© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Commentary on "Effects of ischaemia and reperfusion on calcium exchange and mechanical function in isolated rabbit myocardium"
National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse Street, London SW3 6LY, UK
* Corresponding author. Tel.: +44-1207-351-8179; fax: +44-1207-351-8113 p.poole-wilson{at}ic.ac.uk
KEYWORDS Calcium (cellular); Ischemia; Reperfusion; Ventricular function
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1 Introduction |
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 Top 1 Introduction 2 Methods3 RationaleReferences |
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Rereading and commenting upon an article written 18 years ago is almost to intrude on the prerogatives of history. Wallowing in nostalgia may be a satisfying activity but is not a plausible method for identifying, posing or answering scientific questions. I reread this paper with trepidation but was relieved, perhaps the correct sentiment, to find that it still addressed an important issue. More surprising was the realisation of how limited has been the progress made over the last two decades.
In 1974, as a young research worker, I was fortunate to work in the laboratories of Glen Langer in Los Angeles, supported by the British Heart Foundation. Previously in London I had been investigating the net change in potassium homeostasis in cardiac and skeletal muscle, in vivo, in response to alterations of acid–base balance. Glen Langer had developed a technique which allowed the measurement not just of tissue content of an ion but of ion fluxes. The technique allowed examination of the effect of acid–base changes on potassium fluxes so as to explain the previously observed net changes [1]. Later this same technique was adapted to measure the flux of calcium isotopes (45Ca2+ and 47Ca2+) into and out of the myocardium [2]. Bourdillon developed the system further so that measurements could be made during and after a period of ischaemia. That formed the basis of the paper published in Cardiovascular Research [3].
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2 Methods |
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Top1 Introduction2 Methods3 RationaleReferences |
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The technique used was the arterially perfused intraventricular septum of the rabbit heart. A small cannula is inserted into the first septal artery which in the rabbit comes off shortly after the origin of the left anterior descending coronary artery. Non perfused tissue is cut away so that a small triangular of piece of myocardium weighing approximately 1 g can be held at two corners and tension recorded with a transducer attached to the apex. The same technique can be applied to heart muscle from other animals such as the rat and guinea pig. The advantage of such a preparation is that there is no residual fluid in a chamber such as the atria or ventricle, the radioactivity in the preparation can be measured by placing a probe immediately in front of the tissue and the effluent can be collected in order to measure the efflux of any labelled moiety. Using 42K+ it is relatively simple to record at the same time the total tissue potassium and the efflux of potassium, allowing a detailed analysis of potassium exchange in the total tissue [1]. With calcium the technique is more complex [2]. Efflux can be measured using 45Ca2+ and tissue content using 47Ca2+. 45Ca2+ is a beta emitter and not a suitable isotope for direct and continuous measurement of total tissue calcium. The analysis of signals with calcium is substantially more difficult than with potassium because calcium is compartmentalised within the total heart and the efflux is curvilinear, that is the efflux does not follow single order kinetics. Initially work was undertaken on the effect of acidosis [2] on calcium exchange but on returning to London I and colleagues investigated calcium exchange during and after a period of hypoxia [4] or ischaemia [3,5].
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3 Rationale |
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Top1 Introduction2 Methods3 RationaleReferences |
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In this particular paper [3] we measured continuously the total uptake of calcium into the myocardium after different periods of total ischaemia and after low flow ischaemia. Earlier work by several groups had indicated that there was a large rise in tissue calcium on reperfusion after a period of ischaemia and this was believed to be related to the onset of tissue necrosis. Calcium accumulation was implicated in the poor recovery of function after a period of hypoxia [6,7] or after perfusion with zero external calcium (the oxygen and calcium paradoxes [8]). We demonstrated clearly the changes which occurred in the extracellular space during ischaemia and the uptake of calcium which occurred immediately on reperfusion. A later paper went on to examine the effect of interventions [5]. The equipment for making measurements during ischaemia was more complex than for hypoxia. The muscle was placed inside a temperature controlled chamber. It is often not appreciated how quickly cardiac muscle cools when it is made totally ischaemic and is exposed to the external environment.
The method was not without problems and the criticisms made at the time still apply to our experiments and many which are undertaken today. The tissue was perfused with a physiological fluid and this resulted in oedema and a larger extra cellular space than in vivo. The temperature was held at 32°C and not at 37°C. Since whole tissue was being studied there was the possibility that the uptake of calcium was heterogeneous (and indeed that was shown to be true) so that the signal being measured was the sum of a large uptake into some cells and a smaller uptake into others. It was debated whether the calcium uptake was an indicator of cell death and that the recovery of function was an indicator of the number of cells in which there had been almost no uptake of calcium. Controversy arose because of confusion between those who were attempting to measure cytosolic calcium at the time and this technique which measured total tissue calcium. There was debate concerning whether the rise in resting tension was due to a lack of ATP, to an increase in cytosolic calcium or both. That argument still continues to this day.
