Nmr Studies of Myocardial Energy Metabolism and Ionic Homeostasis During Ischemia and Reperfusion

J.H. Kirkels NMR STUDIES OF MYOCARDIAL ENERGY METABOLISM AND IONIC HOMEOSTASIS DURING ISCHEMIA AND REPERFUSION

A BASIS FOR PROTECTIVE MECHANISMS NMR STUDIES OF MYOCARDIAL ENERGY METABOLISM AND IONIC HOMEOSTASIS DURING ISCHEMIA AND REPERFUSION

A BASIS FOR PROTECTIVE MECHANISMS

NMR studies naar de energie- en ionenhuishouding van het hart tijdens ischemie en reperfusie

Grondslagen voor protectie

(met een samenvatting in het Nederlands)

Proefschrift ter verkrijgmg van de graad van doctor aan de Rijksuniversiteit te Utrecht op gezag van de Rector Magnificus, Prof. dr. J A. van Ginkel ingevolge het besluit van het College van Dekanen in het openbaar te verdedigen op dinsdag 24 oktober 1989 des namiddags te 230 uur

door

JOHANNES HUBERTUS KIRKELS

geboren op 11 juli 1958, te Hoensbroek promotores: Prof. dr. TJ.C. Ruigrok Prof. dr. F.L. Meijler co-promotor: Dr. CJ-A. van Echteld

beoordelingscommissiei

Prof. dr. B. de Kruijff (Rijksuniversiteit Utrecht) Professor PA. Poole- Wilson, MD, FRCP (University of London) Prof. dr. E.O. Robles de Medina (Rijksuniversiteit Utrecht) Prof. dr. T. van der Werf (Katholieke Universiteit Nijmegen) Prof. dr. N. Westerhof (Vrije Universiteit Amsterdam)

This thesis was prepared in the Laboratory of Experimental Cardiology, Heart Lung Institute, University Hospital Utrecht and formed part of Project 10 of the Inter- university Cardiology Institute of The Netherlands. The study was supported by the Wijnand M. Pon Stichting, Leusden, The Netherlands.

Financial support by the Heart Lung Institute of the University Hospital Utrecht, the Interuniversity Cardiology Institute of The Netherlands and The Netherlands Heart Foundation for the publication of this thesis is gratefully acknowledged. aan mijn ouders voorRian Experiments should be planned to answer important questions, but unexpected results may lead to discovery

Serendipity in cardiac metabolism H.E. Morgan CONTENTS

page

Chapter 1. General introduction 9 Aim of the study 30

Chapter 2. Protective effect of pretreatment with the 31 calcium antagonist anipamil on the ischemic- reperfused rat myocardium: a phosphorus-31 nuclear magnetic resonance study.

Chapter 3. Possible mechanisms of the protective effect 45 of pretreatment with the calcium antagonist anipamil in ischemic-reperfused isolated rat hearts.

Chapter 4. Low Ca + reperfusion and enhanced susceptibility of 61 the postischemic heart to the calcium paradox.

Chapter 5. Intracellular sodium during ischemia and calcium-free 75 perfusion: a ^fa NMR study.

Chapter 6. Intracellular magnesium during myocardial ischemia and 91 reperfusion: possible consequences for postischemic recovery.

Chapter 7. In vivo 31P NMR study of acute regional myocardial 105 ischemia and reperfusion in the rabbit presentation of an animal model and preliminary results.

Chapter 8. Human donor heart preservation studied with 125 P nuclear magnetic resonance spectroscopy.

Chapter 9. General discussion 139

Chapter 10. Summary 147 Samenvatting 151 This thesis is partially based upon the work described in the following papers:

TJ.C. Ruigrok, J.H. Kirkels, C J.A. van Echteld, C. Borst, F.L. Meijler. 31P NMR study of intracellular pH during the calcium paradox. J Mol Cell Cardiol 1987;19:135-139.

TJ.C. Ruigrok, J.H. Kirkels. Metabolic aspects of myocardial reperfusion. In: Spaan J Ail., Bruschke A.V.G., Gittenberger-de Groot A.C. (Eds), Coronary Circulation. From Basic Mechanisms to Clinical Implications. Dordrecht, Martinus Nijhoff Publ. 1987:185-193.

T.J.C. Ruigrok, JJL Kirkels, CJ.A. van Echteld, C. Borst, FJL. Meijler. Phosphorus nuclear magnetic resonance measurements of intracellular pH in isolated rabbit heart during the calcium paradox. In: Dhalla N.S., Innes I.R., Beamish R.E. (Eds), Myocardial Ischemia. Boston, Martinus Nijhoff Publ. 1987:257-263.

JJH. Kirkels, TJ.C. Ruigrok, CJA. van Echteld, F.L.Meijler. Protective effect of pretreatment with the calcium antagonist anipamil on the ischemic- reperfused rat myocardium: a phosphorus-31 nuclear magnetic resonance study. J Am Coll Cardiol 1988;ll:1087-1093.

TJ.C. Ruigrok, J.H. Kirkels, CJA. van Echteld. Energy depletion due to the calcium paradox. In: de Jong J.W. (Ed), Myocardial Energy Metabolism. Dordrecht, Martinus Nijhoff Publ. 1988:215-222.

JH. Kirkels, T.J.C. Ruigrok, CJA. van Echteld, F.L. Meijler. Low Ca2+ reperfusion and enhanced susceptibility of the postischemic heart to the calcium paradox. Circ Res 1989,64:1158-1164.

J Ji. Kirkels, TJ.C. Ruigrok, CJA. van Echteld, C. Ceconi, R. Ferrari. Possible mechanisms of the protective effect of pretreatment with the calcium antagonist anipamil in ischemic-reperfused isolated rat hearts. Submitted to Cardiovasc Drugs Ther.

J.H. Kirkels, CJA. van Echteld, TJ.C. Ruigrok. IntraceUular magnesium during myocardial ischemia and reperfusion: possible consequences for postischemic recovery. J Mol Cell Cardiol, in press.

CJA. van Echteld, JM. Kirkels, M. Eijgelshoven, P. van der Meer, TJ.C. Ruigrok. Intracellular sodium during ischemia and calcium-free perfusion: a ^a NMR study. Submitted to S Mol Cell Cardiol. General introduction

GENERAL INTRODUCTION •m «yocardial ischemia leads to a cascade of interrelated events, which can be *•* ^-explained by a reduced availability of oxygen and substrate, and accumulation of metabolic waste products . In order to briefly review the consequences of ischemia it may be helpful to artificially distinguish between primary disturbances, including metabolic changes , altered ionic homeostasis , and reduced defence mechanisms against (oxygen) free radicals , and secondary consequences, mainly membrane damage and cell necrosis . Timely restoration of flow to the ischemic tissue (reperfusion) may completely reverse the detrimental effects of ischemia, but when reperfusion occurs too late, the ischemic cells will be beyond help . However, at intermediate ischemic periods 7 R reperfusion may either initiate slow metabolic and functional recovery ' , or, paradoxically, accelerate the manifestations of ischemic cell damage or give rise to specific reperfusion damage . Unfortunately, the transition from reversible to irreversible cell damage is difficult to assess. It may depend on the species studied, the degree of residual blood supply through a collateral circulation and metabolic demands during ischemia. Furthermore, if the specific conditions of reperfusion can also contribute to tissue salvage, the concept of expressing the extent of ischemic damage in terms of (ir)reversibility may even be inadequate. With the advent of PTCA and thrombolysis (early) reperfusion is becoming a clinically feasible intervention " . Additional basic knowledge is needed to further improve biochemical and functional recovery of previously ischemic myocardial tissue. In this chapter a concise introduction will be given on the consequences of myocardial ischemia and reperfusion, with an overview of the literature. Finally, the specific aim of this study will be presented.

10 General introduction

A. 1 Energy metabolism during ischemia and reperfusion The heart is dependent on aerobic metabolism for energy (ATP) production, necessary for contraction, maintenance of ionic gradients and (macro)molecular synthesis . When the supply of oxygen becomes insufficient for oxidative phosphorylation in mitochondria, the heart is said to be ischemic . The laboratory situation, in which delivery of substrate is continued in the absence of oxygen, is referred to as hypoxia. Although there are some common features in ischemia and hypoxia, there are also many differences, like the accumulation or removal of metabolic waste products ' . If oxygen is no longer available, anaerobic glycolysis will continue to produce energy ' , but at a much lower efficiency (2 mol of ATP per mol glucose as opposed to an additional 36 ATP during oxidative phosphorylation 4). The maximum rate of glycolytic ATP production is less than 10 % necessary to maintain cardiac function . Due to the imbalance between energy production and energy utilization, reserve stores of ATP will be depleted during ischemia, with a concomi- 91 9S tant rise in inorganic phosphate " .However, during the initial phase of ischemia, ATP levels are temporarily maintained by delivery of high-energy phosphate bonds from creatine phosphate (CP) through the reaction catalyzed by creatine kinase: 26-28 CP2" + MgADP' + H+ CT + MgATP2" Contractile work is reduced shortly after aerobic metabolism ceases ^ , prob- 31 34 ably due to acidosis or accumulation of inorganic phosphate ' j and as a result energy expenditure is reduced. Nevertheless, there is a persistent deficit between ADP phosphorylation and ATP hydrolysis that causes ATP content to decrease as the duration of ischemia is prolonged 2>16^3.25-35. When ATP levels reach a critically low level, rigor complexes will be formed by the contractile apparatus, leading to contracture ^ . ATP hydrolysis leads to a transient elevation in ADP content, a more prolonged increase in AMP, followed by the production of adenosine, inosine and hypoxanthine, which may leave the cell ^ .Adenosine is a potent vasodilator , which may cause coronary artery dilation when it has been released in sufficient amounts to the extracellular space in the vicinity of coronary arterioles. ATP hydrolysis also 21 45 46 causes acidification of the cytosol - - > as does, to a lesser extent, the production of lactate by anaerobic glycolysis 17'26-47. The source of anaerobic glycolysis, glycogen, will be gradually depleted during ischemia ' , until one of the key enzymes of glycolysis, phosphofructokinase is inhibited, possibly by the low pH and the high 14 5 I51 [NADH] ' ° J or, more likely, until the activation of phosphorylaseb is inhibited by ADP and glucose 6-phosphate, or glycogen is no longer accessible for degrada-

11 Chapter 1 tion52 Adenine nucleotide degradation during ischemia results primarily in the accumulation of inosine ", which during reperfusion may either be converted to nucleotides (salvage pathway) or washed out of the heart2>53. However, the capacity of myocytes to convert inosine to nucleotides is limited ' , and most of the inosine will be washed out during reperfusion, resulting in a loss of the adenine pool 2 '5 5. Other ways to regenerate ATP include the ribophosphorylation of adenine, the phosphorylation of adenosine and de novo synthesis 4. However, these are also rate limited, either by lack of substrate 56, or by lack of sufficient enzyme activity

57,58 Therefore) virtually all of the increase in ATP during reperfusion comes from phosphorylation of ADP and AMP of the ischemic tissue. On the other hand, creatine cannot easily leave the intact cell and will be readily rephosphorylated to creatine phosphate when mitochondria resume oxydative phosphorylation after reversible ischemia. After brief periods of ischemia an over- shoot in CP may even occur .

A.2 Ionic homeostasis Maintenance of ion gradients is an ATP-dependent process. Intracellular Ca + and Na+ concentrations are maintained at levels far below their extracellular concentrations by the sarcolemmal Na+/K+-ATPase, Ca +-ATPases in the sarcolemma, sarcoplasmic reticulum and mitochondria, and finally by sarcolemmal Na+/Ca + exchange, which uses the Na+ gradient established by the Na+/K+- ATP-ase . Soon after the onset of ischemia, interstitial K+ and intracellular Na+ increase 3'60, suggesting that the Na+/K+-ATPase is inhibited. The increase in extracellular K+ may also result from increased efflux 61 rather than from reduced influx, as a volume regulatory response to the production of intracellular osmotic particles or due to exchange with lactate . As pointed out before, ATP hydrolysis may lead to an increase in H+ (acidification). This may enhance Na+/H+ exchange , leading to a further increase in intracellular Na+. Cytosolic Ca + rises during ischemia " , mainly as a result of failure of energy dependent sequestration of Ca in the sarcoplasmic reticulum 60. At the time of reperfusion, intracellular Ca2+ may further increase, due to exchange for Na+ 72'76 or due to increased membrane permeability for Ca . Influx through the slow channels seems to be less likely 75>77"79. Cellular Ca2+ overload may cause considerable damage •67>77, as mitochondria may take up Ca2+ at the expense of energy production, high cytosolic Ca2+ stimulates ATP utilization by the contractile elements, and contracture may occur. The contracture, resulting from high levels of cytosolic Ca may compress blood vessels, and thus contribute to restriction of

~~12 General introduction~~

the capacity for reperfusion ("no-reflow")2. Another important intracellular ion is Mg , which is an essential cofactor in all on Ofj enzymatic ATP producing and utilizing processes . Since most of the cellular Mg2+ is complexed to ATP, depletion of ATP during ischemia will give rise to intracellular liberation of Mg . Loss of Mg to the extracellular space might limit utilization of newly produced ATP during reperfusion 70<83, but the fate of Mg2"1" liberated from ATP is still unclear. Due to the accumulation of small, osmotically active particles like H+, lactate, and inorganic phosphate, cellular osmolarity will increase, which may lead to cell swelling and eventually to membrane disruption .

A3 Oxygpn derived free radicals '• Generation of free radicals in the high oxygen environment of reperfusion could I kill potentially viable myocytes, damaged by ischemia . During ischemia the enzyme \ xanthine dehydrogenase is converted to xanthine oxidase . Xanthine oxidase in turn catalyzes the conversion of hypoxanthine to xanthine, which may lead to the formation of superoxide (O2") radicals and hydrogen peroxide (H2O2) ana, in the presence of iron, to hydroxyl (OH) radicals , which are known to damage cell membranes by lipid peroxidation. Furthermore, they may cause damage to proteins OO on (enzymes) and DNA . Since hypoxanthine is known to be increased during ischemia, and the naturally occurring defence mechanisms (superoxide dismutase and catalase) are known to be reduced under these circumstances ' , reperfused myocardial tissue is susceptible to injury by free radicals. Damage by oxygen derived free radicals is assumed to play a role in "stunned myocardium" '91. However, differences between species with respect to the presence of xanthine oxidase, have been subject of many discussions for review see **. in particular the reported M absence (or very low levels) of xanthine oxidase in man might limit the eventual •/ importance of this mechanism. t '- Since oxygen free radicals are highly reactive, they are difficult to demonstrate. The majority of studies therefore present indirect evidence by interfering with free . sj radical production or increasing cellular defence mechanisms. The only exception % '": is formed by studies using electron spin resonance to directly demonstrate free 5 ; ' radicals . In these studies reperfusion was found to be associated with a burst ;", of free radical production. Importantly, the (technically difficult) addition of free ,. H radicals to normally perfused hearts could partly mimick ischemia-reperfusion ,| j§- damage 95, suggesting that the concept of free radical mediated damage may be -^ §§ valid. f K In addition to free radical production by xanthine oxidase in myocytes or ^J 11 5 13 Chapter 1 endothelial cells M, other, less important ^ sites of free radical production might be the mitochondria 96, arachidonic acid metabolism, which is stimulated by Ca +- activated phospholipases , catechc.amine degradation by monoamine oxidase , and blood granulocyies 89. Granulocytes are trapped in ischemic myocardial tissue, causing "plugging" of the capillaries and thereby contributing at a microvascular OQ QQ QQ level to the "no-reflow" phenomenon ' '" . Moreover, activated granulocytes can OQ QQ QQ produce free radicals ' ' . A.4 Transition to irreversible damage The exact cause of the transition from reversible to irreversible ischemic damage is difficult to assess , but ultrastructural membrane damage is likely to be of major importance. Several proteases and (phospho)lipases are activated by Ca2+, resulting in ultrastructural changes in membranes , leading to altered sarcolemmal integrity and permeability 5'107 and sarcoplasmic reticulum dysfunction 108. Membrane integrity may also be jeopardized by intermediates of fatty acid metabolism , and free radical mediated mechanisms ' . Eventually, membrane disturbances may lead to further ionic disturbances and cell death, with loss of intracellular constituents .

