© 2001 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
Na+ entry during ischemia, reperfusion and preconditioning
Department of Physiology and Institute for Biomedical Research, University of Sydney F13, NSW 2006, Australia
* Corresponding author. Tel.: +61-2-9351-4602; fax: +61-2-9351-4602
Received 23 January 2001; accepted 23 January 2001
In our recent series of papers [1–3] we have measured intracellular sodium and pH ([Na+]i and pHi) in rat hearts during ischemia and reperfusion and draw the following conclusions about the activity of the cardiac Na+/H+ exchanger (NHE1). (i) NHE1 is inactive during ischemia. (ii) Normally NHE1 reactivates rapidly on reperfusion causing a large Na+ influx which is the precursor to much of the cellular damage. (iii) However in the preconditioned heart NHE1 remains inactive during early reperfusion and this underlies much of the protective effects of preconditioning. Our evidence and interpretation are different to many earlier studies, as Avkiran et al. [4] point out, and we are pleased to have this opportunity to debate the reasons for these differences and consider the implications of our novel interpretation.
We would first like to comment on the statement by Avkiran et al. ‘Much of this evidence, which suggests that NHE activity is sustained during ischemia...., has not been acknowledged by Xiao & Allen [3]’. In our paper we stated ‘... there is dispute about the extent to which Na+ entry occurs during ischemia.... Recent reviews by Murphy [5] and Karmazyn [6] discuss this evidence in detail and support the view that Na+ entry during ischemia by NHE1 is important...’ Thus we believe we have appropriately referenced alternative view points. Our perspective, as holders of a minority view point, is that the majority view represented by Avkiran et al. has been widely promulgated in a series of reviews [5–9] and a more serious difficulty in the field is for alternative view points to receive appropriate attention.
One crucial issue is whether or not the NHE1 is active during ischemia and the extent of the contribution of NHE1 to the rise of [Na+]i in ischemia. One view, to which Avkiran et al. subscribe, is that there is a large rise in [Na+]i during ischemia which is partly blocked by amiloride and its analogues. Their interpretation of these observations is that NHE1 is active during ischemia. There are several difficulties with this interpretation; one is that the pHi during ischemia does not seem to be affected by amiloride analogues [6,7]; another is the substantial body of work showing that metabolic inhibition or absence of ATP inhibits the exchanger [10,11]. However there is another interpretation of these observations which is not mentioned in the letter by Avkiran et al. It is well known that amiloride analogues also block Na+ channels [12] and there is good evidence that much of the rise in [Na+]i during ischemia occurs either by inhibition of the Na+ pump [13] or by increased Na+ entry through persistent Na+ channels [2,14,15]. On this interpretation the reduction in [Na+]i produced by amiloride analogues is caused by blocking Na+ entry through a class of persistent Na+ channels which are preferentially activated during ischemia [16]. Another prediction of this interpretation is that amiloride analogues should reduce Na+ entry throughout ischemia and therefore optimum protection would require their presence throughout ischemia. Thus on this interpretation much of the experimental data which Avkiran et al. argue supports activity of NHE1 during ischemia can be explained by Na+ entry through channels and the fact that amiloride analogues have some blocking effect on these channels. To resolve this issue requires a specific NHE1 inhibitor with no Na+ channel blocking effects. For this reason the study by Hartmann and Decking [17] is of interest because they used cariporide (HOE 642), which is more specific than amiloride analogues, and still observed a fall in [Na+]i during ischemia. However there are other studies which reach the opposite conclusion [18] and effects of cariporide on Na+ channels have not been investigated in detail [19].
