The calcium paradox revisited: An artefact of great heuristic value

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1 Cardiovascular Research 45 (2000) locate/ cardiores locate/ cardiores Update review The calcium paradox revisited: An artefact of great heuristic value H.M. Piper* Physiologisches Institut, Klinikum der Justus-Liebig-Universitat, Aulweg 29, D Giessen, Germany Keywords: Calcium (cellular); Histo(patho)logy; Ischemia; Reperfusion. Introduction that the calcium paradox represents a paradigm for the pathomechanism of severe ischemia-reperfusion injury. In The 967 paper by Zimmerman et al. [] is a sequel to the words of Albrecht Fleckenstein [7]: Certainly, with one published in the previous year [2] in which Ariaen restoring the blood perfusion of the previous ischemic Zimmerman and Willem Hulsmann had described an region, an unlimited Ca supply is re-established, posartefact produced on an isolated rat heart preparation. It is sibly comparable with the calcium paradox described by exceptional that an artefact receives so much attention and Zimmerman and Hulsmann [966]. is not forgotten three decades later. The observation they Another reason for the interest in the calcium paradox is made is that, when an isolated heart is perfused for 2 min that this phenomenon is of obvious relevance to the design with a Ca free, otherwise normal Krebs-Henseleit buffer of crystalloid cardioplegic solutions on which much work and then with buffer containing a physiological Ca had been done since the 960s. The opposing poles for the concentration, it rapidly deteriorates. Massive enzyme best strategy to achieve myocardial protection were arrest release occurs and the heart becomes pale due to myoglo- by Na and Ca removal or arrest by K and Mg bin loss. The 967 paper demonstrates that these changes elevation with physiological Na concentration in the are accompanied with dramatic alterations of myocardial perfusion medium. Prominent proponents were Hansultrastructure, i.e. membrane disruption, myofibrillar hy- Jurgen Bretschneider and collaborators for the first strategy percontracture and mitochondrial damage. The discoverers (intracellular type of solution) [8,9] and Mark Braimnamed this impressive artefact calcium paradox. Since bridge, David Hearse and collaborators [0,] for the the original description this phenomenon has fascinated second (extracellular type of solution). Among other hundreds of researchers, with the highest research activity arguments, the first strategy claimed a larger reduction in in the 980s. This brief review is an attempt to explain 33 ischemic energy expenditure when Ca is lowered in years after the original description the main reasons for myocardial tissue, the second warned against the peril of a this long-lasting fascination. It is not intended to duplicate calcium paradox. previous scientific reviews [3 6]. Fascination of the calcium paradox has continued to date. In the past decade the term has increasingly been used in studies on myocardial calcium overload caused by 2. Why has the calcium paradox been so fascinating? reverse mode activation of the sarcolemmal Na / Ca exchanger. It is debatable if this broadened use of the term In the 970s and 980s the pathophysiological impor- calcium paradox is not misleading as it was originally tance of calcium for the heart was in the centre of scientific introduced for an experimental situation in which reverseawareness. The calcium paradox was soon regarded as a mode activation of the Na / Ca exchanger may not play paradigm in this area of research as it became clear that a causal role. repletion of the once Ca depleted heart leads to massive Ca influx into the myocardial cells, a phenomenon also observed in other situations of severe myocardial cell injury. In particular, it was a widely accepted hypothesis 3. The causal mechanism Downloaded from by guest on 08 January 209 *Tel.: ; fax: address: michael.piper@physiologie.med.uni-giessen.de (H.M. Piper) In the 967 paper Zimmerman et al. [] described the morphological correlates of the biochemical and functional changes occurring during the calcium paradox protocol / 00/ $ see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S (99)00304-