B.I Protective interventions before and during ischemia From the above it will be clear that maintenance of the balance between energy production and energy expenditure is of crucial importance for the survival of ischemic tissue. In the setting of cardioplegia during cardiopulmonary bypass for cardiac surgery, this condition can be ideally met by rapidly inducing cardiac arrest, followed by hypothermia to keep energy expenditure as low as possiblefor review see . It will also be clear that outside the operating room the problem needs a different approach. As recovery of myocardial tissue after ischemia (or infarct size limitation) in the end can only be achieved by restoration of blood flow to the 1 10 ischemic area , most protective interventions intend to slow down the changes during ischemia, in order to buy time for reperfusion. In animals and isolated heart preparations several possible approaches have been studied, mainly focussed on energy metabolism and calcium homeostasis. These include the administration before and during ischemia of drugs such as calcium antagonists 113'124 and beta- 118 12i 125 129 130 132 blockers > . - ; prostaglandins " changing the ionic composition of the perfusion fluid 133-135, and blocking specific enzymes of glycolysis 136 or ATP degradation ' .The overall conclusion is that slowing down ischemic alterations may improve biochemical and functional recovery on reperfusion. Only a limited

~~14 General introduction~~

~~Table 1. Resonance frequencies and nuclear magnetic properties of some nuclei of importance for biomedicine.~~

~~Nucleus natural relative resonance abundance sensitivity* frequency (at 1T) (%) (%) (MHz)~~

'H 99.98 100.0 4Z58 19F 100.0 83.0 40.05 31p 100.0 6.6 17.24 17.237257 inorganic phosphate 17.237172 creatine phosphate 17.237131 y-ATP 17.237042 a-ATP 17.236895 /S-ATP BNa 100.0 93 11.26 1.1 1.6 10.71

* The relative sensitivity is defined as the NMR sensitivity of a number of nuclei as compared to the NMR sensitivity of the same number of *H nuclei. •* The most abundant carbon isotope aC (98.9 %) is not magnetically active, in contrast to the non-radioactive isotope UC.

number of papers reports protection in the absence of energy sparing effects during ischemia 131-139-142. Improving free radical scavenging capacity of the heart by the administration of superoxide dismutase and catalase or blocking radical production e.g. by allopurinol, was found to be protective in some J 3*14 , but not all studies 146-148

B.2 Reperfusion damage and protective interventions at the time ofreflow Although reperfusion is a prerequisite for metabolic and functional recovery of ischemic myocardium, it may also accelerate the manifestations of ischemia, or initiate specific reperfusion damage 9. Therefore, with the advent of PTCA and thrombolysis, interest has diverted from ischemia to reperfusion . There is a growing body of evidence that recovery during reperfusion is not only determined by the extent of ischemic cell damage, but may be modulated by the conditions of reperfusion, confirming the idea of reperfusion damage. Successful interventions at the time of reperfusion include (temporary) changes in the availability of Ca +

124,150-152 aQ(j Mg2+ 150,153 ^ tbe administration of substances interfering with free radical mediated damage (such as superoxide dismutase, catalase, deferoxamine and oxypurinol) 154>155. Calcium antagonists mostly failed to offer any n3 150 156 protection when given during reperfusion only - ' > indicating that the harmful influx of Ca2+ during reperfusion does not primarily occur through the slow

~~15 Chapter 1~~

Figure 1. ilP-NMR spectrum (81.015 MHz; 4.7T) of an isolated rabbit heart, perfused according to Langendorff. Thespectrum was obtained on a BrukerMSL 200 spectrometer in five minutes, from 128 accumulated free induction decays. Peak assignment is as follows: (1) methylene diphosphonate (external standard); (2) phosphomonoesters (sugarphosphates, AMP etc); (3) extra-cellular inorganic phosphate (Pi-ext); (4) intracellular inorganic phosphate (P\-mt); (5) phosphodiesters (gtycerophosphocholine); (6) creatine phosphate (CP); (7) y-ATP; (8) a-ATP; (9) NAD(H); (10) /8-ATP.

~~channels75'77"79'113.~~

C. A concise introduction to NMR spectroscopy Most atomic nuclei behave like tiny magnets due to a property called "spin" (a rotational movement around an axis). In a strong magnetic field these nuclear magnets will align, like a compass needle in the earth magnetic Beld. However, the most important nuclei for medical applications, 1H, 13C and 31P, may align either parallel or antiparallel to the magnetic field. The parallel orientation is energetically favourable, and will therefore be slightly more populated. By applying a radiofrequency pulse, a transition of spins from the parallel to the antiparallel orientation can be induced. After cessation of the radiofrequency pulse, the equilibrium condition will be regained ("relaxation", characterized by two time constants, Ti, the "longitudinal" relaxation time, and T2, the "transversal" relaxation time), while a radiofrequency signal is produced. The decay of this signal is characterized by T2. I The frequency of the radio pulse necessary to induce the transition of the spin r I orientation is equal to the rotational frequency of the spin, and also equal to the frequency of the emitted radio signal during relaxation. This "resonance frequency" is different for different nuclei (see table) and is linearly dependent on the magnetic

~~I 16 General introduction~~

Figure 23 1 P-NMR (a) "pee induction decay" and (b) spectrum of a solution containingfiuctose 6-phosphate (1) and glucose 1-phosphate (2) atpH7.3. In (a) the two superimposed sinuses can be seen. In (b) the resulting (frequency) spectrum is shown, obtained after Fourier transformation of the free induction decay.

Geld strength. When looking more closely at the resonance frequency of a given nucleus, e.g. 31P, it appears that the exact resonance frequency depends on the local magnetic field strength, experienced by that specific nucleus. For any nucleus the external magnetic field is shielded by electrons, surrounding that nucleus. Therefore, the exact resonance frequency of a nucleus is dependent on the number of surrounding electrons and the nature of neighbouring nuclei. This property is essential for nuclear magnetic resonance (NMR) spectroscopy and is called "chemical shift". In short, it indicates the different resonance frequencies of similar nuclei in a different e chemical environment These (small) differences in resonance frequency are the I basis of (bio)chemical analysis with NMR spectroscopy. To compare resonance I frequencies measured at different magnetic field strengths, chemical shift is expressed in dimensionless units, parts per million (ppm), defined as:

~~17 Chapter 1~~

In a 31P NMR spectrum of a heart, a number of chemically different phosphorus nuclei can be distinguished (Fig.l). The area under each resonance peak is linearly related to the number of nuclei with that specific resonance frequency. Binding of H+ to inorganic phosphate also changes the chemical environment of the nucleus, and pH changes will therefore give rise to a change in resonance frequency of Pj. Alternatively, the (intracellular) pH can be calculated from the chemical shift of the (intracellular) Pi resonance frequency. Binding of Mg2+ to ATP also changes the chemical environment of the phosphate groups of ATP, and this principle can be used to estimate the fraction of MgATP and, based on this, the concentration of intracellular free Mgr+. By applying a broad band radiofrequency excitation pulse (e.g. 5,000 Hz) with a central frequency corresponding to the nucleus of interest (e.g. 81.015 MHz for P at 4.7 T), all nuclei within the detection volume and with a resonance frequency within the range of the radiofrequency pulse will resonate. After cessation of the radiofrequency pulse all nuclei will relax while emitting their specific radiofrequency signal. Fourier transformation of this complex pattern of decaying, superimposed sinuses ("free induction decay"), results in a frequency spectrum, with the resonance frequencies on the X-axis and signal intensity of the resonancy frequencies on the Y-axis (Fig.2). Due to the small differences in energy between the two spin orientations, NMR spectroscopy has a low inherent sensitivity. The signal to noise ratio of a resonance peak is determined by numerous factors, some of which will be discussed below. 1. Each nucleus has its own intrinsic sensitivity, expressed relative to that of 1H, and natural abundance (see table). 2. In general, the signal intensity increases with increasing Geld strength. 3. The signal intensity (peak area) is linearly dependent on the number of nuclei within the detection volume. 4. Narrow resonance peaks (= high signal to noise ratio) will only be obtained from nuclei in mobile molecules. Magnetic field inhomogeneity will give rise to spatial- dependent differences in the experienced magnetic field strength by nuclei, and consequently will lead to differences in resonance frequency and broad resonance peaks (with a low signal to noise ratio). Differences in magnetic susceptibility within the sample will also lead to broad resonance peaks. 5. Signal averaging will improve the signal to noise ratio (N scans improve the signal to noise ratio by a factor of VN, but the measuring time increases by a factor of N). However, if a measurement is repeated before complete relaxation has occurred, the subsequent signal intensity will be reduced, due to "saturation". Short repitition times (0.25 s) can only be applied with nuclei possessing a short Ti, like ^Na (0.05

18 General introduction s),whereasfor ^(Ti 1-6 s) repetition times of a few seconds are normally applied. 6. The efficiency of the transmitter and receiver coil is very important for the signal intensity. Radiofrequency coils can either enclose the object (e.g. an isolated perfused heart) or they can be designed as a flat circular coil (surface coil), placed over the region or organ of interest. The detection volume of such a surface coil is roughly equal to a (half) sphere with the radius of the coil. For further information and review of applications of NMR spectroscopy in biomedicine, see references 157-159.

{

~~19 Overview of the experimental setup for isolated perfused rat hearts~~

~~1 and 2: reservoirs containingperfusate~~

~~3: heat-controlled chambers for saturation of the perfusate with 95% 0% 15% CO2~~

~~4: heat-controlled perfusion lines to the heart~~

~~5: NMR magnet~~

~~&• glass tube containing the isolated heart, connected through a bubble trap to the perfusion line~~

~~7: electrical stimulation pulses transmitted to the right ventricle of the heart through Nad orKCl wick electrodes~~

~~& pressure recording from an isovolumic balloon in the left ventricle, connected through a fluid-filled line (9) to a pressure transducer~~

~~10: collection of the coronary effluent for determination of coronary flow~~

~~11: operating and viewing console General introduction~~

~~References~~

1. Neely JR, Feuvray D. Metabolic products and myocardial ischemia. Am J Pathol 1981;102:282-291. 2. Jennings RB, Steenbergen C. Nucleotide metabolism and cellular damage in myocardial ischemia. Ann Rev Physiol 1985;47:727-749. 3. Shine KI. Ionic events in ischemia and anoxia. Am J Pathol 1981;102:256-261. 4. Ferrari R, Ceconi C, Curello S, Guarnieri C, Caldarera CM, Albertinj A, Visioli O. Oxygen-mediated myocardial damage during ischaemia and reperfusion: role of the cellular defences against oxygen toxicity. J Mol Ceil Cardiol 1985;17:937-945. 5. Jennings RB, Reimer KA. Lethal myocardial ischemic injury. Am J Pathol 1981;102:241- 255. 6. Hearse DJ. Reperfusion of the ischemic myocardium. J Mol Cell Cardiol 1977;9:605- 616. 7. Kusuoka H, Porterfield JK, Weisman HF, Weisfeldt ML, Marban E. Pathophysiology and pathogenesis of stunned myocardium. J Clin Invest 1987;79:950-961. 8. Braunwald E, Kloner RA. The stunned myocardium: prolonged, postischemic ventricular dysfunction. Circulation 1982;66:1146-1149. 9. Poole-Wilson PA. Reperfusion damage in heart muscle: still unexplained but with new clinical relevance. Clin Physiol 1987;7:439-453. 10. Schaper W, Binz K, Sass S, Winkler B. Influence of collateral blood flow and of variations in MVO2 on tissue-ATP content in ischemic and infarcted myocardium. J Mol Cell Cardiol 1987;19:19-37. 11. Kiibler W, Schuler G, Schwarz F. Intervention in acute myocardial infarction. Am J Cardiol 1987;60:2A-5A. 12. O'Neill WW. Impact of different reperfusion modalities on ventricular function after acute myocardial infarction. Am J Cardiol 1988;61:45G-53G. 13. Hugenholtz PG. To reperfuse or not to reperfuse, which is the question? J Mol Cell Cardiol 1988;20:367-369. 14. Opie LH. The Heart. Physiology, Metabolism, Pharmacology, Therapy. London, Grune & Stratton, 1984. 15. Jennings RB. Myocardial ischemia - observations, definitions and speculations (editorial). J Mol Cell Cardiol 1970;l:345-349. 16. Reimer KA, Jennings RB. Myocardial ischemia, hypoaa, and infarction. In: The Heart and Cardiovascular System. Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE (Eds). New York, Raven Press, 1986. 17. Richards TL, Terrier F, Sievers RE, Iipton MJ, Moseley ME, Higgins CB. Lactate accumulation in ischemic- and anoxic- isolated rat hearts assessed by H-l spectroscopy. Invest Radiol 1987,22:638-641. 18. Hearse DJ, Chain EB. The role of glucose in the survival and 'recovery1 of die anoxic isolated perfused rat heart Biochem J 1972;128:1125-1133. 19. Liedtke AJ. Alterations of carbohydrate and lipid metabolism in the acutely ischemic heart. Progr Cardiovasc Dis 1981;23:321-336. 20. Kobayashi K, Neely JR. Control of maximum rates of gh/colysis in rat cardiac muscle. Ore Res 1979;44:166-175.

