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Cardiovascular Research 2000 45(1):100-106; doi:10.1016/S0008-6363(99)00294-1
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Copyright © 2000, European Society of Cardiology

Cellular calcium homeostasis during ischemia; a thermodynamic approach

J.W.T Fiolet* and A Baartscheer

Department of Experimental Cardiology, Cardiovascular Research Institute Amsterdam (CRIA), Academic Medical Center, University of Amsterdam, and the Inter University Cardiology Institute of the Netherlands (ICIN), Amsterdam, The Netherlands

* Corresponding author. Tel.: +31-020-566-3254; fax: +31-020-697-5458 J.W.Fiolet{at}AMC.UVA.NL

KEYWORDS Calcium (cellular); Ischemia; Reperfusion; Ventricular function


   1 Introduction
Top
 1 Introduction
2 Frequently cited early...
 3 Transmembrane ion transport...
 4 Na/Ca-exchange and...
 5 Ischemia and anoxia
 6 Concluding remarks
 References
 
The pivotal role of calcium cycling and homeostasis has long been recognized in contractile, metabolic, electrical and ionic alterations associated with myocardial ischemia and anoxia, as well as in hibernation, stunning and mitochondrial dysfunction associated with reperfusion. However, the lack of adequate techniques seriously hampered measurement of cellular calcium in the low and narrow range of physiological concentrations, particularly in the cytoplasm. Uninterrupted measurement of the dynamics of calcium in the cytoplasm and in organelles such as the sarcoplasmic reticulum and mitochondria proved impossible for a long time.

Bourdillon and Poole-Wilson [1] were the first to accomplish this goal in 1981. They devised an elegant technique to continuously monitor mechanical function, calcium uptake and release during ischemia and reperfusion using calcium isotopes and radioactive labeling of the extracellular space. Their basic observations (see below) attracted much attention and the study remains frequently cited. Since then, new techniques have become available, superior in sensitivity and time resolution, among which are intracellular calcium specific fluorescent indicators [2–4] and 19F NMR [5,6]. Consequently, an abundance of original papers and many review articles have been published over the last 2 decades addressing the issue of calcium handling during ischemia and reperfusion.

It is beyond the scope of this short update to present a full record of all literature on calcium handling during ischemia, anoxia and reperfusion over the past 2 decades. For an overview of the reperfusion related literature the reader is referred to recent review articles dealing with: the role of the Na/H-exchanger [7–9], mitochondria [10–12], sarcoplasmic reticulum [13], e–c-coupling in stunning [14], hibernation [15] and clinical implications [16].

The aim of the present contribution is to put the available data on calcium handling and homeostasis during ischemia and anoxia in a thermodynamic perspective with particular emphasis on the role of the sarcolemmal Na/Ca-exchanger. Ischemia or anoxia may be characterized as an acute metabolic energy crisis. Within minutes myocardial energy supply becomes severely reduced and transmembrane ion gradients must inevitably adapt (see below). Accordingly, ion transport activities will change, which has fundamental implications for contractile dysfunction and arrhythmogenesis [17].


   2 Frequently cited early observations of Bourdillon and Poole-Wilson [1]
Top
 1 Introduction
 2 Frequently cited early...
3 Transmembrane ion transport...
 4 Na/Ca-exchange and...
 5 Ischemia and anoxia
 6 Concluding remarks
 References
 
Bourdillon and Poole-Wilson were the first to provide continuous and simultaneous measurements of mechanical function and uptake and release of calcium in the ischemic and reperfused intact heart. They made a number of fundamental observations. Following the onset of ischemia there was a rapid decline of developed tension and a rapid loss of whole tissue calcium, which was associated with a decrease of the extracellular compartment. Subsequently, resting tension gradually increased. Upon reperfusion developed tension partly recovered and resting tension transiently increased. Reperfusion was associated with immediate large and prolonged uptake of calcium and a transient increase of the rate of efflux. Notably, they found that the reperfusion-associated calcium uptake was related to the degree of mechanical recovery, and depended on the severity and duration of ischemia. Their conclusions on the reperfusion related calcium movements are indisputable and remain valid to date. However, their contention that the early loss of tissue calcium was entirely due to reduction of the extracellular space may be disputed, in part because their technique was less suited to assess redistribution of calcium between tissue compartments during ischemia.