2 24 The calcium paradox physical contact creating at these sites excessive mechanical tension. This results in disruption of the sarcolem- ma. It is possible that the mechanical stability of the sarcolemma is also reduced after Ca depletion. Accord- ing to this theory, all other functional and structural features of the calcium paradox are sequels of this me- chanical damage. In particular, the secondary massive Ca influx leads to hypercontracture of the myofibrils, further disruption of the primarily affected and of adjacent cells and extrusion of the constituents from the disrupted cells. Mitochondrial structural damage and amorphous depositions are also due to this massive secondary Ca overload. Other theories on the pathomechanism regard an altered sarcolemmal permeability developing during Ca depletion as being crucial for the damage provoked by sub- sequent re-calcification. According to these theories Ca - free conditions increase sarcolemmal permeabilities for electrolytes and/ or water [6,6,7]. This leads either to forced excessive calcium uptake or to excessive osmotic swelling upon calcium repletion. The first type of argu- ment is based on experiments where Ca depletion was found accompanied by Na overload and on evidence that re-admission of calcium to the extracellular media caused massive calcium influx through activation of the reverse mode of the Na /Ca exchanger. The second type of argument is based on experiments where increased extracellular oncotic pressure and/ or reduced ionic strength could attenuate cell damage during Ca repletion. In favour of the first theory it has been argued that (i) Na overload is not necessary to cause the Ca repletioninduced [4,8,9] injury, (ii) inhibition of contractile activation either by pharmacological inhibition [20] or energy depletion [] upon Ca repletion can prevent this injury and, alternatively, contractile activation without external Ca influx is sufficient to cause severe cell injury after Ca depletion [22], and (iii) calcium paradox injury is not seen in isolated cardiomyocytes submitted to an analogous protocol of Ca depletion and repletion [5,23]. In favour of the second theory speak experiments indicating a strong activation of the reverse mode of the Na / Ca exchanger. These latter findings are pro- nounced when Ca depletion is combined with Mg depletion [6,24]. It seems that in absence of both Ca and Mg from the extracellular milieu, L-type Ca channels become permeable to Na and that this leads to a cellular Na overload [25]. Re-calcification causes then an abrupt reverse mode activation of the Na / Ca exchanger, leading to Ca overload and hypercontracture, in intact They reported that the ultrastructural changes during Ca depletion are minimal, but become drastic immediately after Ca repletion. The prevalent picture consists of hypercontracted cells or cells having lost most of their structural components. Mitochondria contain electrondense material on which the authors rightly assume that it contains calcium precipitations. Later studies on myocar- dial ultrastructure have described that, during Ca free perfusion, the intercalated discs are partly separated and part of the glycocalyx, covering the sarcolemmal surface, can be lost [2 5]. Separation of intercalated discs is now known to be due to the disassembly by cadherin complexes in macula and fascia adherens junctions. Other cell cell contacts remain structurally intact, e.g. gap junctions. Upon Ca removal, adjacent cells become thus incompletely mechanically uncoupled. One of the theories on the causal mechanism of the calcium paradox regards this incomplete mechanical un- coupling during Ca depletion as being crucial for the events occurring during subsequent Ca repletion (Fig. ). Ca repletion causes Ca influx into the cells and contractile activation. Because of the weakening of cell cell contacts the force developed in the cardiomyocytes is transmitted to the small areas where adjacent cells retain Fig.. Mechanism of the calcium paradox, according to the mechanical myocardium as well as in isolated cardiomyocytes [26]. theory. Ca depletion causes partial separation of adjacent car- Mg depletion was not part of the original protocol of the diomyocytes at intercalated discs. Contractile force generated upon Ca calcium paradox, however [,2]. It has been argued repletion disrupts cells at these weakened sites of cell cell contact. therefore [4,5] that the pathomechanism initiated by com- Terminal Ca overload and hypercontracture follow. Since mechanical cell cell interaction is a key element of this pathomechanism, it does not bined Ca and Mg depletion should not be subsumed occur in isolated cardiomyocytes. under the term calcium paradox since the latter occurs in Downloaded from by guest on 08 January 209