~~21 Chapter 1~~

21. Flaherty JT, Weisfeldt ML, Bulkley BH, Gardner TJ, Gott VL, Jacobus WE. Mechan- isms of ischemic myocardial cell damage assessed by phosphorus-31 nuclear magnetic resonance. Circulation 1982;65:561-571. 22. Hoffmeister HM, Storf R, Thiedemann KU, Seipel L. High-energy phosphates, myocardial contractile function and material properties after short periods of oxygen deficiency. Basic Res Cardiol 1989;84:77-90. 23. Jennings RB, Reimer KA, Hill ML, Mayer SE. Total ischemia in dog hearts, in vitro. 1. Comparison of high energy phosphate production, utilization, and depletion, and of adenine nucleotide catabolism in total ischemia in vitro vs severe ischemia in vivo. Circ Res 1981;49:892-900. 24. Neill WA, Ingwall JS, Andrews E, Gopal MA, Klein K, Kramer M, Oxendine JM, Piotrowski ZH, Reiss I. Stabilization of a derangement in adenosine triphosphate metabolism during sustained, partial ischemia in the dog heart. J Am Coll Cardiol 1986;8:894-900. 25. Nishioka K, Jarmakani JM. Effect of ischemia on mechanical function and high-energy phosphates in rabbit myocardium. Am J Physiol 1982;242:H1077-H1083. 26. Gevers W. Generation of protons by metabolic processes in heart cells (editorial). J Mol Cell Cardiol 1977:9:867-874. 27. Kammermeier H. Why do cells need phosphocreatine and a phosphocreatine shuttle? J Mol Cell Cardiol 1987;19:115-118. 28. McClellan G, Weisberg A, Winegrad S. Energy transport from mitochondria to myofi- bril by a creatine phosphate shuttle in cardiac cells. Am J Physiol 1983;245:C423-C427. 29. Clarke K, O'Connor AJ, Willis RJ. Temporal relation between energy metabolism and myocardial function during ischemia and reperfusion. Am J Physiol 1987;253:H412- H421. 30. Hearse DJ. Oxygen deprivation and early myocardial contractile failure: reassessment of the possible role of adenosine triphosphate. Am J Cardiol 1979;44:1115-1121. 31. Cooke R, Franks K, Luciani GB, Pate E. The inhibition of rabbit skeletal muscle contraction by hydrogen ions and phosphate. J Physiol 1988;395:77-97. 32. Couper GS, Weiss J, Hiltbrand B, Shine KI. Extracellular pH and tension during ischemia in the isolated rabbit ventricle. Am J Physiol 1984;247:H916-H927. 33. Krause SM, Hess ML. The effect of short term normothermic global ischaemia and acidosis on cardiac myofibrillar Ca2+-Mg2+ ATPase activity. J Mol Cell Cardiol 1985;17:523-526. 34. Kubler W, Katz AM. Mechanism of early 'pump' failure of the ischemic heart: possible role of adenosine triphosphate depletion and inorganic phosphate accumulation. Am J Cardiol 1977;40:467-47L 35. Jennings RB, Reimer KA, Steenbergen C. Myocardial ischemia revisited. The osmolar load, membrane damage, and reperfusion. J Mol Cell Cardiol 1986;18:769-780. 36. Hearse DJ, Garlick PB, Humphrey SM. Ischemic contracture of the myocardium: mechanisms and prevention. Am J Cardiol 1977^9:986-993. 37. Katz AM. Physiology of the heart. New York, Raven Press 1977. 38. van Belle H, Goossens F, Wynants J. Formation and release of purine catabolites during hypoperfusion, anoxia, and ischemia. Am J Physiol 1987;252:H886-H893. 39. Zoref-Shani E, Kessler-Icekson G, Sperling O. Pathways of adenine nucleotide catabolism in primary rat cardiomyocyte cultures. J Mol Cell Cardiol 1988^0:23-33.

~~22 General introduction~~

40. Clarke B, Coupe M. Adenosine: cellular mechanisms, pathophysiological roles and clinical applications. Int J Cardiol 1989;23:l-10. 41. Olafsson B, Forman MB, Puett DW, Pou A, Cates CU, Friesinger GC, Virmani R. Reduction of reperfusion injury in the canine preparation by intracoronary adenosine: importance of the endothelium and the no-reflow phenomenon. Circulation 1987;76:1135-1145. 42. Sparks HV, Bardenheuer H. Regulation of adenosine formation by the heart. Circ Res 1986;58:193-20L 43. Vial C, Owen P, Opie J-H, Posel D. Significance of release of adenosine triphosphate and adenosine induced by hypoxia or adrenaline in perfused rat heart J Mol Cell Cardiol 1987;19:187-197. 44. Zucchi R, Limbruno U, Poddigbe R, Mariani M, Ronca G. The adenosine hypothesis revisited: relationship between purine release and coronary flow in isolated rat heart Cardiovasc Res 1989;23:125-131. • 45. Jacobus WE, Pores JH, Lucas SK, Weisfeldt ML, Flaherty JT. Intracellular acidosis and contractility in the normal and ischaemic heart as examined by P NMR. J Mol Cell Cardiol 1982;14(Suppl3):13-20. \ 46. Takach TJ, Glassman LR, Ribakove GH, Clark RE. Continuous measurement of intramyocardial pH: correlation to functional recovery following normothermic and hypothermic global ischemia. Ann Thorac Surg 1986;4231-36. 47. McFalls EO, Pantely GA, Ophuis TO, Anselone CG, Bristow JD. Relation of lactate production to postischaemic reduction in function and myocardial oxygen consumption after partial coronary occlusion in swine. Cardiovasc Res 1987^1:856-862. 48. Lagerstrom CF, Walker WE, Taegtmeyer H. Failure of grycogen dentation to improve left ventricular function of the rabbit heart after hypothermic ischemir arrest Circ Res 1988;63:81-86. 49. Neely JR, Grotyohann LW. Role of glycolytic products in damage to ischemic myocardium. Dissociation of adenosine triphosphate levels and recovery of fuact-on of reperfused ischemic hearts. Circ Res 1984^5:816-824. 50. Rovetto MJ, Lamberton WF, Neely JR. Mechanisms of glycolytic inhibition in ischemic rat hearts. Circ Res 1975^7:742-751. 51. Opie LH. Metabolism of free ratty adds, glucose and catecholamines in .-CL-.S myocardial infarction. Am J Cardiol 1975^6:938-953. 52. Bailey IA, Radda GK, Seymour AML. The effects of insulin on myocardial axetabolism and acidosis in nonnoxia and hypoaa. A P-NMR study. Biochim Biopkys Acta 1982;720:17-27. , 53. vanderMeerP, de Jong JW. Regeneration of adenine nucleotides in the hean. In: de JongJW(Ed).Myocara^alEnergyMetabonsm.Dordrecht/Boston/Lancester,Martinus Nijhoff Publishers, 1988, p 283-289. 54. Dow JW, Bowditch J, Nigdikar SV, Brown AK. Salvage mechanisms for regeneration of adenosine triphosphate in rat cardiac myocytes. Cardiovasc Res 1987;21:188-196. 55. Vary TC,AngelakosET,SchafferSW. Relationship between adenine nudeotidemeta- | bolism and irreversible ischemic tissue damage in isolated perfused rat heart Circ Res J[ 1979;45:218-225. j-

~~23 Chapter 1~~

56. Swain JL, Hines JJ, Sabina RL, Holmes EW. Accelerated repletion of ATP and GTP pools in postischemic canine myocardium using a precursor of purine de novo synthesis. Circ Res 1982;51:102-105. 57. Glower DD, Spratt JA, Newton JR, Wolfe JA, Rankin JS, Swain JL. Dissociation between early recovery of regional function and purine nucleotide content in postischaemic myocardium in the conscious dog. Cardiovasc Res 1987;21:328-336. 58. Reibel DK, Rovetto MJ. Myocardial adenosme salvage rates and restoration of AVP content following ischemia. Am J Physiol 1979;237:H247-H252. 59. Ichihara K, Abiko Y. Rebound recovery of myocardial creatine phosphate with reperfusion after ischemia. Am Heart J 1984;108:1594-1596. 60. Walsh LG, Tormey JM. Subcellular electrolyte shifts during in vitro myocardial ischemia and reperfusion. Am J Physiol 1988;255:H917-H928. 61. K16ber AG, Riegger CB, Janse MJ. Extracellular K+ and H+ shifts in early ischemia: mechanisms and relation to changes in impulse propagation. J Mol Cell Cardiol 1987;19(suppl V):35-44. 62. Crake T, Kirby MS, Poole-Wilson PA. Potassium efflux from the myocardium during hypoxia: role of lactate ions. Cardiovasc Res 1987;21:886-891. 63. Lazdunski M, Frelin C, Vigne P. The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. J Mol Cell Cardiol 1985;17:1029-1042. 64. Pierce GN, Philipson KD. Na+-H + exchange in cardiac sarcolemmal vesicles. Biochim Biophys Acta 1985;818:109-116. 65. Piwnica-Worms D, Jacob R, Shigeto N, Horres CR, Lieberman M. Na/H exchange in cultured chick heart cells: secondary stimulation of electrogenic transport during recovery from intracellular acidosis. J Mol Cell Cardiol 1986;18:1109-1116. 66. Murphy JG, Marsh JD, Smith TW. The role of calcium in ischemic myocardial injury. Circulation 1987;75(suppl V):V15-V24. 67. Nayler WG. The role of calcium in the ischemic myocardium. Am J Pathol 1981;102:262- 270. 68. Nayler WG, Elz JS, Buckley DJ. Dissociation of hypoxia-induced calcium gain and rise in resting tension in isolated rat hearts. Am J Physiol 1988;254:H678-H685. 69. Steenbergen C, Murphy E, Levy L, London RE. Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart Circ Res 1987;60:700- 707. 70. Shen AC, Jennings RB. Myocardial calcium and magnesium in acute ischsmic injury. Am J Pathol 1972;67:417-440. 71. Shen AC, Jennings RB. Kinetics of calcium accumulation in acute myocardial ischemic injury. Am J Pathol 1972;67:441-452. 72. Dixon JMC, Eyolfson DA, Dhalla NS. Sarcolemmal Na+-Ca2+ exchange activity in hearts subjected to hypoxia reoxygenation. Am J Physiol 1987;253:H1026-H1034. 73. Grinwald PM, Brosnahan C. Sodium inbalance as a cause of calcium overload in post-hypoxic reoxygenation injury. J Mol Cell Cardiol 1987;19:487-495. 74. Haworth RA, Goknur AB, Hunter DR, Hegge JO, Berkoff HA. Inhibition of calcium influx in isolated adult rat heart cells by ATP depletion. Circ Res 1987;60:586-594. 75. Murphy JG, Smith TW, Marsh JD. Mechanisms of reoxygenation-induced calcium overload in cultured chick embryo heart cells. Am J Physiol 1988;254:H1133-H1141.

~~24 General introduction~~

76. Pridjian AK, LevitskyS, Krukenkamp I, Silverman NA, Feinberg H. Intracellular sodium and calcium in the postischemic myocardium. Ann Thorac Surg 1987;43:416-419. 77. Poole-Wilson PA, Harding DP, Bourdillon PDV, Tones MA. Calcium out of control. J Mol Cell Cardioi 1984;16:175-187. 78. Sperelakis N. Regulation of calcium slow channels of cardiac muscle by cyclic nucleotides and phosphorylation. J Mol Cell Cardioi 1988;20(Suppl n):75-105. 79. Trautwein W, Cavalid A, Flockend V, Hofmann F, Pelzer D. Modulation of calcium channel function by phosphorylation in guinea pig ventricular cells and phospholipid bilayer membranes. Circ Res 1987;61(suppl I):I17-I23. 80. Garfinkel L, Altschuld RA, Garfinkel D. Magnesium in cardiac energy metabolism. J Mol Cell Cardioi 1986;18:1003-1013. 81. Polimeni PI, Page E. Magnesium in heart muscle. Circ Res 1973;33:367-374. 82. Shine KI. Myocardial effects of magnesium. Am J Pliysiol 1979;237:H413-H423. 83. Hearse DJ, Stewart DA, Braimbridge MV. Myocardial protection during ischemic cardiac arrest. The importance of magnesium in cardioplegic infusates. J Thorac Cardiovasc Surg 1978;75:877-885. 84. Downey JM, Hearse DJ, Yellon DM. The role of xanthine oxidase during myocardial ischemia in several species including man. J Mol Cell Cardioi 1988;20(suppl n):55-63. 85. Bolli R. Oxygen-derived free radicals and postischemic myocardial dysfunction ("stunned myocardium"). J Am Coll Ca,diol 1988;12:239-249. 86. Granger DN. Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol 1988;255:H1269-H1275. 87. Hearse DJ, Manning AS, Downey JM, Yellon DM. Xanthine oxidase: a critical mediator of myocardial injury during ischemia and reperfusion? Acta Physiol Scand 1986^48:65- 78. 88. Kako KJ. Free radical effects on membrane protein in myocardial ischemia/reperfusion injury. J Mol Cell Cardioi 1987;19:209-211. 89. Forman MB, Puett DW, Virmani R. Endothelial and myoeardial injury during ischemia and reperfusion: pathogenesis and therapeutic implications. J Am Coll Cardioi 1989;13:450-459. 90. Ferreira R, Llesuy S, Milei J, Scordo D, Hourquebie H, Molteni L, dePalma C, Boveris A. Assessment of myocardial oxidative stress in patients after myocardial revascularization. Am Heart J 1988;115:307-312. 91. Farber NE, Vercellotti GM, Jacob HS, Pieper GM, Gross GJ. Evidence for a role of iron-catalyzed oxidants in functional and metabolic stunning in ths canine heart. Circ Res 1988;63:351-360. 92. Garlick PB, Davies MJ, Hearse DJ, Slater TF. Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy. Circ Res 1987;61:757- 760. 93. Zweier JL. Measurement of superoxide-derived free radicals in the reperfused heart. J Biol Chem 1988;263:1353-1357. 94. Zweier JL, Kuppusamy P. Electron paramagnetic resonance measurements of free radicals in the intact beating heart: a technique for detection and characterization of free radicals in whole biological tissues. Proc Natl Acad Sci USA l988;85:5703-5707. f 95. Miki S, Ashraf M, Salka S, Sperelakis N. Myocardial dysfunction and ultrastructural J alterations mediated by oxygen metabolites. J Mol Cell Cardioi 1988-^0:1009-1024. -«=•

~~25 Chapter 1~~

96. Shlafer M, Myers CL, Adkins S. Mitochondrial hydrogen peroxide generation and activities of glutathione peroxidase and superoxide dismutase following global ischemia. J Mol Cell Cardiol 1987;19:1195-1206. 97. Hammond B, Hess ML. The oxygen free radical system: potential mediator of myocardial injury. J Am Coll Cardiol 1985;6:215-220. 98. Engler RL, Dahlgren MD, Morris DD, Peterson MA, Schmid-Schonbein GW. Role of leukocytes in response to acute myocardial ischemia and reflow in dogs. Am J Physiol 1986;251:H314-H322. 99. Engler R. Consequences of activation and adenosine-mediated inhibition of granulocytes during myocardial ischemia. Fed Proc 1987;46:2407-2412. 100. Schaper J, Mulch J, Winkler B, Schaper W. Ultrastructural, functional, and biochemical criteria for estimation of reversibility of ischemic injury: a study on the effects of global ischemia on the isolated dog heart. J Mol Cell Cardiol 1979;11:521-541. 101. Das DK, Engelman RM, Rousou JA, Breyer RH, Otani H, Lemeshow S. Role of membrane phospholipids in myocardial injury induced by ischemia and reperfusion. Am J Physiol 1986;251:H71-H79. 102. Frank JS, Beydler S, Wheeler N, Shine KI. Myocardial sarcolemma in ischemia: a quantitative freeze-fracture study. Am J Physiol 1988;255:H467-H475. 103. Harper IS, Lochner A. Sarcolemmal integrity during ischaemia and reperfusion of the isolated rat heart. Basic Res Cardiol 1989;84:208-226. 104. Post JA, Ruigrok TJC, Verkleij AJ. Phospholipid reorganization and bilayer destabili- zation during myocardial ischemia and reperfusion: a hypothesis. J Mol Cell Cardiol 1988;20(suppl II):107-lll. 105. Steenbergen C, Jennings RB. Relationship between lysophospholipid accumulation and plasma membrane injury during total in vitro ischemia in dog heart. J Mol Cell Cardiol 1984;16:605-621. 106. van Bilsen M, van der Vusse GJ, Willemsen PHM, Coumans WA, Roemen THM, Reneman RS. Lipid alterations in isolated, working rat hearts during ischemia and reperfusion: its relation to myocardial damage. Circ Res 1989;64:304-314. 107. Sen A, Miller JC, Reynolds R, Willerson JT, Buja LM, Chien KR. Inhibition of the release of arachidonic acid prevents the development of sarcolemmal membrane defects in cultured rat myocardial cells during adenosine triphosphate depletion. J Clin Invest 1988;82:1333-1338. 108.Franson R, Gamache D, Blackwell W, Eisen D, Hess ML. Sarcoplasmic reticulum dysfunction: phospholipid alterations induced by lysosomal phospholipase C. Am J Physiol 1986;251:H1017-H1023. 109. Lamers JMJ, de Jonge-Stinis JT, VerdouwPD, Hulsmann WC. On the possible role of long chain fatty acylcarnitine accumulation in producing functional and calcium permeability changes in membranes during myocardial ischaemia. Cardiovasc Res 1987;21:313-322. 110. Liedtke AJ. Lipid burden in ischemic myocardium. J Mol Cell Cardiol 1988;20(suppl H):65-74. 111. Hearse DJ, Braimbridge MV, Jynge P. Protection of the Ischemic Myocardium, Car- dioplegia. New York, Raven Press, 1981. 112. Hearse DJ, Yellon DM, Downey JM. Can betablockers limit myocardial infarct size? Eur Heart J 1986,7:925-930.

