have shown that activity of NHE1 is reduced by metabolic inhibition of various sorts. Studies of metabolic inhibition are technically much easier than ischemia because NHE1 activity can be studied by standard acid loading techniques produced by extracellular solution changes. For instance Weissberg et al. showed that inhibition of glycolysis in isolated myocytes using deoxyglucose produced a substantial reduction in the activity of NHE1 [47]. Similar results have been found with cyanide and deoxyglucose in Purkinje fibres [48] and with anoxia in perfused hearts [25] and equivalent results have been reported in many other cell types (for review, see Ref. [15]). The mechanism by which metabolic inhibition reduces NHE1 activity is not known but it is often attributed to ATP depletion. The NHEs are classical exchangers that do not require metabolic energy for their operation and in isolated vesicles the exchanger can operate in the absence of ATP [49]. Nevertheless in intact cells ATP depletion does prevent activity and the effective half maximal concentration of ATP can be high (2 mM [50]; 5 mM [51]). The exact role of ATP is not clear; no phosphorylation site dependent on ATP has been identified [52] and it may be that an ancillary factor dependent on ATP is necessary [53]. Whatever the cause, it seems possible that the metabolic inhibition associated with ischemia may contribute to the reduced activity of NHE1. However it is unlikely that a fall in ATP is the sole mechanism of NHE1 inhibition during ischemia because the ATP concentration falls quite slowly during ischemia [54].

	8. Reconciling the disparate results

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 
We propose that a key variable is the magnitude of the rise of [Na⁺]_i during ischemia. In our experiments, the rise of [Na⁺]_i is relatively small and seems to be largely caused by Na⁺ influx on I_Na,P with perhaps a late contribution by Na⁺ pump inhibition. Conversely the rise of [Na⁺]_i during reperfusion is large and seems to be almost entirely caused by activation of NHE1 since it is largely eliminated by NHE1 inhibitors [25,32]. In these circumstances it would be predicted that NHE1 inhibitors exert their main effect during reperfusion and have little effect during ischemia.

In the experiments in which [Na⁺]_i shows a very large increase during ischemia, the evidence suggests that this is caused by I_Na,P activation and late inhibition of the Na⁺ pump but early Na⁺ entry on NHE1 may play some role. When reperfusion occurs, Na⁺ entry on NHE1 will be reduced by the reduction of the chemical gradient for Na⁺ [17] and, in addition, the Na⁺ pump will be maximally stimulated by the large [Na⁺]_i and will tend to disguise any rise of [Na⁺]_i associated with NHE1 activity. In these circumstances, since the rise of [Na⁺]_i occurs principally during ischemia, it would be predicted that drugs which prevent Na⁺ entry would be effective during ischemia. Note that this prediction is not dependent on whether NHE1 inhibitors act by inhibiting NHE1 or by blockade of I_Na,P.

Why is the rise of [Na⁺]_i during ischemia so variable between different studies? In our own studies we believe the small rise of [Na⁺]_i during ischemia is because we stimulate the hearts at 2 Hz, significantly slower than the physiological rate of around 5 Hz at 37 °C. Our reasons for doing this relate to the problem of O₂ supply in the isolated heart. The in vivo heart has haemoglobin in the blood supply and under physiological conditions Hb carries about 200 ml O₂/l blood whereas only 3 ml is carried in physical solution in the plasma. O₂ supply to the haemoglobin-free perfused heart is increased about sixfold by increasing the PO₂ from 100 to 600 mmHg and about fourfold by increasing the flow rate from 2.5 to 10 ml/g per min but this still leaves a substantial reduction in O₂ supply. In order to match O₂ consumption to the reduced O₂ supply, we therefore reduce the stimulation frequency. Such hearts will have a better metabolic status before the start of ischemia [55] and would be expected to have reduced metabolic changes in the early period of ischemia [56]. In support of this conclusion, we observed that the ischemic contracture developed more quickly in hearts stimulated at 5 Hz compared to 2 Hz [32].

What is the evidence that reducing the heart rate to 2 Hz influences [Na⁺]_i during ischemia? We have shown that the increase in [Na⁺]_i during ischemia is larger when the stimulation rate is 5 Hz compared to 2 Hz [32,57]. The question then becomes why does metabolic stress associated with a faster heart rate cause the [Na⁺]_i to rise more rapidly during ischemia? The most likely explanation is that the source of Na⁺ entry is via I_Na,P which is known to be activated by metabolic stress [36]. Another possibility is that inhibition of the Na⁺ pump occurs earlier in the 5 Hz stimulated hearts. Cross et al. [8] used an NMR method that allowed them to assess Na⁺ pump function during low-flow ischemia. Their experiments showed that the Na⁺ pump became inhibited shortly after the time that the ischemic contracture developed. Since [Na⁺]_i rises in our experiments well before the ischemic contracture we suspect that slowing of the Na⁺ pump makes only a small contribution in our experiments though it is likely that the contribution of this mechanism is greater at 5 Hz.