3 Key publication: A.N.E. Zimmerman et al. / Cardiovascular Research presence of a normal extracellular Mg concentration and without a marked Na overload [9,27]. Instead, Ca and Mg depletion represents a special cause of (Na -in- duced) Ca overload (Fig. 3). The beneficial effects of an elevation of oncotic pressure and reduction of ionic strength throughout Ca depletion, by replacement of NaCl by sucrose, are difficult to interpret [6,7]. They have been taken to indicate in- creased sarcolemmal permeability after Ca depletion rendering cardiomyocytes susceptible to osmotic swelling [7]. It is conceivable that cell swelling is a partial cause of sarcolemmal disruption. It has also been speculated that at low ionic strength there is less Ca removed from cellular binding sites which are critical for membrane integrity [6]. A further observation not readily explainable with one or the other of the above theories is that the calcium paradox can be attenuated by protocols imitating ischemic preconditioning [28 30]. Activation of protein kinase C seems involved in this mode of protection against calcium paradox injury. One could speculate that preconditioning reduces membrane fragility caused by Ca depletion, possibly via changes in the phosphorylation pattern of strategic cytoskeletal elements. It has also been shown that heat stress reduces myocardial susceptibility to Ca paradox injury [3]. Again, the protective mechanism is not known. 4. Comparison with the pathomechanism of acute Fig. 2. Mechanism of the oxygen paradox (acute lethal reperfusion injury). Oxygen depletion leads to energy loss and consecutive disreperfusion injury turbance of cellular cation control. Cytosolic Ca concentration rises, the sarcolemma remains physically intact. When cells are reoxygenated, oxidative energy production is re-initiated. Energy plus Ca overload For many, the calcium paradox has been a fascinating leads to excessive contractile activation. This causes hypercontracture. In research topic as it seems to imitate and exaggerate the tissue, but not in isolated cardiomyocytes, hypercontracture leads to cell mechanism of cell injury provoked in myocardium by rupture, due to the forces acting between adjacent cells, and a terminal acute reoxygenation after anoxia or ischemia. The term further increase in Ca overload. oxygen paradox, introduced by Hearse et al. [32] for acute lethal reperfusion injury, was explicitly chosen for this reason. In the early 980s it has been common sense pathomechanisms which is characterised by energy-depenthat the two paradoxes are closely related biological dent contractile hyperactivation, mutual cell disruptions phenomena. and calcium overload. The similarity in temperature depen- Indeed, calcium paradox injury and acute lethal reperfu- dency seems, however, coincidental. The protective effect sion injury (Fig. 2) share a number of features, such as: (i) of hypothermia during myocardial ischemia/ anoxia is They both result in hypercontracted cells with disrupted based on a slower decrease of the high energy phosphate membranes and a consecutive loss of cell constituents and stores, whereas different protective mechanisms are in- massive uptake of Ca. (ii) They may both be inhibited volved during hypothermic Ca depletion, since Ca by pharmacological blockade [20,33 35] of the contractile depletion per se is not accompanied by a decrease of machinery or by lowering cellular ATP reserves to an myocardial energy stores. extent where contractile activation is no longer possible It is also clear now that there are distinct differences [,36]. (iii) Both phenomena also exhibit a strong tem- between the two paradoxes, such as: (i) Ca overload in perature dependency, related to the priming conditions of anoxic-reoxygenated cardiomyocytes seems always pre- either ischemia/ anoxia or Ca depletion []. (iv) In ceded by cellular Na overload, but under the classical isolated cells imitations of the classical calcium paradox calcium paradox protocol Na overload does not occur protocol and of ischemia-reperfusion conditions do not prior to massive Ca overload. (ii) Cardiomyocytes in cause cell lysis [5,23]. The identity of symptoms (i), (ii) tissue and in the isolated state can be rescued from the and (iv) can be explained by the common end stage of both oxygen paradox by a temporary interference at the time of Downloaded from by guest on 08 January 209