At first sight this conclusion seems based on the kinetic similarity between calcium loss and the reduction of the extracellular space. There are, however, some clues in the data that might indicate loss of intracellular calcium. Given their statement that the extracellular compartment comprises approximately 30% of total tissue calcium (discussion page 128), the data presented (e.g. Fig. 3) suggest that the total loss of whole tissue calcium exceeds the amount of calcium associated with loss of extracellular fluid. Simultaneous measurement of extracellular space and calcium with ion sensitive electrodes support the conclusion of early loss of intracellular calcium [18]. In addition, potential effects of osmotically driven water movement between intra- and extracellular compartments should not be disregarded. This is especially relevant because the authors suggest that the extracellular calcium concentration may have increased during ischemia (discussion page 129).

The authors place their observations in the perspective of disturbances in energy metabolism and ATP availability during ischemia and reperfusion. If this indeed would be the ultimate cause for the mechanical behavior and calcium movements observed, potential early extrusion of calcium from the intracellular compartment should also be considered in this perspective.


   3 Transmembrane ion transport and thermodynamics
Top
 1 Introduction
 2 Frequently cited early...
 3 Transmembrane ion transport...
4 Na/Ca-exchange and...
 5 Ischemia and anoxia
 6 Concluding remarks
 References
 
The activity of a particular ion transport system or ion channel is the product of both its ‘conductivity’ and its driving force. The ‘conductivity’ is a rather complex function of the kinetic properties of the protein such as ion binding affinities, pH, temperature, ligand binding characteristics of regulatory compounds and the characteristic time and voltage dependence. The driving force, or free energy, of ion channels and exchangers is a function of the transmembrane electrochemical potential differences ({Delta}µion) of the respective ions (for a schematic diagram see Fig. 1).


Figure 1
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  		Fig. 1 Diagram of thermodynamic regulation of transmembrane ion gradients. The size of the respective ion symbols indicates direction and magnitude of ion gradients. Glossary: {Delta}G{equiv}free energy difference (expressed in kJ/mol); {Delta}µion{equiv}electrochemical potential difference (expressed in volts); n{equiv}valence of a particular ion; R{equiv}universal gas constant; T{equiv}absolute temperature; F{equiv}Faraday constant; Vm{equiv}‘average’ membrane potential; ln{equiv}natural logarithm; [ADP], [Pi], and [ATP]: cytoplasmic activities of ADP, inorganic phosphate and ATP; [Ca], [Na] and [K]: activities of calcium, sodium and potassium; Subscripts: c{equiv}cytoplasmic, e{equiv}extracellular, SR{equiv}sarcoplasmic reticular; {Delta}Gion=nF{Delta}µion; {Delta}GATP=RTln [ADP][Pi]/[ATP]; {Delta}Gpump={Delta}GATP–(3RTln [Na]c/[Na]e–2RTln [K]c/[K]e+FVm); {Delta}GSERCA={Delta}GATP–2RTln [Ca]c/[Ca]SR (assuming zero SR membrane potential); {Delta}Gexch=3RTln [Na]c/[Na]e–RTln [Ca]c/[Ca]e+FVm.

 
Maintenance of {Delta}µion requires continuous input of metabolic energy by ATP driven ion pumps. The driving force of these pumps is provided by the free energy of hydrolysis of ATP, called the cytosolic phosphorylation potential ({Delta}GATP), which is coupled to energetically ‘uphill’ ion-transport. The magnitude of {Delta}GATP is determined by the free concentrations of ATP, ADP and inorganic phosphate (see legend to Fig. 1) resulting from the balance between the rate of oxidative and glycolytic ATP generation and ATP hydrolysis by energy consuming processes [19–24]. Therefore, {Delta}GATP is either directly or indirectly the ultimate driving force for any ion transport system and forms an intrinsic part of its transport rate.