~~26 General introduction~~

113. Bourdillon PD, Poole-Wilson PA. The effects of verapamil, quiescence, and cardioplegia on calcium exchange and mechanical function in ischemic rabbit myocardium. Circ Res 1982;50:360-368. 114. de Jong JW, Harmsen E, de Tombe PP. Diltiazem administered before or during myocardial ischemia decreases adenine nucleotide catabolism. J Mol Cell Cardiol 1984;16:363-370. 115. Hamm CW, Opie LH. Protection of infarcting myocardium by slow channel inhibitors. Comparative effects of verapamil, nifedipine, and diltiazem in the coronary-ligated, isolated working rat heart. Circ Res 1983;52(suppl I):129-138. 116. Hugenholtz PG, Serruys PW, Fleckenstein A, Nayler W. Why Ca2+ antagonists will be most useful before or during early myocardial ischaemia and not after infarction has been established. Eur Heart J 1986;7:270-278. 117. Huizer T, de Jong JW, Achterberg PW. Protection by bepridil against myocardial ATP-catabolism is probably due to negative inotropy. J Cardiovasc Pharmacol 1987;10:55-61. 118. Ichihara K, Abiko Y. Effects of diltiazem and propranolol on irreversibility of ischemic cardiac function and metabolism in the isolated perfused rat heart. J Cardiovasc Pharmacol 1983;5:745-751. 119. Kloner RA, Braunwald E. Effects of calcium antagonists on infarcting myocardium. Am J Cardiol 1987;59:84B-94B. 12O.Knabb RM, Rosamond TL, Fox KAA, Sobel BE, Bergmann SR. Enhancement of salvage of reperfused ischemic myocardium by diltiazem. J Am Coll Cardiol 1986;8:861- 871. 121. Nayler WG, Ferrari R, Williams A. Protective effect of pretreatment with verapamil, nifedipine and propranolol on mitochondria] function in the ischemic and reperfused myocardium. Am J Cardiol 1980;46:242-248. 122.Takio S, Tanonaka K, Tazuma Y, Fukao N, Yoshikawa C, Fukumoto T, Tanaka T. Diltiazem and verapamil reduce the loss of adenine nucleotide metabolites from hypoxic hearts. J Mol Cell Cardiol 1988;20:443-456. 123. van der Vusse GJ, van der Veen FH, Prinzen FW, Coumans WA, van Bilsen M, Reneman RS. The effect of diltiazem on myocardial recovery after regional ischemia in dogs. Eur J Pharmacol 1986;125:383-394. 124. Watts JA, Koch CD, LaNoue KF. Effects of Ca2+ antagonism on energy metabolism: Ca2+ and heart function after ischemia. Am J Physiol 198023&H909-H916. 125. Lavanchy N, Martin J, Giacomelli M, Rossi A. Evaluation by 31P NMR of the effects of acebutalol on the ischaemic isolated rat heart. Eur J Pharmacol 1986;125:341-35L 126. Malloy CR, Matthews PM, Smith MB, Radda GK. Influence of propranolol on acidosis and high energy phosphates in ischaemic myocardium of the rabbit Cardiovasc Res 1986;20:710-720. 127. Nakazawa M, Katano Y, ImaiS, Matsushita K, Ohuchi M. Effects of 1- andd-propranolol on the ischemic myocardial metabolism of the isolated guinea pig heart, as studied by 31P-NMR. J Cardiovasc Pharmacol 1982;4:700-704. 128. Ochi S, Ogawa Y, Imai H, Nakazana M, Imai S. Effects of B-blockers on the fall of pH in the early phase of ischemia in the isolated perfused rat heart: a nuclear magnetic resonance study. J Cardiovasc Phannac 1988;11:326-331.

~~27 Chapter 1~~

129. Veronee CD, Lewis WR, Takla MW, Hull-Ryde EA, Lowe JE. Protective metabolic effects of propranolol during total myocardial ischemia. J Thorac Cardiovasc Surg 1986;92:425-433. 130. Darius H, Osbome JA, Beibel DK, Lefer AM. Protective actions of a stable prostacyclin analog in ischemia induced membrane damage in rat myocardium. J Mol Cell Cardiol 1987;19:243-250. 131. Pieper GM, Wu ST, Salhany JM. A polymeric prostaglandin (PGBX) attenuates adenine nucleotide loss during global ischemia and improves myocardial function during reperfusion. J Mol Cell Cardiol 1985;17:775-783. B2.van der Giessen WJ, Schoutsen B, Tijssen JGP, Verdouw PD. Iloprost (ZK 36374) enhances recovery of regional myocardial function during reperfusion after coronary artery occlusion in the pig. Br J Phannac 1986;87:23-27. 133. Bersohn MM, Shine KI, Sterman WD. Effect of increased magnesium on recovery from ischemia in rat and rabbit hearts. Am J Physiol 1982;242:H89-H93. 134.Renlund DG, Gerstenblith G, Lakatta EG, Jacobus WE, Kallman CH, Weisfeldt ML. Perfusate sodium during ischemia modifies post-ischemic functional and metabolic recovery in the rabbit heart. J Mol Cell Cardiol 1984;16:795-801. 135. Watts JA, Maiorano LJ, Maiorano PC. Comparison of the protective effects of verapamil, diltiazem, nifedipine, and buffer containing low calcium upon global myocardial ischémie injury. J Mol Cell Cardiol 1986;18:255-263. 136. Allen DG, Morris PG, Orchard CH, Pirolo JS. A nuclear magnetic resonance study of metabolism in the ferret heart during hypoxia and inhibition of glycolysis. J Physiol 1985;361:185-204. 137. Achterberg PW, de Jong JW. Adenosine deaminase inhibition and myocardial adenosine metabolism during ischemia. Adv Myocardiol 1985;6:465-472. 138. Dhasmana JP, Digerness SB, Geckle JM, Ng TC, Glickson JD, BLackstone EH. Effect of adenosine deaminase inhibitors on the hearts functional and biochemical recovery from ischemia; a study utilizing the isolated rat heart adapted to 31P nuclear magnetic resonance. J Cardiovasc Pharmacol 1983;5:1040-1047. 139. Brezinski ME, Darius H, Lefer AM. Cardioprotective actions of a new calcium channel blocker in acute myocardial ischemia. Drug Res 1986;36:464-466. 140. Higgins AJ, Blackburn KJ. Prevention of reperfusion damage in working rat hearts by calcium antagonists and calmoduline antagonists. J Mol Cell Cardiol 1984;16:427-438. 141. Kirkels JH, Ruigrok TJC, van Echteld CJA, Meijler FL. Protective effect of pretreatment with the calcium antagonist anipamil on the ischemic-reperfused rat myocardium: a phosphorus-31 nuclear magnetic resonance study. J Am Coll Cardiol 1988;ll:1087- 1093. 142. Watts JA, Whipple JP, Hatley AA. A low concentration of nisoldipine reduces ischémie heart injury, enhanced reflow and recovery of contractile function without energy preservation during ischemia. J Mol Cell Cardiol 1987; 19:809-816. 143. Lasley RD, Ely SW, Berne RM, Mentzer RM. Allopurinol enhanced adenine nucleotide repletion after myocardial ischemia in the isolated rat heart. J Clin Invest 1988;81:16-20. 144.Werns SW, Simpson PJ, Mickelson JK, Shea MJ, Pitt B, Lucchesi BR. Sustained limitation by Superoxide dismutase of canine myocardial injury due to regional ischemia followed by reperfusion. J Cardiovasc Pharmacol 1988;ll:36-44.

~~28 General introduction~~

145. Zucchi R, Poddighe R, Limbruno U, Maiîani M, Ronca-Testoni S, Ronca G. Protection of isolated rat heart from oxidative stress by exogenous creatine phosphate. J Mol Cell Cardiol 1989;21:67-73. 146. Gallagher KP, Buda AJ, Pace D, Gerren RA, Shlafer M. Failure of Superoxide dismutase and catalase to alter size of infarction in conscious dogs after 3 hours of occlusion Mowed by reperfusion. Circulation 1986;73:1065-1076. 147. Kinsman JM, Murry CE, Richard VJ, Jennings RB, Reimer KA. The xanthine oxidase inhibitor oxypurinol does not limit infarct size in a canine model of 40 minutes of ischemia with reperfusion. J Am Coll Cardiol 1988;12:209-217. 148. Richard VJ, Murry CE, Jennings RB, Reimer KA. Therapy to reduce free radicals during early reperfusion does not limit the size of myocardial infarcts caused by 90 minutes of ischemia in dogs. Circulation 1988;78:473-480. 149 Nayler WG, Elz JS. Reperfusion injury, laboratory artifact or clinical dilemma? Circu- j lation 1986;74:215-221. 150. Ferrari R, Albertini A, Curello S, Ceconi C, DiLisa F, Raddino R, Visioli O. Myocardial recovery during post- ischaemic reperfusion: effects of nifedipine, calcium and magnesium. J Mol Cell Cardiol 1986;18:487-498. 151. Kirkels JH, Ruigrok TJC, van Echteld CJA, Meijler FL. Low Ca2+ reperfusion and enhanced susceptibility of the postischemic heart to the calcium paradox. Circ Res 1989;64:1158-1164. 152. Kuroda H, Ishiguro S, Mori T. Optimal calcium concentration in the initial reperfusate for post-ischemic myocardial performance (calcium concentration during reperfusion). J Mol Cell Cardiol 1986;18:625-633. 153. Ferrari R, Ceconi C, Curello S, Cargnoni A, Condorelli E, Belloli S, Albertini A, Visioli O. Metabolic changes during post-ischaemic reperfusion. J Mol Cell Cardiol 1988;20(suppl n):119-133. 154. Ambrosio G, Zweier JL, Jacobus WE, Weisfeldt ML, Flaherty JT. Improvement of postischemic myocardial function and metabolism induced by administration of deferoxamine at the time of reflow: the role of iron in the pathogenesis of reperfusion injury. Circulation 1987;76:906-915. 155. Badylak SF, Simmons A, Turek J, Babbs CF. Protection from reperfusion injury in the isolated rat heart by postischaemic deferoxamine and oxypurinol administration. Car- diovasc Res 1987;21300-506. 156. Chappell SP, Lewis MJ, Henderson AH. Myocardial reoxygenation damage: can it be circumvented? Cardiovasc Res 1985;19:299-303. ;' 157.Gadian DG. Nuclear Magnetic Resonance and its Applications to Living Systems. \ Oxford University Press, 1982. > 158. van Echteld CJA, Meijler FL. Nucleaire magnetische resonantie-spectroscopie in de f, geneeskunde: biochemie van het intacte weefsel. Ned Tîjdschr Geneesk 1986;130:1595- 1602. ,. 159. van Echteld CJA. Cardiac energy metabolism probed with nuclear magnetic resonance. ~.\ In: de Jong JW (Ed), Myocardial Energy Metabolism, Dordrecht/Boston/Lancaster, Martinus Nijhoff Publishers, 1988;127-144.

~~29 Chapter 1~~

Aim of the study The object of this study was to gain more insight in myocardial energy metabolism and ionic homeostasis during ischemia and reperfusion; nuclear magnetic resonance spectroscopy was used to monitor these metabolic and ionic changes and to evaluate the effect of protective interventions. The protective effect of pretreatment with the new calcium antagonist anipamil is reported (Chapter 2) and some possible mechanisms of this protection were further investigated (Chapter 3). In an attempt to circumvent reperfusion damage, the extracellular Ca2+ concentration during reperfusion was temporarily altered (Chapter 4). This study revealed a relationship between ischemia and reperfusion, and the calcium paradox, which led to a comparative sodium NMR study of these conditions (Chapter 5). The changes in intracellular Mg + during ischemia and reperfusion were studied, and possible consequences for recovery are discussed (Chapter 6). To bridge the gap between isolated heart studies and large intact animal studies, an in vivo model of regional ischemia and reperfusion in the rabbit was developed (Chapter 7). Initial experiments are presented in which the relationship between the duration of the ischemic period and recovery was studied, as well as the possible beneficial effects of the anti-granulocyte agent AICA-riboside. A practical application of phosphorus NMR spectroscopy in the assessment of metabolic changes during human heart transplant preservation is presented in Chapter 8.

~~30 Protection by anipamil~~

~~PROTECTIVE EFFECT OF PRETREATMENT WITH THE CALCIUM ANTAGONIST ANIPAMIL ON THE ISCHEMIC-REPERFUSED RAT MYOCARDIUM: A PHOSPHORUS-31 NUCLEAR MAGNETIC RESONANCE STUDY.~~

~~Abstract~~

To assess whether the prophylactic administration of anipamil, a new calcium antagonist, protects the heart against the effects of ischemia and reperfusion, rats were injected intraperitoneally twice daily for 5 days with 5 mg/kg body weight of this drug. The hearts were then isolated and perfused by the Langendorff technique. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to monitor myocardial energy metabolism and intracellular pH during control perfusion and 30 min of total ischemia (37 °C), followed by 30 min of reperfusion. Pretreatment with anipamil altered neither left ventricular developed pressure under normoxic conditions nor the rate and extent of depletion of adenosine triphosphate and creatine phosphate during ischemia. Intracellular acidification, however, was attenuated. On reperfusion, hearts from anipamil-pretreated animals recovered significantly better than untreated hearts, with respect to replenishment of ATP and creatine phosphate stores, restitution of low levels of intracellular inorganic phosphate and recovery of left ventricular function and coronary flow. Intracellular pH recovered rapidly to preischemic levels, whereas in untreated hearts a complex intracellular inorganic phosphate peak indicated the existence of areas of different pH within the myocardium. It is concluded that anipamil pretreatment protects the heart against some of the deleterious effects of ischemia and reperfusion. Because this protection occurred in the absence of a negative inotropic effect during normoxia, it cannot be attributed to an energy sparing effect during ischemia. Therefore, alternative mechanisms of action are to be considered.