Is inhibition of NHE1 during reperfusion an important mechanism of ischaemic protection produced by preconditioning? In support of this novel hypothesis we have three independent lines of evidence. (i) Measurements of [Na+]i and of pHi in preconditioned hearts first suggested that NHE1 was inhibited in early reperfusion [2] (ii). We therefore measured activity of the NHE1 directly by the acid loading technique which confirmed the original conclusion [3]. (iii) Finally we measured recovery of developed pressure and showed that the enhanced functional recovery caused by preconditioning was unaffected by an NHE1 inhibitor [3]. With respect to (ii) Avkiran et al. raise a number of points. They propose that activity of the Na+/HCO3– cotransporter may confuse the situation although we have previously shown that under control conditions both the rise of [Na+]i and the recovery of pHi are greatly reduced by NHE inhibitors [1,3]. They comment that NHE1 is relatively inactive at pH 7.2 though since we can measure its activity it is not clear why this is regarded as a problem. They note that the initial acidosis caused by Na lactate was small but this is simply proportional to the concentration of Na lactate used and the intracellular pH buffering and does not invalidate our experimental approach. With respect to (iii), Avkiran et al. comment that other studies under different conditions have reached different conclusions. This is not surprising since it is widely accepted that mitochondrial KATP channels have an important role in the protection provided by preconditioning [20] but this in no way reduces the interest of our finding.
We do not think that the comments of Avkiran et al. cast any serious doubt on our main experimental findings and interpretation but we agree with them that ‘the validity of these conclusions beyond the specific conditions is debatable’. One way in which our experimental conditions differ from most others is that we use a low rate of stimulation (2 Hz) whereas the spontaneous heart rate at 37°C is 
5 Hz. Langendorff-perfused hearts are normally perfused with haemoglobin-free perfusate whose O2 carrying capacity is grossly reduced compared to blood. This reduced O2 carriage is only partly compensated by increasing the flow rate and increasing the PO2; thus by stimulating at a low rate the metabolic consumption can be more closely matched to the O2 supply. Consequently we believe our hearts start with a better metabolic status and the consequences of ischemia develop more slowly. For instance, we have shown that the rise of [Na+]i during ischemia is faster in hearts beating at 5 Hz when compared to 2 Hz [2]. Thus we suggest that in our experimental conditions, Na+ entry through persistent Na+ channels is smaller than in many other studies and this explains both the relatively small rise of [Na+]i during ischemia and also the relatively large rise in [Na+]i caused by NHE1 on reperfusion. This interpretation can also explain the different results concerning the timing of application of NHE1 inhibitors in various studies. This raises the general question; what are the most appropriate experimental conditions for studies of the consequences of ischemia? We argue that an appropriate ‘gold standard’ for experiments on ischemia is that the results prove relevant to the understanding or treatment of human ischemia. This point is examined below.
It is important to understand, as Avkiran et al. also emphasize, that this debate is not simply an academic one about the details of the regulation of NHE1. If, as Avkiran et al. maintain, NHE1 is active throughout ischemia and the protective effect of NHE1 inhibition arises through blocking Na+ influx through the exchanger, then to obtain maximal protection it is necessary for an NHE1 inhibitor to be present throughout the period of ischemia. Clinically this is impractical because most patients experience ischemia initially in the community and, even when medical attention becomes available, access of a drug to an ischaemic region is obviously problematic. If, as we propose, the key time for myocardial protection is the first few minutes of reperfusion then a cardiologist who is performing primary angioplasty (reperfusion) has the perfect opportunity to apply the drug at the right moment and to target the ischemic region. To our knowledge this important clinical experiment has not been performed but the first successful trial of cariporide has recently been published [21]. In this trial patients entering hospital with acute myocardial infarction were randomly assigned to a treatment group, who received intravenous cariporide immediately before angioplasty, or a control group who had only angioplasty. The treated group had improved outcome by several criteria; functional measurements after 3 weeks showed ejection fraction increased from 40% (control) to 50% and end systolic volume was reduced from 97 ml (control) to 69 ml; also myocardial enzyme release was reduced in the treated group. The key point is that the treated group only received the drug immediately before reperfusion so it is clear that benefits arose from the drug during reperfusion. This is exactly the outcome our experiments predict and strongly suggests that our experimental approach and interpretation are appropriate for understanding human ischemia and reperfusion. More important, this trial marks the successful transition of NHE1 inhibitors from laboratory to clinic and shows that several decades of work on isolated hearts is now leading to clinical benefits.
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