4 26 The calcium paradox reoxygenation [37]. It seems not possible, however, to prevent calcium paradox injury by only temporary interventions during the early phase of re-calcification. (iii) Even though the complete pictures of calcium paradox and oxygen paradox injury are both missing in isolated car- diomyocytes, there is a marked difference. Brief Ca depletion with normal Mg and subsequent Ca repletion does not cause any of the typical features of the calcium paradox, such as Ca overload, hypercontracture and cytolysis in isolated cardiomyocytes. Anoxia-reoxygenation of isolated cardiomyocytes, however, causes all typical aspects of the oxygen paradox except one: cytolysis [38]. After prolonged exposure to simulated ischemic conditions isolated cardiomyocytes develop hypercontracture upon reoxygenation, caused by re-energisa- tion in presence of Ca overload. They do not become disrupted because mechanical cell cell interactions remain absent. 5. What is left? Most articles on the calcium paradox were published in the 980s. Searching MEDLINE for calcium paradox, one finds 74 articles in , 74 in and 20 in These figures show that the interest in this phenomenon is declining. This is understandable Fig. 3. Mechanism of calcium overload. Cytosolic calcium overload can considering the progress in those fields of research which result from Ca influx across the sarcolemma, e.g. via cation channels or stimulated initially the interest in the calcium paradox, i.e. the Na / Ca exchanger in reverse mode. It may also be due to release research on cardioplegia and ischemia-reperfusion. of Ca from endogenous stores, in particular the sarcoplasmic reticulum Bretschneider s cardioplegic solutions which are nomi- (SR). Ca overload can be secondary to Na overload. Na influx from nally Ca -free have been used successfully and in large extracellular space can occur, e.g., through opening of cation channels, 2 activation of the Na / H exchanger or the Na / HCO3 symporter. It can numbers in the clinic, predominantly in Central Europe. be aggravated by inactivation of the Na / K -ATPase. In a well- These crystalloid cardioplegic solutions are applied at cold energised muscle cell Ca overload causes excessive contractile activatemperature (4 88C). They also contain trace amounts of tion and, consecutively, hypercontracture. In tissue, but not in isolated Ca, due to the production process. As the calcium cardiomyocytes, hypercontracture leads to cell rupture, due to the forces paradox has a strong temperature dependence and is acting between adjacent cells, and a terminal further increase in Ca eliminated by trace amounts of Ca, it is understandable overload. why the calcium paradox has not turned out as a practical risk for Bretschneider s cardioplegia. World-wide, cardiop- of acute lethal reperfusion injury, i.e. the oxygen paradox, legic principles with near-physiological Ca concentra- is ischemic Ca overload and this represents a true tion are, nevertheless, used much more frequently. jeopardy for ischemic myocardium upon reperfusion. Research on ischemia-reperfusion injury has profited The artefact called calcium paradox has been of great indirectly from the work on the calcium paradox even heuristic value. though its pathomechanism is different. This is because analysis of the calcium paradox has led to many insights in the multiple functions of Ca in the heart that helped also Acknowledgements the research on ischemia-reperfusion. To avoid conceptual confusions one should draw a clear line between the This work was supported by a grant of the BIOMED-2 calcium paradox and other conditions creating a rapid program of the European Union. myocardial calcium overload (Fig. 3). The calcium paradox is a laboratory artefact that does not occur under any natural pathophysiological circumstance. Dangerous myocardial Ca overload is, however, a real and everyday References problem in cardiac pathophysiology as it occurs whenever [] Zimmerman AN, Daems W, Hulsmann WC et al. Morphological myocardium becomes ischemic. One of the determinants changes of heart muscle caused by successive perfusion with Downloaded from by guest on 08 January 209

5 Key publication: A.N.E. Zimmerman et al. / Cardiovascular Research calcium-free and calcium-containing solutions (calcium paradox). [] Ruigrok TJ, Boink AB, Spies F et al. Energy dependence of the Cardiovasc Res 967;: calcium paradox. J Mol Cell Cardiol 978;0: [2] Zimmerman AN, Hulsmann WC. Paradoxical influence of calcium [22] Vander Heide RS, Altschuld RA, Lamka KG, Ganote CE. Modiions on the permeability of the cell membranes of the isolated rat fication of caffeine-induced injury in Ca -free perfused rat hearts. heart. Nature 966;: Relationship to the calcium paradox. Am J Pathol 986;23:35 [3] Altschuld RA, Ganote CE, Nayler WG, Piper HM. What constitutes 364. the calcium paradox? [editorial]. J Mol Cell Cardiol 99;23:765 [23] Piper HM, Spahr R, Hutter JF, Spieckermann PG. The calcium and 767. the oxygen paradox: non-existent on the cellular level. Basic Res [4] Ruigrok TJ, Van Echteld CJ. 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Sarcolem- [20] Daly MJ, Elz JS, Nayler WG. Contracture and the calcium paradox mal integrity and metabolic competence of cardiomyocytes under in the rat heart. Circ Res 987;6: anoxia-reoxygenation. Am J Physiol 990;258:H285 H29. Downloaded from by guest on 08 January 209