~~31 Chapter 2~~

~~Introduction~~

e myocardial ischemia results in a number of events, including depletion of high-energy phosphate stores ' , intracellular acidification ' , loss of ionic homeostasis, and mitochondrial and membrane damage 5 ultimately leading to cell death. To date the exact sequence of events and the relative importance of several contributing mechanisms are still unclear. The only way to stop this process, however, is by timely reestablishing coronary flow , although after a critical period c n Q of ischemia this may paradoxically extend or accelerate ischemic cell damage ' ' . Reperfusion before this critical period will cause either uncomplicated recovery or delayed restoration of metabolism and function ("stunned myocardium") . Because ischemia-reperfusion damage in myocardial cells is associated with the accumulation of calcium (Ca ) ions in the cytoplasm and mitochondrial matrix ' ' , calcium antagonists have been used to protect the myocardium during ischemia 2>12>13 and subsequent reperfusion 14'15. Several mechanisms for their effectiveness have been proposed including afterload reduction by peripheral arterial vasodilation, negative inotropic and chronotropic effects reducing myocardial oxygen consumption, and coronary vasodilation and enhancement of collateral flow improving blood supply to the jeopardized tissue . The combination of these effects would help to maintain the balance of energy expenditure and supply under circumstances of impaired oxygen and substrate availability. More recently, additional direct effects of calcium antagonists have been postulated on myocardial metabolism , the myocardial cell membrane or cellular viability ' during ischemia and reperfusion. The aim of this study was to determine whether the prophylactic treatment of rats with anipamil, a new calcium antagonist, protects their isolated heart during ischemia and subsequent reperfusion with regard to high-energy phosphate metabolism, intracellular pH and cardiac function. Anipamil is a highly lipophilic verapamil-type calcium antagonist, intended for long-lasting antihypertensive and cardioprotective action . The potential of phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy to evaluate the protective effect of a calcium antagonist was first demonstrated by Nunnally and Bottomley 23. We have used this technique to nondestructively follow the time course in phosphorus-containing metabolites and intracellular pH 2>4>24 with a simultaneous determination of left ventricular function.

~~32 Protection by anipamil~~

~~Methods~~

Animal preparations Male Wistar rats, weighing 300 to 350 g received twice daily intraperitoneal injections containing either 5 mg/kg body weight anipamil (Knoll AG) dissolved in 5% glucose solution or the solvent without the drug. On the fifth day of this regimen, 2 h after the last injection, the rats were anesthetized with ether and heparinized; the heart was then rapidly excised. Perfusion was started by the Langendorff technique at a constant pressure of 75 mm Hg as previously described . The heart was stimulated throughout the experiment at 300 beats/min by two sodium chloride wick electrodes sutured to the right ventricle. Left ventricular pressure was measured by way of a perfusate-filled open catheter, inserted through the apex £3^°. The difference between peak-systolic and end-diastolic pressure was taken to be the left ventricular developed pressure. The heart was placed in a 20 mm NMR tube with a capillary containing methylene diphosphonate for spectral reference. The glass tube with the submerged heart was then lowered into the magnet The effluent was collected in 5 min fractions for determination of coronary flow. Myocardial temperature was carefully maintained at 37 °C.

NMR methods P-NMR spectra were obtained on a Broker MSL 200 spectrometer equipped with a 4.7 tesla vertical bore magnet. No field frequency lock was used. Five minute spectra were obtained from 128 accumulated free induction decays following 90 D pulses repeated at 2.3 s intervals. The data were accumulated using a 2K timetable and 5,000 Hz spectral width. Exponential multiplication resulting in 10 Hz line broadening was applied and baseline correction was performed on thespectra. Fig.l shows typical examples of spectra obtained during preischemic control perfusion of a heart from an untreated (A) and an anipamil-pretreated rat (B). Intra- and w extracellular pH were calculated from the chemical shift of the respective inorganic h phosphate peaks relative to methylene diphosphonate. Zero parts per million was assigned to creatine phosphate ^ Quantitation of metabolites was achieved by integrating the areas under individual peaks of interest in each spectrum, with the Z beta-phosphate peak of adenosine triphosphate (ATP) representing ATP levels. | Data on high energy phosphate metabolites are presented as changes relative H to the average of three successive control measurements preceding ischemia. Data §| were processed in a blinded fashion. Levels of intracellular inorganic phosphate Bf (Pi) are expressed as a percentage of the total amount of phosphate groups from P creatine phosphate (CP), ATP and intracellular Pi during preischemic control §£- I 33 Chapter 2 perfusion:

~~Pi/[CP + 3ATP + Pi]control perfusion x 100%.~~

~~Creatine phosphate and intracellular Pi were corrected for partial saturation; the saturation factors 1.5 and 1.1, respectively, were determined using a 10 s recycle time.~~

Experimental protocol After 30 to 35 min of control perfusion all hearts were made totally ischemic for 30 min by cross-clamping the perfusion line, this period was followed by 30 min of reperfusion. During the control period the hearts were allowed to stabilize for about 15 min. Subsequently, three spectra were recorded during control perfusion, six during ischemia and six during reperfusion.

Statistical analysis Results are presented as mean ± standard deviation (SD) of 25 anipamil and 13 control experiments. Because consecutive measurements were performed on each heart to establish the time course in phosphorus containing metabolites and pH, differences between treated and untreated hearts were statistically evaluated by "analysis of variance with repeated measurements"27>28. A test result with a p-value < 0.05 was considered significant. Data on ischemia and reperfusion were treated separately. In addition, data on creatine phosphate, ATP and intracellular Pi during reperfusion were analyzed for the occurrence of a steady state, as were pH data during ischemia.

Table 1. Effects of pretreatment with anipamil on myocardial function and coronary flow of isolated rat hearts during control perfusion and after 30 min of reperfusion following 30 min of total ischemia.

~~Pre-ischemic Reperfusion~~

LVDP CF LVEDP LVDP CF LVEDP (mmHg) (ml/min) (mmHg) (mmHg) (ml/min) (mmHg) untreated 85.4+3.0 13.2+3.2 3.4±0.9 32.4+24.8 8.2+23 323+243 (n = 13) anipamU- 82.4+6.8 12.6+2.0 33+0.8 79.8+10.9* 14.6+2.1* 7.6+6.0* pretreated (n = 25)

*p<0.0001 vs. untreated; CF=coronary flow, LVDP=teft ventricular developed pressure; LVEDP: left ventricular end-diastolic pressure. 34 Protection by anipamil

~~Results~~

Myocardicd function and coronary flow Typical examples of left ventricular pressure recordings obtained during control perfusion, ischemia and reperfusion from an untreated and an anipamil pretreated heart are shown in Figure 1. Table 1 summarizes values for left ventricular de-

~~mntig 100 r~~

~~5 0 -S -10 -15 ppm~~

~~untrtattd /pv nnHg 100~~

~~e v 7 V **~VV^.Mty>-,~~

~~5 0-5 -10 -15 ppm 5 0 -S -10 -JS pp»~~

~~nfaml m mmHg lamHg 100. 100~~

~~5 0-5 -10 -15 5 0-5 -JO -15 ppm~~

Figure 1. 3lP-NMR spectra and simultaneous left ventricular pressure recordings from an untreated (A,QE)andan anipamilpretreated (B,D,F) rat heart during control perfusion (A,B), between 15 and 20 min of ischemia (QD), and between 25 and 30 min of reperfitsion (E,F). Numbered peaks are: 1, extracellular inorganic phosphate (Pi); 2, intracellular Pi 3, creatine phosphate; 4,5,7, y-, a-and ^-phosphate group of adenosinetriphosphate; and 6, nicotine-amide adenine dinucleotide.

~~35 Chapter 2~~

~~ISCHEMIA~~

~~20 30 40 50 60 Time (mm)~~

Figure 2. Effect ofpretreatment of ruts with anipamil on the time course in levels of (A) creatine phosphate (CP); (B) adenosine triphosphate (ATP); and (C) intracellular inorganic phosphate (Pi) as measured with 3lP-NMR in isolated perfused hearts submitted to 30 min ofnormother- mic global ischemia followed by 30 min of reperfusion. CP and ATP levels are expressed as a percent of their respective preischemic values. Intracellular P\ is expressed as a percent of the amount of phosphate groups from CP, ATP and intracellular Pi during preischemic control perfiision: Ptl[CP + 3ATP + Pi/p«ischemic x 100%. Measurements were obtained from consecutive 5 min 3lP-NMR spectra. Each point represents the mean ± SD of 25 anipamil-pretreated or!3 untreated hearts. During ischemia treated and untreated hearts were not different, whereas during reperfusion recovery ofCPand ATPwere significantly better in treatedhearts and intracellular Pi levels were lower. *p < O.OOOl versus untreated.

~~36 Protection by anipamil~~

~~10 20 30 50 60 Time (mm)~~

Figure3. Effect of pretreatment of rats with anipamilon intracellularpHoftheir isolatedperfused heart during 30 min of global ischemia followed by 30 min of reperfusion, as assessed with P-NMR spectroscopy. During reperfusion of the untreated heart, a complex pattern in the intracellular Pi region was observed, corresponding with a range of intracellularpH values (see Fig.le). The highest and lowest values are indicated by the upper and lower dashed curves, respectively. Each point represents the mean ± SD of 25 pretreated or 13 untreated hearts. After 10 min of ischemia, pH in treated hearts was significantly higher than in untreated hearts. *p < 0.0001 versus untreated. veloped pressure and end-diastolic pressure during preischemic control perfusion and at the end of reperfusion. During control perfusion there was no significant difference in contractile performance between anipamil-pretreated and untreated hearts, whereas functional recovery after ischemia and reperfusion was significantly better in the anipamil group. Moreover, contracture, as indicated by the increase in end-diastolic pressure, was less pronounced in treated hearts. During ischemia left ventricular developed pressure decreased rapidly to zero in both groups and V," the period of still detectable contractile activity was equal (25 to 3 min). On reperfusion, contraction usually began with a period of ventricular fibrillation or chaotic ventricular arrhythmia, which gradually gave way to stable contractile function. As this process showed large variations, only values for left ventricular developed pressure at the end of reperfusion are presented. m Coronary flow during preischemic control perfusion in treated hearts was not I significantly different from flow in untreated hearts (Table 1), indicating that there was no vasodilation due to anipamil pretreatment. like recovery of mechanical function, restoration of coronary flow on reperfusion was more complete in the anipamil group and even exceeded flow rates during control perfusion. In untreated

~~37 Chapter 2 hearts a considerable reduction in coronary flow was observed after ischemia.~~

NMR spectroscopy: creatine phosphate, ATP and inorganic phosphate (P\) Figure 1 shows examples of 31P-NMR spectra with simultaneous left ventricular pressure recordings during control perfusion (A and B), between 15 and 20 min of ischemia (C and D), and at the end of reperfusion (E and F), obtained from an untreated heart (A.C.E) and a heart from an anipamil pretreated rat (B,D,F). They illustrate the similarity of untreated and anipamil pretreated hearts in phosphorus containing metabolites during control perfusion and ischemia, but not during reperfusion. Figure 2 summarizes creatine phosphate, ATP and intracellular Pi levels in hearts from untreated and anipamil pretreated rats during ischemia and reperfusion. The depletion of high energy phosphates during ischemia, which was balanced by a simultaneous accumulation of intracellular Pi, was similar in both groups. Reperfusion induced a rapid and complete restoration of creatine phosphate in anipamil pretreated hearts, whereas in untreated hearts only about 40% of preischemic levels were replenished, the difference between treated and untreated hearts being significant (p < 0.0001). Recovery of ATP in anipamil-pretreated hearts was also better than in untreated hearts (p < 0.0001), but no complete restoration of ATP was found. Statistical analysis indicated that after 10 min of reperfusion, no further improvement of high energy phosphate metabolites occurred. Pretreatment with anipamil also helped to restore low levels of intracellular inorganic phosphate; in untreated hearts intracellular Pj levels remained elevated even after 30 min of reperfusion and were significantly higher than in treated hearts (p < 0.0001). The major changes in intracellular Pj levels took place during the first 10 min of reperfusion, but no real steady state was reached.

IntracelUularpH During preischemic control perfusion intracellular pH was not affected by pretreatment with anipamil (Fig.3), but after 10 min of ischemia acidification of the cytosol was attenuated as compared to untreated hearts (p < 0.0001). Unlike depletion of high-energy phosphates during ischemia, which was unaffected by anipamil pretreatment, pH values stabilized at a higher level in hearts from anipamil-pretreated rats than in untreated hearts. The final pH in both groups was reached after 15 min of ischemia. During reperfusion intracellular pH rapidly recovered to pre-ischemic values in treated hearts, whereas in untreated hearts the 31P-NMR spectra were often characterized by the presence of multiple intracellular Pi peaks (Fig. IE), corre-

~~38 Protection by anipamil sponding with different pH values within the myocardium . Despite this incomplete or inhomogeneous reperfusion, the main Pj peak corresponded with a pH of 7.00 to 7.10.~~

~~Discussion~~

These results demonstrate that the isolated perfused heart of the rat pretreated with the new calcium antagonist anipamil is protected against some of the effects of total ischemia and reperfusion. Intracellular acidosis during ischemia was attenuated. On reperfusion, the heart in the anipamil pretreated animals showed a significantly better recovery of biochemical and functional variar/ *~ *han did the untreated heart.

Previous studies It is generally accepted that for optimal protection against ischemia-reperfusion- induced damage, calcium antagonists should be present before or, at the latest, during the ischemic event ^ . The protective effect of calcium antagonists is commonly attributed to the energy-sparing effect during ischemia due to their 11 13 15 2 35 negative inotropic properties ' . > ' ^ - ^ fnat wav more energy would remain available for the maintenance of cellular ionic homeostasis, particularly with respect to sodium (Na+) and calcium (Ca2+) ions. On the other hand there is evidence that mechanisms unrelated to reduction of cardiac work may play a role in the protection exerted by calcium antagonists. Henry and Wahl demonstrated that hypoxic contracture in electrically and mechanically quiescent myocardium was suppressed by diltiazem and nifedipine, thereby exclud- ing a protective effect in terms of energy conservation. Others 18^° reported protection of isolated rat hearts in the presence of diltiazem, nifedipine and verapamil at such a low concentration that no negative inotropic effect could be observed. They suggested a direct preservation of cellular viability and a direct 18 beneficial effect on the energy metabolism of the ischemic heart . To date, only a few studies on the cardioprotective action of anipamil are available. Raschack and Kirchengast demonstrated that ST elevation and potassium release after coronary artery occlusion in pigs were attenuated by anipamil. Brezinski et al 21, who ligated the left anterior descending artery of cats in vivo, found cardioprotection without reduced oxygen demand in the presence of anipamil. They proposed a direct cytoprotective action of anipamil during ischemia. Curtis et al 38 studied the effects of anipamil in rats in vivo after coronary artery 39 Chapter 2

~~ligation and observed an antiarrhythmic effect during ischemia without changes of heart rate and blood pressure during the preischemic period.~~

Biochemical and functional effects of anipamil In our study, no negative inotropic effect was observed in the isolated heart of anipamil-pretreated rats. It is in accordance with this finding that neither the rate nor the extent of depletion of adenosine triphosphate (ATP) and creatine phosphate during ischemia was attenuated. Despite the lack of an energy-sparing effect during ischemia, recovery of high energy phosphates and cardiac function during reperfusion was better in treated than in untreated hearts and contracture was prevented. Anipamil pretreatment also improved restoration of coronary flow of the isolated heart during reperfusion as compared with the untreated hearts. It is questionable whether this is a direct consequence of vasodilation by anipamil, because coronary flow under preischemic conditions was not different in treated and untreated hearts. In our experimental model, impaired reperfusion after transient ischemia (no reflow phenomenon) may be due to endothelial cell swelling and vasoconstriction and myocardial cell swelling and contracture of myocytes ' . Although prevention of contracture by anipamil (Table 1) may contribute to a better reperfusion, it may not be a complete explanation because nifedipine was found to improve coronary flow during reperfusion without reduction of contracture . We propose that either direct cytoprotective effects ' or a rapid recovery of cardiac metabolism enables a better control of transmembrane ionic currents and cytosolic osmolarity in both vascular cells and myocytes, thereby preventing the formation of edema and contracture. This in turn will enhance tissue perfusion and may further facilitate aerobic metabolism.