A second, not always fully appreciated consequence of {Delta}GATP, is that it sets a thermodynamic upper limit to {Delta}µion in energetically coupled ion transport processes. By thermodynamic law the free energy of the ion gradient involved can never exceed {Delta}GATP. Myocardial energy consuming processes are usually characterized by high efficiency [20]; i.e. the energetically coupled process operates not too far from thermodynamic equilibrium. Thus, {Delta}GATP is only moderately higher than the {Delta}µion it is coupled to. As a consequence, whenever {Delta}GATP decreases, such as during ischemia or anoxia, any directly or indirectly coupled {Delta}µion inevitably adapts, whatever time it takes [20,21]. The degree of adaptation, however, not only depends on {Delta}GATP but also on potential changes in thermodynamic efficiency, which may result from alterations in the ‘conductivity’ properties of the respective ion transport proteins and leak reactions [23].

The sarcolemmal and mitochondrial Na/Ca-exchangers, the sarcolemmal L-type calcium channel and the SR calcium release channel are the most significant calcium transport systems indirectly coupled to {Delta}GATP. The sarcolemmal Na/K-ATPase and the SR Ca-ATPase (SERCA) are the directly {Delta}GATP coupled ion transport systems of relevance. Because of its low capacity the sarcolemmal Ca-ATPase is probably of minor importance from a quantitative point of view [25].


   4 Na/Ca-exchange and thermodynamics
Top
 1 Introduction
 2 Frequently cited early...
 3 Transmembrane ion transport...
 4 Na/Ca-exchange and...
5 Ischemia and anoxia
 6 Concluding remarks
 References
 
The sarcolemmal Na/Ca-exchanger transports one calcium ion against three sodium ions [26] across the sarcolemma in an electrogenic and energetically ‘downhill’ manner. Its free energy is determined by the difference between three times {Delta}µNa and once {Delta}µCa. Accordingly, its magnitude depends on the respective transmembrane gradients as well as on the membrane potential. The Na/K-ATPase must continuously restore {Delta}µNa at the expense of ATP hydrolysis in order to fuel the Na/Ca-exchanger and prevent collapse of {Delta}µNa and {Delta}µCa. Because on a molar basis cytosolic sodium is 105 to 106 times larger than calcium, it is sodium that buffers the driving force of the Na/Ca-exchanger and thus determines its ‘capacity’.

In resting cardiac muscle, estimates of the reversal potential of the Na/Ca-exchanger range, species dependently, from about –20 to –40 mV [27–29]. At a resting membrane potential of –80 to –90 mV this corresponds to a free energy of 6 to 4 kJ/mol, which is confirmed by direct measurement of {Delta}µNa and {Delta}µCa in isolated quiescent rat ventricular myocytes [30]. At membrane potentials negative to the reversal potential the driving force is associated with outward calcium transport.

During the electrically stimulated cardiac cycle the free energy of the Na/Ca-exchanger continuously changes; both {Delta}µNa and {Delta}µCa change dynamically due to the action potential and the cytosolic calcium transient. Early during the action potential the driving force of the Na/Ca-exchanger becomes even shortly reversed. The associated calcium influx may contribute to excitation contraction coupling [31–33], although possibly with relatively low efficiency [34]. Thus, apart from the magnitude of {Delta}µNa, the ‘average’ free energy and the net calcium flux of the Na/Ca-exchanger during one cardiac cycle depends on resting membrane potential, cycle length, action potential amplitude and duration as well as on diastolic calcium and the amplitude and duration of the calcium transient. All of these parameters may be subject to alteration during metabolic inhibition, ischemia and reperfusion.