The study by Cross et al. [8] provides further evidence to support our hypothesis that the metabolic state determines whether the rise of [Na⁺]_i is large or small. In their experiments, in the absence of glucose, [Na⁺]_i showed a large rise during ischemia and a monotonic fall on reperfusion. Interestingly, when glucose was added to the perfusate, [Na⁺]_i showed only a small rise during ischemia but a transient rise on reperfusion. This important result shows that the two types of [Na⁺]_i behaviour can be observed in the heart by manipulating the metabolic state.

If this analysis is correct, it is important to decide whether there is a large or small rise of [Na⁺]_i in the human heart during ischemia as this will determine the best strategy for the timing of application of an NHE1 inhibitor. At present there is little evidence to resolve this point though in the future Na⁺ measurements in the infarcted regions of human hearts may be possible [58].

	9. Current status of clinical trials

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 
The fact that NHE1 inhibitors applied during ischemia and/or reperfusion protect the heart against ischemic damage is extremely well established in animal studies. This result has been repeated in many animal species, with a variety of NHE1 inhibitors and using a wide range of end points including infarct size, mechanical recovery and various biochemical and histological markers. Despite this uniformity of results from animal models, human trials have not shown unequivocal evidence of protection.

The GUARDIAN trial [59] was a large (11 000 patients) multinational trial of cariporide whose underlying philosophy was that the exchanger rapidly activates during ischemia and therefore the inhibitor needs to be present before the onset of ischemia. Three groups of patients at risk of myocardial ischemia were studied; a group with unstable angina, a high risk group about to undergo angioplasty and patients undergoing coronary artery bypass surgery. These patients were given one of three doses of cariporide or placebo for 2–7 days to cover the period of high risk and assessed for death or myocardial infarct after 4–5 weeks. Only the coronary artery surgery group showed evidence of some protection at the highest dose of cariporide. This study showed no benefit of cariporide under most circumstances but since the animal studies have only demonstrated protection on reperfusion following established ischemia, the number of patients who fulfilled this criterion may have been very small.

The second trial by Rupprecht et al. was a small study of 100 patients with established infarcts [60]. All were treated with angioplasty and half received an i.v. dose of cariporide 10 min before the reperfusion occurred. In the treated group cardiac enzyme release was reduced and cardiac function assessed by ejection fraction at 3 weeks showed significant improvement. Presumably in this study the drug did not reach most of the ischemic region until after reperfusion had started so the implication is that the benefits arose from inhibition of NHE1 during reperfusion.

The third trial was on 1400 patients with established infarcts (ESCAMI trial) [61] and followed essentially the same approach as the Rupprecht et al. study except that eniporide was the NHE1 inhibitor. Unfortunately, this study found no significant benefit on infarct size or patient outcome in the therapy group. The only sub-group which showed slight benefit were those who were reperfused relatively late (>4 h). The authors were unable to identify reasons for the difference between their study and the smaller earlier study and were forced to the conclusion that uncontrolled factors in the earlier small study may have led to an artefactually positive result.

Many commentators assessing these trials deduce that for effective use of NHE1 inhibitors they must be present during ischemia [62,63]. This is based on an interpretation of the animal literature that concludes that NHE1 is active during ischemia and that clinical trials need to reflect this fact. As reviewed above, we feel the evidence on this point is unresolved and a strong case can be made that the NHE1 is largely inhibited during ischemia and that evidence to the contrary arises from the multiple actions of most NHE1 inhibitors. If the argument that NHE1 is inactive during ischemia but active during reperfusion is correct for the human heart, one may ask why the ESCAMI trial did not give a positive result? Possible reasons for its failure are as follows: (i) The i.v. NHE1 inhibitors may not have reached the critical ischemic site sufficiently rapidly to exert maximum protection. (ii) The exclusion of patients with symptoms of greater than 6 h may have been a strategic error as it may be this group who have a greater degree of damage on reperfusion and who are more sensitive to the effect of NHE1 inhibition.

These considerations lead us to advocate a small trial in which the NHE1inhibitor is given via the coronary catheter directly into the ischemic region prior to full reperfusion. This would overcome the possible objection to the ESCAMI trial that the drug did not reach the target regions sufficiently quickly during reperfusion. Because the period of Na⁺ influx on NHE1 occurs principally in the first few minutes of reperfusion [16,31,32] it is essential to ensure that the drug reaches the ischemic region at the beginning of this window of opportunity for cardiac protection. This approach would be particularly effective if other damage pathways that contribute to reperfusion damage could be identified and could also be inhibited therapeutically. Furthermore, it would seem logical to focus on a group of patients who have had a relatively long period of ischemia and might be expected to have a less good response to angioplasty alone.

Time for primary review 35 days.

	Acknowledgements

 
We are grateful to the National Heart Foundation of Australia for research support.

	References

Top Abstract 1. Introduction 2. The coupled exchanger... 3. Properties of NHE1 4. Activity of NHE1... 5. Measurements of [Na+]i... 6. Timing of the... 7. Possible mechanisms of... 8. Reconciling the disparate... 9. Current status of... References

 

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