Intracellular pH v. In our experiments the major difference during ischemia between treated and i untreated hearts appeared to be the intracellular pH, which stabilized at a significantly higher level in treated hearts. A limitation of the amount of hydrogen ions generated during ischemia may reduce the cellular uptake of Ca2+ at the onset of C reperfusion by way of the sarcolemmal H+/Na+ and Na+/Ca2+ exchange mechan- | isms . Because uncontrolled influx of Ca2+ into the cells is one of the crucial steps 1; in the development of reperfusion damage 6'10111, limitation of the production of £ hydrogen ions during ischemia would eventually facilitate recovery of the myocar- fi dium. f| There are several possible explanations for the attenuation of intracellular r 40 Protection by anipamil acidosis during ischemia in the anipamil-treated hearts. Because Ca is known to stimulate the conversion of phosphorylase b into phosphorylase a, which is necessary for the breakdown of glycogen , limitation of Ca2+ influx during ischemia would contribute to a reduction of glycogen degradation and a concomitant attenuation of in tracellular acidosis . It has been shown for several calcium antagonists ' that a limitation of Ca influx during ischemia can occur even in the absence jf energy-sparing effects. It is conceivable that such a limitation of Ca influx is not necessarily the result of a direct interaction between calcium antagonists and the slow channels, but may also be mediated by a reduced release of catecholamines 43, which activate the slow channels and augment Ca2+ influx. It is also possible that catecholamine stores were already depleted because of the pretreatment of the animals, as has been shown for other verapamil-type calcium antagonists • ' . A decreased availability of catecholamines during ischemia will also reduce the cyclic adenosine monophosphate- (cAMP)-mediated degradation of glycogen.

Alternative mechanism It is uncertain whether the influx of Ca2+ early during reperfusion is mediated by the slow channels because these may be inactivated by acidosis and dephosphorylation . In most studies l~ the presence of calcium antagonists only during reperfusion failed to reduce reperfusion damage. However, Weishaar and Bing suggested a limitation of the massive Ca influx during reperfusion by diltiazem. More likely routes for Ca + entry are the Na+/Ca + exchange mechanism , as mentioned before, and an increased membrane permeability for Ca + ' • . Therefore, the protection exerted by anipamil may also be related to effects on the cell membrane. In particular, the phenylalkylamines (like verapamil and anipamil) are supposed to exert their effects by initially dissolving into the phospholipid bilayer and subsequently migrating toward the slow channels . Anipamil would be a perfect candidate for such a mode of action because of its highly lipopbilic properties. In addition, its presence in the phospholipid bilayer could make the "r, sarcolemma less sensitive to Ca -induced conformational changes during ischemia ,^g and reperfusion . By maintaining sarcolemmal integrity during ischemia and '^ reperfusion, excessive entry of Ca2+and irreversible cell damage would then be (| prevented. Additional experiments will be needed to elucidate the exact mechanism >• of myocardial protection by anipamil.

~~Conclusions s Pretreatment of rats with anipamil protected their isolated heart against ischemia-reperfusion-induced damage. In contrast to most in vitro studies with calcium~~

~~41 Chapter 2~~

antagonists, in which protection can be attributed to a negative inotropic effect that in the in vivo situation might be overcome by compensatory mechanisms, in our experiments pretreatment with anipamil did not impair contractile performance of the heart. Because anipamii appeared to be an effective drug to prevent the aggravation or acceleration of ischemic damage by reperfusion, it may be a promi- sing tool for clinical practice in which reperfusion is of increasing importance.

~~We thank Pieter van der Meer and Dirk de Moes for excellent technical assistance and Ingeborg van der Tweel for statistical advice. Anipamil was kindly supplied by Knoll AG, Ludwigshafen, F.R.G.~~

~~References~~

1. Hearse DJ, Garlick PB, Humphrey SM. Ischemic contracture of the myocadium: mechanisms and prevention. Am J Cardiol 1977; 39:987-993. 2. Ruigrok TJC, Van Echteld CJA, De Kruijff B, Borst C, Meijler FL. Protective effect of nifedipine in myocardial ischemia assessed by phosphorus-31 nuclear magnetic resonance. Eur Heart J 1983; 4(suppl C): 109-113. 3. Cobbe SM, Poole-Wilson PA. The time of onset and severity of acidosis in myocardial ischaemia. J Mol Cell Cardiol 1980; 12:745-760. 4. Garlick PB, Radda GK, Seeley PJ. Studies of acidosis in the ischaemic heart by phosphorus nuclear magnetic resonance. Biochem J 1979; 184:547-554. 5. Jennings RB, Rehner KA, Steenbergen C. Myocardial ischemia revisited. The osmolar load, membrane damage, and reperfusion. J Mol Cell Cardiol 1986; 18:769-780. 6. Hearse DJ. Reperfusion of the ischemic myocardium. J Mol Cell Cardiol 1977; 9:605-616. 7. Bourdillon PD, Poole-Wilson PA. The effects of verapamil, quiescence, and cardioplegia on calcium exchange and mechanical function in ischemic rabbit myocardium. Circ Res 1982; 50:360-368. 8. Manning AS, Hearse DJ. Reperfusion-induced arrhythmias: mechanisms and prevention. J Mol Cell Cardiol 1984; 16:497-518. 9. Braunwald E, Kloner R. The stunned myocardium: prolonged, postischemic dysfunction. Circulation 1982; 66:1146-1149. 10. Jennings RB, Ganote CE. Mitochondrial structure and function in acute myocardial ischemic injury. Circ Res 1976; 38(suppl 1): 80-89. 11. Nayler WG, Ferrari R, Williams A. Protective effect of pretreatment with verapamil, nifedipine and propranolol on mitochondrial function in the ischemic and reperfused myocardium. Am J Cardiol 1980; 46:242-248. 12. Henry PD, Shuchleib R, Davis J, Weiss ES, Sobel BE. Myocardial contracture and accumulation of mitochondrial calcium in ischemic rabbit heart. Am J Physiol 1977; 233: H677-684.

~~42 Protection by anipamil~~

13. De Jong JW, Huizer T. Reduced glycolysis by nisoldipine treatment of ischemic heart. J Cardiovasc Phannacol 1985; 7:497-500. 14. Sashida H., Abiko Y. Protective effect of diltiazem on ultrastmctural alterations induced by coronary occlusion and reperfusion in dog hearts. J Mo] Cell Cardiol 1986; IS: 401-411. 15. Ichihara K, Abiko Y. Effects of diltiazem and propranolol on irreversibility of ischemic cardiac function and metabolism in the isolated perfused rat heart. J Cardiovasc Phar- macol 1983; 5:745-751. 16. Braunwald E. Mechanism of action of calcium-channel- blocking agents. N EngI J Med 1982; 307:1618-1627. 17. Crome R, Hearse DJ, Manning AS. Ischemia- and reperfusion-induced arrhythmias: beneficial actions of nifedipine. J Cardiovasc Phannacol 1986; 8:1249-1256. 18. Lavanchy N, Martin J, Rossi A. Effects of diltiazem on the energy metabolism of the isolated rat heart submitted to ischaemia: a 31P NMR study. J Mol Cell Cardiol 1986; 18: 931-941. 19. Drake-Holland AJ, Noble MIM. Myocardial protection by calcium antagonist drugs. Eur Heart J 1983; 4:823-825. 20. Higgins AJ, Blackburn KJ. Prevention of reperfusion damage in working rat hearts by calcium antagonists and caunodulin antagonists. J Mol Cell Cardiol 1984; 16:427-438. 21. Brezinski ME, Darius H, Lefer AM. Cardioprotective actions of a new calcium channel blocker in acute myocardial ischemia. Drug Res 1986; 36:464-466. 22. Raschack M, Gries J. Cardioprotective and anti-hypertensive effects of the new calcium antagonist anipamil in rats and hamsters. J Mol Cell Cardiol 1986; 18 (suppl I): 119 (abstract). 23. Nunnally RL, Bottomley PA. Assessment of pharmacological treatment of myocardial infarction by phosphorus-31 NMR with surface coils. Science 19814211:177-180. 24. Flaherty JT, Weisfeldt ML, Bulkley BH, Gardner TJ, Gott VL, Jacobus WE. Mechan- isms of ischemic myocardial cell damage assessed by phosphorus-31 nuclear magnetic resonance. Circulation 1982; 65:561-571. 25. Neely JR, Liebenneister H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol 1967; 212:804-814. 26. Neely JR, Grotyohann LW. Role of glycolytic products in damage to ischemic myocardium. Circ Res 1984; 55:816-824. 27. Dixon WJ. BMDP Statistical Software. Berkely: University of California Press, 1985. 28. Fleiss JL. The Design and Analysis of Clinical Experiments. New York: John Wiley and Sons, 1986:220-235. 29. Bailey IA, Seymour AML, Radda GK. A 31P-NMR study of the effects of reflow on the ischaemic rat heart. Biochim Biophys Acta 1981; 637:1-7. 30. Nayler WG.PanagiotopoulosS, ElzJS, Sturrock WJ. Fundamental mechanisms of action of calcium antagonists in myocardial ischemia. Am J Cardiol 1987; 59:75B-83B. 31. Fleetwood G, Poole-Wilson PA. Diastolic coronary resistance after ischaemia in the isolated rabbit heart: effect of nifedipine. J Mol Cell Cardiol 1986; 18:139-147. 32. Ferrari R, Albertini A, Cureilo S, Ceconi C, Dilisa F, Raddino R, Visioli O. Myocardial recovery during post- ischaemic reperfusion: effects of nifedipine, calcium and magnesium. J Mol Cell Cardiol 1986; 18:487-498. 33. Watts JA, Koch CD, LaNoue KF. Effects of Ca2+ antagonism on energy metabolism: Ca2+ and heart function after ischemia. Am J Physiol 1980; 238: H909-H916.

~~43 Chapter 2~~

34. Watts J A, Maiorano LJ, Maiorano PC. Comparison of the protective effects of verapamil, dilliazein, nifedipine, and buffer containing low calcium upon global myocardial ischemic injury. J Mol Cell Cardiol 1986; 18:255-263. 35. Weishaar RE, Bing RJ. The beneficial effect of a calcium channel blocker, diltiazem, on the ischemic-reperfused heart. J Mol CeU Cardiol 1980; 12:993-1009. 36. Henry PD, Wahl AM. Diltiazem and nitrendipine suppress hypoxic contracture in quiescent ventricular myocardium. Eur Heart J 1983; 4:819-822. 37. Raschack M, Kirchengast M. Inhibition by anipamil of epicardial ST-elevation and K+ liberation after coronary artery occlusion in pigs. J Mol Cell Cardiol 1986; 18 (suppl I): 142 (abstr). 38. Curtis MJ, Walker MJA, Yuswack T. Actions of the verapamil analogues anipamil and ronipamil against ischaemia-induced arrhythmias in conscious rats. Br J Pharmacol 1986; 88:355-361. 39. Apstein CS, Mueller M, Hood WB. Ventricular contracture and compliance changes with global ischemia and reperfusion, and their effect on coronary resistance in the rat. Ore Res 1977; 41:206-217. 40. Lazdunski M, Frelin C, Vigne P. The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. J Mol Cell Cardiol 1985; 17:1029-1042. 41. Drummond GI, Duncan L. The action of calcium ion on cardiac phosphorylase b kinase. J Biol Chem 1966; 241:3097-3103. 42. Van der Vusse GJ, Van der Veen FH, Prinzen FW, Coumans WA, Van Bilsen M, Reneman RS. The effect of diltiazem on myocardial recovery after regional ischemia in dogs. Eur J Pharmacol 1986; 125:383-394. 43. Nayler WG, Sturrock WJ. An inhibitory effect of verapamil and diltiazem on the release of noradrenaline from ischaemic and reperfused hearts. J Mol Cell Cardiol 1984; 16: 331-344. 44. Chaudhry A, Vohra MM. A reserpine-like action of verapamil on cardiac sympathetic nerves. Eur J Pharmacol 1984; 97:156-158. 45. Chaudhry A, Vohra MM. Depletion of cardiac noradrenaline stores by the calcium- channel blocker D-600. Can J Physiol Pharmacol 1984; 62:640-644. 46. Sperelakis N. Properties of calcium-dependent slow action potentials: their possible role in arrhythmias. In: Opie LH ed. Calcium Antagonists and Cardiovascular Disease. New York: Raven Press, 1984:277-291. 47. Poole-Wilson PA, Harding DP, Bourdillon PD V, Tones MA. Calcium out of control. J Mol Cell Cardiol 1984; 16:175-187. 48. Katz AM, Pappano AJ, Messineo FC, Smilowitz H, Nash-Adler P. Calcium-channel- blocking drugs. In: Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE eds. The Heart and Cardiovascular System. New York: Raven Press, 1986:1597-1611. 49. Post JA, Leunissen-Bijvelt J, Ruigrok TJC, Verkleij AJ. Ultrastructural changes of sarcolemma and mitochondria in the isolated rabbit heart during ischemia and reperfusion. Biochim Biophys Acta 1985; 845:119-123.