   5 Ischemia and anoxia
Top
 1 Introduction
 2 Frequently cited early...
 3 Transmembrane ion transport...
 4 Na/Ca-exchange and...
 5 Ischemia and anoxia
6 Concluding remarks
 References
 
5.1 Phosphorylation potential, {Delta}GATP
Control values of {Delta}GATP range from 55 to 60 kJ/mol [19–24,35,36]. Immediately following onset of ischemia or anoxia the rate of ATP supply is substantially reduced by cessation of oxidative metabolism. The much less efficient anaerobic glycolysis, although up-regulated, takes over oxidative ATP production at a much lower rate during a limited period of time until cellular glycogen stores are depleted and ATP production entirely stops. Accordingly, {Delta}GATP is expected to decrease in a biphasic manner. Indeed, {Delta}GATP rapidly decreases by about 10 to 12 kJ/mol following the onset of ischemia or hypoxia [19] and by about another 20 kJ/mol following glycogen depletion [21]. The first phase entirely depends on anaerobic glycolysis; blockade of the glycolytic pathway abolishes the first phase and immediately forces energy metabolism into the second phase. A major difference between ischemia and anoxia, possibly related to absence of cellular acidosis and continuous clamping of extracellular ionic conditions by perfusion in anoxia, is the duration of the first phase, which is substantially shorter during anoxia.

The biphasic decrease of {Delta}GATP is closely followed by changes in other (electro-) physiologic parameters: membrane depolarization [37], ischemic extracellular potassium accumulation [38–42], anoxic potassium loss [21], early contractile failure [43], contracture development and electrical cellular uncoupling [43,44] and depletion of the adenine nucleotide pool [21,45,46]. From a thermodynamic point of view the latter is of special relevance, because depletion of the adenine nucleotide pool, which determines the ‘capacity’ of the phosphorylation potential, may affect functional recovery upon reperfusion or reoxygenation.

5.2 Whole cell calcium
Early loss of whole tissue calcium was originally attributed to loss of extracellular fluid by collapse of the vasculature [1]. However, not only loss of extracellular fluid occurs, but also redistribution of water between the intra- and extracellular compartments due to osmotic effects [18,42,47]. Reduction of the extracellular and increase of the intracellular space, even without net membrane ion transport, affects ion concentrations, transmembrane ion gradients and {Delta}µion by osmotic effects. In addition to osmotic effects net sarcolemmal calcium transport occurs. In the intact rat heart early after the onset of ischemia there is energy dependent efflux for several minutes, followed by reuptake in association with contracture development [18]. Inhibition of anaerobic glycolysis completely prevented the efflux and greatly accelerated development of contracture. The early intracellular calcium loss at least in part originated from SR as concluded from depletion of SR calcium measured in isolated anoxic myocytes [18,23].

5.3 Cytoplasmic calcium
Data available at present on the time course of change of cytosolic free calcium following cessation of oxidative metabolism are not entirely unanimous. On the one hand, early diastolic and systolic increase of calcium was reported followed by a gradual decline and a secondary increase [48] confirming earlier reports using aequorin [49,50] or indo-1 [51]. During ischemia the early changes in cytosolic calcium did not correlate with the rapid decline in contractility, which was claimed to be caused by desensitization of contractile proteins by acidosis and increase of magnesium and inorganic phosphate [41,49,52]. In comparison with ischemia, anoxic changes of diastolic and systolic tension correlate better with changes in systolic calcium [50,53]. On the other hand, in the ischemic isolated blood perfused rabbit papillary muscle, diastolic calcium remained stable up until the moment of contracture development, systolic calcium rapidly decreased and an initial transient increase was not observed. These changes correlated with stable end diastolic tension and decline of developed tension [50,54]. Diastolic calcium and tension were also stable up until the moment of rigor development in anoxic isolated myocytes [18,55,56].

The discrepancies may at least in part result from differences in experimental conditions. The pH and magnesium sensitivity of aequorin should be considered in ischemia [57]. In intact preparations an early increase of endothelial calcium with too excessive endothelial compartmentalization of fluorescent dyes could substantially contribute to the overall fluorescent signals [58]. Cytosolic calcium measured with NMR also demonstrates stable levels until an abrupt increase occurs in association with contracture development [59–61]. It also has been shown that ischemic preconditioning prolongs the period of diastolic calcium stability [43,62,63]. Regardless of the quantitatively diverse estimates on the early stability of end diastolic calcium, there is general agreement that it remains relatively low in comparison with the eventual large increase during contracture development.