An interesting point to note over the last 20 years is the change of terminology which has taken place. Early papers identified many of the biochemical and morphological changes which occurred in the myocardium after ischaemia whether it had been perfused with blood or physiological fluid. These changes included cell swelling and blebbing, mitochondrial swelling (and other mitochondrial changes) and disruption of the cell membrane. More recently words such as hibernation, stunning, apoptosis, necrosis and oncosis have been used and applied when particular morphological, biochemical or functional criteria are fulfilled. I suspect the process being studied by us so many years ago was a combination of what is now called apoptosis, oncosis and necrosis. Undoubtedly some of the cells in this preparation were stunned or hibernating. Perhaps the name does not matter.
A further debate in the 1980s concerned the mechanism for the presumed benefit from calcium antagonists in the context of myocardial ischaemia. Many researchers had shown that calcium antagonists given before a period of ischaemia in high doses resulted in greater recovery of many biochemical and functional measurements on reperfusion. There were at least three possible explanations. One was that there was an increased perfusion of the tissue during low flow ischaemia or on reperfusion. A second was that the negative inotropic effect of the rather high doses of calcium antagonists resulted in a cardioplegic effect. The third was that the calcium antagonists selectively inhibited the uptake of calcium. A subsequent paper [5] to the one in Cardiovascular Research [3] showed that calcium antagonist, verapamil, did not inhibit the immediate uptake of calcium on reperfusion and that the most probable mechanism was a cardioplegic effect. A consequence was that the uptake of calcium on reperfusion was not through the L type calcium channel and that there would need to be other explanations for any clinical benefit from calcium antagonists. That was not a popular view at the time. Indeed the observation contradicted the immediate impact of the attractive phrase ‘calcium antagonist’.
Since 1981 much has been learnt about the alteration of cytosolic calcium during ischaemia and reperfusion. The states of hibernation and stunning have been recognised. The process of apoptosis has been described. But many of the fundamental questions remain the same. Perhaps the most important is that in clinical practice patients with ischaemia or myocardial infarction require interventions which can be applied at the time of reperfusion and not prior to the period of ischaemia. Interventions designed to bring advantage prior to the period of ischaemia are probably better directed towards stabilisation of the atheromatous plaque, prevention of thrombosis and inhibition of trigger factors rather than prophylactic preservation of the myocardium itself. The greatest advance in reducing myocardial infarct size has undoubtedly been thrombolysis, that is the early restoration of normal coronary flow. Indeed the restoration of blood flow whether by intervention, thrombolysis or through collaterals is the key to alleviation of the consequences of ischaemia. The pursuit of a simple intervention, which given at the time of reperfusion would minimise the ultimate size of an infarct, has not been successful. The role of cytosolic, mitochondrial and sarcoplasmic calcium, the control mechanisms for calcium homeostasis and the control of excitation–contraction coupling are still not fully understood but seem to have a key role in heart muscle in relation to ischaemia, cell death, hypertrophy and heart failure.
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References |
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Top1 Introduction2 Methods3 RationaleReferences |
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- Poole-Wilson P.A., Langer G.A. Effect of pH on ionic exchange and function in rat and rabbit myocardium. Am. J. Physiol. (1975) 229:570–581.
[Abstract/Free Full Text] - Poole-Wilson P.A., Langer G.A. Effects of acidosis on mechanical function and Ca2+ exchange in rabbit myocardium. Am. J. Physiol. (1979) 236:H525–H533.[Web of Science][Medline]
- Bourdillon P.D., Poole-Wilson P.A. Effects of ischaemia and reperfusion on calcium exchange and mechanical function in isolated rabbit myocardium. Cardiovasc. Res. (1981) 15:121–130.
[Abstract/Free Full Text] - Harding D.P., Poole-Wilson P.A. Calcium exchange in rabbit myocardium during and after hypoxia: effect of temperature and substrate. Cardiovasc. Res. (1980) 14:435–445.
[Abstract/Free Full Text] - Bourdillon P.D., Poole-Wilson P.A. The effects of verapamil, quiescence, and cardioplegia on calcium exchange and mechanical function in ischemic rabbit myocardium. Circ. Res. (1982) 50:360–368.
[Abstract/Free Full Text] - Poole-Wilson P.A., Harding D.P., Bourdillon P.D., Tones M.A. Calcium out of control. J. Mol. Cell. Cardiol. (1984) 16:175–187.[CrossRef][Web of Science][Medline]
- Nayler W.G., Poole-Wilson P.A., Williams A. Hypoxia and calcium. J. Mol. Cell. Cardiol. (1979) 11:683–706.[CrossRef][Web of Science][Medline]
- Hearse D.J., Humphrey S.M., Bullock G.R. The oxygen paradox and the calcium paradox: two facets of the same problem. J. Mol. Cell. Cardiol. (1978) 10:641–668.[CrossRef][Web of Science][Medline]
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