~~44 Protective mechanisms of anipamil~~

~~POSSIBLE MECHANISMS OF THE PROTECTIVE EFFECT OF PRETREATMENT WITH THE CALCIUM ANTAGONIST ANIPAMIL IN ISCHEMIC-REPERFUSED ISOLATED RAT HEARTS~~

~~Abstract~~

In order to gain more insight in the protective action of pretreatment with the new calcium antagonist anipamfl, rats were administered anipamil (5 mg/kg) or glucose, intraperitoneally twice daily for 5 days. During this period mean arterial blood pressure (MABP) and heart rate (HR) were measured daily. The heart was then isolated and perfused according to Langendorff. P nuclear magnetic resonance ( P NMR) spectroscopy was used to monitor energy metabolism and intracellular pH during 30 min of ischemia followed by 30 min of reperfusion, with a simultaneous isovolumetric measurement of left ventricular function. Myocardial noradrenaline and glycogen content were assayed immediately after excision of the heart, after IS min oxygenated perfusion, at the end of ischemia and at the end of reperfusion. Metabolic and functional recovery during postischemic reperfusion were significantly better in hearts pretreated with anipamil (p < 0.0005 vs controls). However, protection was not preceded by an effect on MABP or HR in vivo, or a negative inotropic effect during control perfusion of the isolated hearts. Accordingly, there was no energy sparing effect during ischemia. Only intracellular pH during ischemia stabilized at a higher level (p < 0.0005 vs controls). Myocardial noradrenaline and glycogen stores were not decreased by pretreatment with anipamil, and the release ^ or degradation due to ischemia and reperfusion were also not different from M controls. -f In conclusion, commonly known mechanisms of myocardial protection by cal- J cium antagonists fail to explain the protection by pretreatment with anipamil as observed in our experiments, and alternative mechanisms arc to be considered. s 1

~~45 Chapter 3~~

~~Introduction~~

-fyetreatment of rats with the new calcium antagonist anipamil was found to •* improve postischemic recovery of their isolated heart *. Protection could not be attributed to a negative inotropic effect during preischemic control perfusion or an energy sparing effect during ischemia. It was suggested that attenuation of ischemic acidosis may be an important mechanism of protection, as has been reported for some /?-blockers in vitro and in vivo ' , and also for calcium antagonists 4. Depletion of glycogen stores before ischemia or a decreased utilization of glycogen during ischemia may have such an effect on the pH during ischemia 6. Since the breakdown of glycogen is enhanced by catecholamine stimulation , reduced catecholamine release may be a mediator of the reduction of ischemic acidosis . However, conflicting results have been reported on a possible protective effect of preischemic glycogen depletion . Alternatively, release of noradrenaline from myocardial tissue during ischemia and reperfusion " will cause several other detrimental effects, including vasoconstriction, platelet aggregation, excessive energy utilization and arrhythmias Therefore, both a depletion of noradrenaline stores prior to the ischemic event and a reduced liberation of noradrenaline during ischemia (and reperfusion) may be beneficial ' ' . Several studies have shown that calcium antagonists may either reduce noradrenaline stores prior to ischemia ' , or reduce the release of noradrenaline due to ischemia and reperfusion l ' ' . Furthermore, calcium antagonists may offer some protection against tissue damage resulting from high levels of catecholamines ' . This study was performed to gain more insight into the mode of myocardial protection after pretreatment with anipamil, a verapamil-type calcium antagonist intended for long term anti hypertensive and cardioprotective action . We therefore measured (1) blood pressure and heart rate in the awake animals during pretreatment, and (2) noradrenaline and glycogen content after pretreatment, and release (degradation) due to ischemia and reperfusion in the isolated heart. In addition, P NMR spectroscopy was used to monitor myocardial energy metabolism and intracellular pH throughout the protocol, combined with an intraventricular balloon to monitor myocardial function.

~~46 Protective mechanisms ofanipamil~~

~~Materials and methods~~

In all experimental series male Wistar rats, weighing 325 to 375 g, were injected for 5 days intraperitoneally twice daily with either anipamil 5 mg/kg in 5% glucose solution or with the solvent only (controls).

Blood pressure and heart rate in the awake animals In a first series of experiments the femoral artery of anesthetized rats was cannulated and silicon tubing tunneled subcutaneously to the neck for later blood pressure measurement. The animals were allowed to recuperate for three or four days. The cannula was then connected for about 45 to 60 min to a Statham P23dB pressure transducer by means of a swivel, allowing almost unrestrained movement of the rat. During the first 15 to 30 min the animal adapted to the situation as appeared from stabilization of the mean arterial blood pressure (MABP) and resumption of normal activity or sleep. During the next 30 min MABP and heart rate (HR) were read from a Gould Brush recorder every 10 min, averaged over 30 heart beats. Three values for MABP and for HR were thus obtained per day and per rat. Baseline measurements of MABP and HR were performed prior to the first injection. During the treatment period measurements were repeated daily, two hours after the morning injection, for four days. On day five of this regimen, two hours after the last injection, the rats were either killed by a blow on the neck or anesthetized with diethylether, prior to blood sampling for determination of plasma levels of anipamiL The heart was then rapidly excised, rinced with ice-cold saline solution, gently blotted on a tissue and the atria were removed. The ventricles were then divided into two parts, each consisting of half of the left and right ventricle. These tissue samples were weighed and stored in liquid nitrogen until determination of glycogen and noradrenaline.

Isolated perfused hearts In a second series the rats were anesthetized with diethylether and heparinized on day five of the pretreatment, two hours after the last injection. After a blood sample had been taken for determination of the plasma level of anipamil, the heart was rapidly excised and perfused according to Langendorff at a constant pressure of 100 cm H2O as previously described . Left ventricular function was monitored by means of a latex balloon inserted through the left atrium and connected to a Statham P23dB pressure transducer. The balloon was filled to give an end-diastolic level of 10 cm H2O. The hearts were stimulated at 300 beats/min throughout the entire protocol by means of two NaCl wick electrodes attached to the right ven-

47 Chapter 3 tricular outflow tract. Coronary flow was determined by fractioned collection of the effluent from the heart. The heart was then assigned to one of three protocols: 15 min control perfusion, control perfusion plus 30 min of total ischemia, or control perfusion and ischemia followed by 30 min of reperfusion. At the end of each protocol the heart was dismounted from the perfusion cannula and the atria were removed. The ventricles were then treated and stored as described above, for determination of glycogen and noradrenaline. During ischemia the balloon was deflated and during reperfusion the balloon volume was adjusted to maintain an end-diastolic pressure of 10 cm H2O. At the end of 30 minutes of reperfusion the balloon was filled again with the same amount of fluid as during preischemic control perfusion. The end-diastolic pressure resulting from this inflation is given as a measure of contracture.

31P NMR spectroscopy The perfusion sequence of the isolated hearts was performed inside a 4.7 T, vertical bore magnet, interfaced with a Bruker MSL 200 NMR spectrometer. The hearts were placed in a 20 mm NMR tube with a capillary containing methylene diphosphonate (MDP) for spectral reference and were lowered into the magnet. Myocardial and perfusate temperature were carefully maintained at 37 °C. P NMR spectra were obtained in five minutes from 128 accumulated free induction decays after 90° pulses repeated at 2.3 s intervals, with 5000 Hz spectral width. Exponential multiplication resulting in 10 Hz line broadening and deconvolution were applied to enhance the signal to noise ratio and to correct baseline distortions, respectively. Intracellular pH was calculated from the chemical shift of the intracellular inorganic phosphate (Pi) peak relative to methylene diphosphonate. Zero parts per million was assigned to creatine phosphate (CP). Quantitation of metabolites was achieved by integrating the areas under individual peaks of interest in each spectrum, with the /^-phosphate peak of adenosine triphosphate (ATP) representing ATP levels. CP and ATP are given as a percentage of their preischemic levels. Pi is expressed as a percentage of the total amount of phosphate from CP, ATP and intracellular Pi during preischemic control perfusion: Pi/{CP + 3ATP + PiWrol perfiision x 100%. CP and intracellular Pj were corrected for partial saturation; saturation factors of U and 1.1, respectively, were determined using a 10 s repetition time.

~~Tissue glycogen and noradrenaline At the end of each experiment ventricular glycogen was determined by a~~

48 Protective mechanisms ofanipamil spectrophotometric method according to Bergmeyer and protein content according to Lowry . Glycogen content was then expressed as nmol glucose equivalents per mg protein. For determination of noradrenaline the tissue samples were defrozen and homogenized for two periods of 15 seconds at 4 °C in a homogenizing solution consisting of 0.05 mol/1 perchloric acid with 0.1 mmol/I EDTA and 50 ng/ml dehydroxybenzylamine as internal standard. The homogenate was centrifuged at 10,000 x g for 15 min at 4 °C. 500 /A of supernatant was pipetted into a tube containing 20 mg of AI2O3. The pH of the mixture was then increased by adding 1 ml of 3 mol/1 Tris buffer, pH 9.2 and the tube was shaken for 15 min at 4 °C; the supernatant was decanted and the residue washed twice with distilled water and transferred to individual microfilters. Catecbolamines were eluted with 500/tl of 0.2 mol/1 acetic acid. Chromatography was performed as described previously .

~~Plasma levels ofanipamil Plasma levels of anipamil were determined by HPLC (Knoll AG, Ludwigshafen) 31~~

Statistical analysis Results are presented as mean ± SEM. Differences between pretreated animals or hearts and controls were statistically evaluated by Student's t test or analysis of variance with repeated measurements, as appropriate. A test result with a p value < 0.05 was considered significant

~~Results~~

In vivo blood pressure and heart rate Figure 1 summarizes data on mean arterial blood pressure (la) and heart rate \ (lb). There were no significant differences between anipamil pretreated animals |jj and controls on any of the time points shown (p > 0.20 for MABP and HR). In both ;| anipamil pretreated animals and controls MABP and HR did not change signifi- 1 cantly during the pretreatment period. •-•

Tissue noradrenaline content Tissue noradrenaline content (Figure 2a) as measured immediately after exci- = sion of the heart (time 0) was not altered by 5 days of pretreatment with anipamil (p = 0.40 vs controls). Isolation of the hearts followed by 15 minutes of Langendorff

~~49 Chapter 3~~

150 Figure 1. (a) Mean arterial blood pressure (MABP), and (b) heart rate (HR) of rats before (baseline) . i J_ and during treatment with anipamil, 5 mglkg, twice daily C* • ) or glucose (o o). Data represent mean ± SEM (n = 7). Measurements were made two hours after the moming injection. Values obtained from anipamil 4 treated rats and controls were not significantly different on any of the time points shown.

270 day 1 day 2 day 3 day time perf usion did not induce any major changes in noradrenaline content in both control hearts and anipamil pretreated hearts. Therefore, the noradrenaline content at the onset of ischemia was not different in pretreated hearts and controls (p = 0.31). After 30 minutes of ischemia the noradrenaline content (as expressed per gram wet weight) was apparently increased to an equal extent in both experimental groups (p = 0.78 vs controls). However, this can be explained by an increased dry to wet weight ratio (0.21 ±0.01 at the end of ischemia as compared to 0.15 ±0.01 after control perfusion and 0.16±0.01 at the end of reperfusion). When taking into account this 30 to 40 % increase in dry to wet weight ratio, and the fact that after total ischemia (without reperfusion) tissue noradrenaline cannot be distinguished from noradrenaline released to the extracellular space, this observation merely confirms the similarity in tissue noradrenaline before ischemia. At the end of 30 minutes of reperfusion, tissue noradrenaline content had decreased in both pretreated and control hearts, the difference between the two groups not being significant (p = 0.22). Therefore, myocardial noradrenaline release due to 30 minutes of ischemia and reperfusion was not diminished by pretreatment of the rats with anipamil.

~~50 Protective mechanisms ofanipamil~~

1000 Figure 2. Myocardial tissue content a of noradrenaline (a) and glycogen 1 (b) after five days of pretreatment I 800 with anipamil (Y%&) or glucose Ol £ 6OO • fHHJ- Time points refer to hearts 8 assayed immediately after excision ra 40° (0), after 15min of oxygenated Lan- u t gendorffperfusion (15), afterWmin ra 200 1 of total ischemia (45) and at the end Hi of'30 min of postischemic reperfusion (75). Data represent mean and I15 45 time (mm) SEM (n = 7 or 10). None of the values from anipamil pretreated hearts differed significantly from the corresponding value from controls. The apparent increase in endischemic tissue noradrenaline content is caused by an increase in the dry to wet weight ratio. For further explanation see text

~~time (mm)~~

Tissue glycogen content Myocardial glycogen content after pretreatment with anipamil (Figure 2b) was not signiGcantily different from controls as measured immediately after excision of the heart (time 0, p = 0.15). Fifteen min of control perfusion did not induce a major change in glycogen content in both pretreated and control hearts, the difference again not being significant (p = 0.22). At the end of 30 minutes of ischemia, and also after 30 minutes of reperfusion, myocardial glycogen stores were depleted in pretreated and control hearts, the difference between the two groups not being significant (p = 0.56 and 0.28, respectively).

Left ventricular Junction and coronary flow Table 1 summarizes data on left ventricular pressure and coronary flow during preischemic control perfusion and at the end of reperfusion. During control perfusion LVDP in anipamil pretreated hearts was not different from controls (p = 0.26), and coronary flow was significantly lower (p = 0.002). In pretreated hearts recovery of LVDP during reperfusion was complete, and coronary flow was higher than during preischemic control perfusion. On the other hand, control hearts showed a

51 Chapter 3 significantly worse recovery of LVDP (p = 0.0006), and postischemic coronary flow was impaired as compared to the preischemic situation ("no reflow") and significantly lower than in pretreated hearts (p = 0.001). Contracture, as indicated by the increase in end-diastolic pressure, was very pronounced in control hearts, but almost completely absent in pretreated hearts.

31PNMRdata Figure 3a summarizes the data on CP, ATP and P, during ischemia and reperfusion. According to the lack of a negative inotropic effect before ischemia in pretreated hearts, the rapid breakdown of CP and ATP, with a concomitant increase in Pi, did not differ significantly from control hearts. However, intracellular pH stabilized at a higher level (p < 0.0005) in pretreated hearts (Figure 3b). Reperfusion induced a rapid and complete restoration of CP and a 50% recovery of ATP in pretreated hearts, unlike control hearts, which recovered only poorly. At the same time, anipamil pretreatment helped to restore low levels of intracellular Pi during reperfusion. All differences were highly significant (p < 0.0005). In control hearts intracellular Pi levels remained elevated even after 30 min of reperfusion, and the spectra were often characterized by the presence of multiple intracellular Pi peaks, corresponding with different pH values within the myocardium . In figure 3b the highest and lowest pH value are shown in the upper and lower dashed curve, respectively. In pretreated hearts intracellular pH recovered homogeneously to preischemic values.