5.4 Mitochondrial calcium
In normoxic myocytes the free mitochondrial calcium concentration does not follow the calcium transient and remains a little lower than end diastolic cytoplasmic calcium [64]. During anoxia, mitochondrial free calcium follows end diastolic cytoplasmic calcium and the gradient across the mitochondrial matrix membrane is maintained both before and during rigor development [65,66].

5.5 SR calcium
Data on SR calcium during ischemia and anoxia are sparse and conflicting. The SR calcium content in the intact heart during ischemia measured with 19F NMR and in isolated myocytes during anoxia measured with indo-1 using a rapid cooling technique has been reported to fall [18,67]. These findings are compatible with the general observation that SERCA activity becomes reduced during ischemia, which has been attributed to a decrease of maximal rate but not to a change of calcium affinity or phospholamban dependent regulation [68] (for review see [13]). Also altered calcium release channel kinetics may be responsible in part [69]. One other study, however, reported that a caffeine induced calcium transient, comparable in magnitude to pre-anoxic values, could still be evoked when calcium transients already had been reduced to zero [53]. No definite explanation for these discrepant findings can be provided. It may be speculated that either the very low stimulation frequency on the one hand or the disturbance of the intracellular milieu in the whole cell patch clamp technique used in the latter study may have been partly responsible.

5.6 Thermodynamics and SR calcium; role of the SR calcium pump
Assuming negligible SR membrane potential [70], a stoichiometry of two calcium ions per ATP, 100% efficiency for SERCA and a normoxic {Delta}GATP of 60 kJ/mol, the thermodynamic upper limit of the SR membrane calcium gradient is about 105. In the perfused rabbit heart, having an end diastolic cytosolic calcium of 10–7 mol/l, the measured overall efficiency was about 80% and the calcium gradient was about 104, which corresponds to an SR calcium content of about 10–3 mol/l [23]. Kinetic regulation by β-adrenergic stimulation enhanced the efficiency removing phospholamban dependent kinetic limitations and increased the SR calcium gradient. Without a change of efficiency the early ischemic or anoxic drop of {Delta}GATP by 10 to 12 kJ/mol would reduce the thermodynamic limit of the SR calcium gradient by a factor of 10. In the globally ischemic rabbit heart the actual gradient was 4·102 at an overall efficiency of 69% [23]. These measurements, however, were made after 30 min when cytosolic calcium had increased to 3.4·10–6 mol/l and, consequently, SR calcium was not depleted. When cytosolic calcium is still rather low before contracture development, the seriously reduced thermodynamic limit of the SR calcium gradient implicates substantial depletion of SR calcium. Indeed, a several fold reduction of the SR calcium content was reported in anoxic myocytes, which occurred in concert with energy dependent extrusion of calcium to the extracellular compartment measured in the intact heart [18]. Besides desensitization of the contractile proteins, the early depletion of SR could at least in part be associated with the rapid loss of contractility.

5.7 Thermodynamics and cytoplasmic calcium; role of the Na/Ca-exchanger
The ability of the cell to maintain low steady state diastolic calcium levels depends on the magnitude and direction of the free energy of the Na/Ca-exchanger ({Delta}Gexch). It is well known that in normoxic conditions the Na/Ca-exchanger is capable to rapidly remove all calcium released from SR with caffeine. In order to maintain low diastolic calcium during the early phase of ischemia and anoxia, the ‘average’ {Delta}Gexch during one cardiac cycle should remain sufficiently high to preserve a large enough calcium efflux rate compensating for L-type Ca-channel related calcium influx and SR calcium loss. Reduced L-type Ca-channel related calcium influx during ischemia [71] may favorably affect the {Delta}Gexch requirements.

Full description of the relationship between free energy and flux of the Na/Ca-exchanger requires knowledge on the kinetic properties of the Na/Ca-exchanger and all components describing the ‘average’ {Delta}Gexch. During early ischemia the kinetic properties do not seem to be greatly affected [71,72]. After prolonged ischemia, however, depressed Na/Ca-exchanger function has been found [71], possibly related to extensive depletion of ATP (for review see [73]). Decreased sodium gradient, depolarization of resting membrane, and decreased calcium transient amplitude (causing an increase of the ‘average’ sarcolemmal calcium gradient) all tend to reduce the ‘average’ {Delta}Gexch. In contrast, shortening of the action potential, decreased action potential amplitude and increased end-diastolic calcium (causing a decrease of the ‘average’ sarcolemmal calcium gradient) all tend to increase ‘average’ {Delta}Gexch. Energy dependent extrusion of calcium in the ischemic rat heart [18] suggests that at least initially {Delta}Gexch remains sufficiently high.

The major pathways involved in changes of cytosolic sodium during metabolic inhibition are the Na/H-exchanger, the Na/HCO3-symporter the Na-channel and the Na/K-ATPase. The Na/H-exchanger and the Na/HCO3-symporter cause electroneutral inward sodium transport to compensate for increased proton activity resulting from anaerobic metabolism (see for review [7,52]). Reduced Na-channel activity may counteract increase of cytosolic sodium, which has been directly demonstrated with sodium channel inhibitors [74,75]. However, it also has been demonstrated that Na-channel characteristics hardly change during ischemia or anoxia [8]. Also decreased Na/K-ATPase activity, either due to kinetic or thermodynamic changes, would result in increased cytosolic sodium (see below).

Several techniques have been used to study cytosolic sodium during ischemia or anoxia including ion-sensitive microelectrodes [76,77], isotopes [78], NMR [79] and fluorescent dyes [80]. Data on cytosolic [Na+] during the early phase of ischemia or anoxia up to the moment of contracture development range from an immediate moderate but progressive increase [55,79,80] to no change or even a small decrease [61,77]. During the later phase of ischemia, associated with contracture development and increase of cytosolic calcium, all studies report a two- to three-fold increase of cytosolic [Na+].

Increased sodium influx does not necessarily implicate an immediate increase of cytosolic sodium. Whether or not such an increase occurs depends on the capability of the free energy of the Na/K-ATPase to maintain a high {Delta}µNa. The drop of the phosphorylation potential, {Delta}GATP, would decrease the ‘average’ free energy of the sodium pump. However, early during ischemia, membrane depolarization would largely compensate for the reduction of {Delta}GATP, leaving the free energy difference of the pump effectively intact. During anoxia there is hardly any depolarization, which may in part explain the more rapid changes observed in anoxia compared to ischemia. At a later stage the Na/K-ATPase may become also kinetically compromised [81] (see for review [82]), which together with a further decrease of {Delta}GATP would initiate the progressive increase of cytosolic sodium. Secondary to this, ‘average’ {Delta}Gexch becomes negative, the Na/Ca-exchanger comes into reversed mode and causes the progressive net calcium influx associated with contracture development.


   6 Concluding remarks
Top
 1 Introduction
 2 Frequently cited early...
 3 Transmembrane ion transport...
 4 Na/Ca-exchange and...
 5 Ischemia and anoxia
 6 Concluding remarks
References
 
It is the great merit of Bourdillon and Poole-Wilson that they described for the first time the entire time course of calcium movements and mechanical function in the intact ischemic and reperfused heart and to put their observations into a bioenergetic perspective.

If the changes in calcium homeostasis and handling observed during ischemia and anoxia were to be attributed mainly to the energy ‘crisis’, the thermodynamics of the Na/Ca-exchanger operating in concert with the Na/K-ATPase are of major relevance. Definite assessment of the validity of this contention requires quantitative description of the free energy of these systems. This implies measurement of the time course of change of metabolic parameters determining the phosphorylation potential, cytosolic sodium and calcium, SR calcium and all electrophysiological parameters related to action potential and resting membrane potential during the course of ischemia.


   References
Top
 1 Introduction
 2 Frequently cited early...
 3 Transmembrane ion transport...
 4 Na/Ca-exchange and...
 5 Ischemia and anoxia
 6 Concluding remarks
 References
 

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J. W.T Fiolet
Reperfusion injury and ischemic preconditioning: two sides of a coin?
Cardiovasc Res, November 1, 2000; 48(2): 185 - 187.
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