~~Table 1. Left Ventricular Function and Coronary Flow~~

~~Freischemic Postischemic~~

~~LVDP EDP flow LVDP EDP flow~~

~~(cm H20) (cmHzO) (ml/min) (on 1*0) (cmH20) (ml/min)~~

~~untreated 117+6 10 14.1+0.5 50±9 164+15 10.9±0.6~~

~~anipamil 109+5 10 11.7+0.5 118±12 35+5 14.7+0.5 pretreated~~

~~LVDP: left ventricular developed pressure EDP: end-diastolic pressure. During control perfusion the intraventricular balloon volume was ad-~~

~~justed to obtain an EDP of 10 cm H2O; at the end of postischemic reperfusion the balloon was filled again with the preischemic volume, to measure contracture. Data are presented as mean+SEM.~~

~~52 Protective mechanisms ofanipamil~~

~~31 140 Figure 3. P AflMK dbfa obtained ischernia reperfus on a from isolated rat hearts during ischemia and reperfusion after five days of presentment with anipamil I 80 (• *) or glucose (o o). (a)~~

\ High-energy phosphates and Ft ""* 40 During ischemia creatine phos- U 20 A phate (CP) and ATP were rapidly depleted with a concomitant in- L-\ ., crease in intracellular inorganic phosphate (Pi). Anipamil pretreated and control hearts were not different On reperfusion CP and ATPrecoveredsignificantfybetterin hearts pretreated with anipamil as compared to controls and lower levels of Pi were restored (p < 0.0005 for all differences), (b) Intracellu- __ o lar pH. During ischemia intracellu- si 100 ii lar pH stabilized at a significantly 5 higher level in hearts pretreated with a 80 anipamil (p < 0.0005 vs controls), and immediately after reperfusion < pH normalized to preischemic

values. In control hearts the Pxpeak was split into several peaks, corre- 20 • IJ sponding with different pH values I 1 I 1 1 within the myocardium. The highest 10 15 20 25 30 35 40 45 50 55 60 and lowest values are represented by time ( the upper and lower dashed curves, 7.40 respectively. Data are presented as 7.20 '. ischemia reperfusion b mean and SEM (n = 14 during 7.00 ischemia; n = 7 during reperfu- I \ 6.80 sion). HI \\\\

~~llu l 6.6O a> * 6.40 650 6.00 5.80 • it* i 10 30 5O 60 f•, • , • ~J 1 1 time (min)~~

~~Plasma levels ofanipamil Plasma samples from 24 animals were analyzed, resulting in 139±11 ng/ml.~~

~~53 Chapter 3~~

~~Discussion~~

This study demonstrates that pretreatment of rats for 5 days with anipamil protects their isolated heart against metabolic and functional damage caused by 30 minutes of ischemia and reperfusion. Protection occurred in the absence of hemodynamic effects during pretreatment, negative inotropism in the isolated hearts, and preservation of high-energy stores during ischemia. Myocardial stores of noradrenaline and glycogen were not decreased by the pretreatment, and the release or degradation during ischemia and reperfusion did not differ from controls. Only myocardial acidosis during ischemia appeared to be attenuated. Protection of the ischemic myocardium by calcium antagonists, as observed in isolated hearts, is mostly related to a large reduction of contractile force and in association with this, to an energy sparing effect during ischemia ' ' " . This may postpone ischemic damage and thereby enhance recovery after a limited period of ischemia. Although this is an important basic mechanism, the relevance of such an effect for the in-vivo situation is questionable, since reflex compensatory mechanisms may overcome these effects. In addition, an excessive reduction in contractile force may be harmful. On the other hand, we and others have shown that calcium antagonists may also protect the ischemic-reperfused heart in the absence of a negative inotropic effect in isolated hearts ' ' or in vivo , and different mechanisms of action have been proposed.

Noradrenaline During ischemia, noradrenaline may be lost from the myocardial sympathetic nerve endings to the extracellular space, either due to inhibition of reuptake of 12 noradrenaline or due to damage of cell membranes "' f depending upon the duration of the ischemic period. Noradrenaline may then accelerate metabolism, calcium influx and cell death, and may enhance vulnerability to arrhythmias ' ' ^ ' . During washout of noradrenaline early during reperfusion its extracellular concentration has been calculated to be even high enough to cause cellular .19 70 DO necrosis ^ . Hence, direct inhibition of noradrenaline release during ischemia and reperfusion, or preischemic depletion of noradrenaline stores (e.g. by chemical sympath- ectomy) is known to be beneficial 9>4°. Other verapamil-type calcium antagonists were found to cause both a (reversible) depletion of noradrenaline stores 15'20>21 and a preservation of noradrenaline during ischemia and reperfusion 16'22. It was proposed that noradrenaline depletion may be due to either a direct effect of the drug on the sympathetic nerves or an increased reflex sympathetic activity secondary

54 Protective mechanisms of anipamil to vasodilation . We therefore measured heart rate and mean arterial blood pressure in the rats during the pretreatment period with anipamil. The absence of any effects of anipamil on both blood pressure and heart rate in this experimental model makes the occurrence of reflex sympathetic stimulation unlikely. This is in accordance with results by Curtis et al 41, who found an impressive anti-arrhythmic action by pretreatment of rats with anipamil, which caused only a slight cardiodepressant effect. The plasma levels of anipamil were much higher than those in our experiments, and the authors speculated that anti-ischemic actions of anipamil may occur at lower plasma levels. However, noradrenaline content in anipamil pretreated hearts, assayed immediately after excision, did not differ from controls (Fig. 2a), which is consistent with observations by others . The endischemic tissue noradrenaline content is difficult to establish in a model of total global ischemia, as, in addition to tissue stores, the noradrenaline released to the extracellular space will be assayed as well16. There- fore, the endischemic values merely represent total preischemic noradrenaline, which confirms the absence of a depletion due to pretreatment with anipamil. The apparent increase in tissue noradrenaline during ischemia must be attributed to an equal increase in dry to wet weight ratio of about 30 to 40%. Finally, after 30 min of reperfusion, tissue noradrenaline was still not different from controls; this eliminates a reduced release of noradrenaline as a possible protective mechanism in in our experiments. Others reported that anipamil attenuated mobilization of cardiac noradrenaline caused by 15 minutes of global ischemia and reperfusion. However, it is conceivable that this effect is limited to short periods of ischemia, since different mechanisms are responsible for early and late release of noradrenaline during ischemia U'n>i9.

Gfycogen During ischemia anaerobic glycolysis is the only potential source of high-energy phosphate, but on the other hand glycogenolysis (with subsequent glycolytic production of lactate, hydrogen ions, and NADH) may contribute to myocardial cell damage **. Attenuation of ischemic acidosis by /8-blockers contributed to their protective effect , suggesting that catecholamines that have been released in the myocardium during ischemia are partiaiiy responsible for the ischemic acidosis, '. through the formation of cyclic AMP (cAMP), which will stimulate glycogenolysis. On the other hand, it has been proposed that attenuation of ischemic acidosis may :j be related to an indirect effect of /?-blockers through depression of cardiac function, 21 rather than to direct effects on myocardial energy metabolism . It has been suggested that postischemic recovery correlates better with low

55 Chapter 3 ischemic lactate levels than with preservation of adenine nucleotides. Accordingly, glycogen depletion before ischemia 6'8 and inhibition of glycogenolysis during ischemia were found to improve postischemic recovery . In contrast, in a different species and at a higher [Ca2+] it was suggested that loss of glycogen is detrimental to the heart and that the amount of glucose provided by glycogen may be critical to myocardial cell survival during ischemia 7. Part of the controversy may depend on the fact that accumulation of lactate in the ischemic myocardium only partly contributes to the fall in pH during ischemia "4 . Furthermore, preischemic glycogen depletion may be accompanied by a depletion of intracellular triglyceride stores, which may limit the accumulation of toxic degradation products during : ischemia . In the present study, in agreement with previous results *• 3, intracellular acidosis during ischemia was less profound in anipamil pretreated than in control hearts, despite an equal depletion in high-energy phosphates and accumulation of inor- f. ganic phosphate. In contrast, others reported a beneficial effect of verapamil on / ischemic acidosis, which was ascribed to a reduced myocardial function and meta- ( bolic demand. Although we cannot explain the attenuation of ischemic acidosis in our experiments on the basis of glycogen metabolism or noradrenaline release (Fig. 2a,b), it may very well contribute to cell protection, since acidosis is correlated to activation of (phospho)lipases and the inactivation of ATPases such as the Na+/K+- ATPase °. In addition, reduction of the amount of hydrogen ions generated during ischemia will limit cellular Ca2+-uptake during reperfusion through H+/Na+ and Na+/Ca2+exchange44.

Heart function and coronary flow As mentioned before, in vivo blood pressure and heart rate during the pretreatment protocol remained unchanged. This observation is supported by findings from others , that anipamil offered cardioprotection in an in vivo cat model of coronary artery occlusion, without an effect on the rate pressure product. On the other hand, rye anipamil was found to reduce blood pressure in hypertensive patients and in spontaneously hypertensive rats , the latter at similar plasma levels of anipamil as in our study (M. Raschack, personal communication). This suggests that the effect of anipamil may depend on the biood pressure (and heart rate) before treatment, as has been sugggested for other calcium antagonists 46'47. Left ventricular developed pressure of the isolated hearts during control perfusion was not significantly different from controls, in agreement with the report by 1R Nayler , that within the therapeutic dose range the negative inotropism of anipamil, unlike verapamil, is negligible. Upon reperfusion LVDP recovered com- 56 Protective mechanisms ofanipamil pletely in pretreated hearts and end-diastolic pressure was only slightly raised as compared to controls (Table 1), indicating that contracture was largely prevented by anipamil. For unknown reasons coronary fiow during control perfusion was lower in pretreated than in control hearts. However, recovery of coronary flow during reperfusion was much better than in controls, where a partial "no-reflow phenomenon" occurred. In addition, coronary flow during reperfusion in pretreated hearts exceeded preischemic values, suggesting that the coronary vasculature was still capable of reacting to ischemic stress ("reactive hyperemia"). It remains unclear whether this improved coronary flow during reperfusion is a result of a reduction of myocardial and endothelial damage, and contracture , or that anipamil has direct vasodilating properties, which become manifest during reperfusion.

Alternative mechanism Although anipamil, being a calcium antagonist , may exert negative inotropic effects, this may be related to the mode of application and the dosage used. Our experiments show that even in the absence of negative inotropic effects, both during the pretreatment period and subsequently in the isolated hearts, anipamil is highly protective against ischemia-reperfusion induced myocardial damage. Since commonly known mechanisms of cardioprotection fail to explain the protective effect of anipamil in this model of ischemia and reperfusion, alternatives have to be considered. These include altering of cellular buffering capacity for protons, which might explain the attenuation of ischemic acidosis while glycogenolysis is unaffected. However, this hypothesis is not supported by any reports on this subject. Next, although anipamil did not alter noradrenaline stores prior to ischemia or noradrenaline release during ischemia and reperfusion, it may well protect the heart against some of the consequences of high levels of catecholamines ^\ Finally, due to the highly lipophilic properties ofanipamil and the long pretreatment period, anipamil may dissolve in the phospholipidbilayer of the cell membrane ' . This could make the sarcolemma less sensitive to Ca2+ induced conformational changes during ischemia and reperfusion , and thereby contribute to maintaining cellular integrity, which is essential for postischemic myocardial recovery.

~~The authors wish to thank Carina Kasbergen and Pieter van der Meer for then- excellent biotechnical and technical assistence and Ingeborg van der Tweel for statistical advice.~~

~~57 Chapter 3~~

~~References~~

1. Kirkels JH, Ruigrok TJC, van Echteld CJA, Meijler FL. Protective effect of pretreatment with the calcium antagonist anipamil on the ischemic-reperfused rat myocardium: a phosphorus-31 nuclear magnetic resonance study. J Am Coll Cardiol l°88;ll:1087-1093. 2. Ochi S, Ogawa Y, Imai H, Nakazawa M, Imai S. Effects of 6-blockers on the fall of pH in the early phase of ischemia in the isolated perfused rat heart: a nuclear magnetic resonance study. J Cardiovasc Pharmacol 1988;11:326-331. 3. Pieper GM, Todd GL, Wu ST, Salhany JM, Clayton FC, Eliot RS. Attenuation of myocardial acidosis by propranolol during ischaemic arrest and reperfusion: evidence with 31P nuclear magnetic resonance. Cardiovasc Res 1980;14:646-653. 4. Hara Y, Ichihara K, Abiko Y. MCI-176, a novel calcium channel blocker, attenuates the ischemic myocardial acidosis induced by coronary artery occlusion in dogs. J Pharmacol Exp Ther 1988;245;305-310 5. Ichihara K, Ichihara M, Abiko Y. Involvement of beta adrenergic receptors in decrease of myocardial pH during ischemia. J Pharmacol Exp Ther 1979;209:275-281. 6. Kupriyanov W, Lakomkin VL, Steinschneider AY, Severina MY, Kapelko VI, Ruuge EK, Saks VA. Relationships between pre-ischemic ATP and glycogen content and post-ischemic recovery of rat heart. J Moi Cell Cardiol 1988;20:1151-116X 7. Lagerstrom CF, Walker WE, Taegtmeyer H. Failure of glycogen depletion to improve left ventricular function of the rabbit heart after hypothennic ischemic arrest. Circ Res 1988;63:81-86. 8. Neely JR, Grotyohann LW. Role of glycolytic products in damage to ischemic myocardium. Dissociation of adenosine triphosphate levels and recovery of function of reperfused ischemic hearts. Circ Res 1984;55:816-824. 9. Carlsson L. Mechanisms of local noradrenaline release in acute myocardial infarction. Acta Physiol Scand 1987;129 (Suppl.559). 10. Carlsson L, Abrahamsson T, Almgren O. Local release of myocardial norepinephrine during acute ischemia: an experimental study in the isolated perfused rat heart. J Cardiovasc Pharmacol 1985;7:791-798. 11. Schomig A. Adrenergic mechanisms in myocardial infarction: cardiac and systemic catecholamine release. J Cardiovasc Pharmacol 1988;12 (SuppI 1):S1-S7. 12. Schomig A, Dart AM, Dietz R, Mayer E, Kubler W. Release of endogenous catecholamines in the ischemic myocardium of the rat; Part A: Locally mediated release. Circ Res 1984;55:689-701. 13. Watts JA, Koch CD, LaNoue KF. Effects of Ca2+ antagonism on energy metabolism: Ca2+ and heart function after ischemia. Am J Physiol 1980;238:H909-H916. 14. Nayler WG, Ferrari R, Williams A. Protective effect of pretreatment with verapamiL nifedipine and propranolol on mitochondrial function in the ischemic and reperfused myocardium. Am J Cardiol 1980;46:242-248. 15. Nayler WG, Panagiotopoulos S, Elz JS, Sturrock WJ. Fundamental mechanisms of action of calcium antagonists in myocardial ischemia. Am J Cardiol 1987;59:75B-83B. 16. Nayler WG, Sturrock WJ. An inhibitory effect of verapamil and diltiazem on the release of noradrenaline from ischaemic and reperfused hearts. J Mol Cell Cardiol 1984;16:331- 344.

~~5R Protective mechanisms ofanipamil~~

17. Opie LH. Metabolism of free fatty adds, glucose and catecholamines in acute myocardial infarction. Relation to myocardial ischemia and infarct size. Am J Cardiol 1975,36:938- 953. 18. Nayler WG. The pharmacology of the calcium antagonists. In: Fleckenstein A, Laragh JH, eds. Hypertension - the Next Decade: Verapamil in Focus. Edinburg: Churchill Livingstone, 1987:53-66. 19. Chaudhry A, Vohra MM. Depletion of cardiac noradrenaline stores by the calcium - channel blocker D-600. Can J Physiol Pharmacol 1984;62:640-644. 20. Chaudhry A, Vohra MM. A reserpine-like action of verapamil on cardiac sympathetic nerves. Eur J Pharmacol 1984;97:156-158. 21. Nayler WG, Dillon JS, Sturrock WJ, Buckley WJ. Effect of chronic verapamil therapy on cardiac norepinephrine and B-adrenoceptor density. J Cardiovasc Pharmacol 1988;12:629-636. 22. Nayler WG, Sturrock WJ. Inhibitory effect of calcium antagonists on the depletion of cardiac norepinephrine during postischemic reperfusion. J Cardiovasc Pharmacol 1985;7:581-587. 23. Ciplea AG, Kretzschmar R, Heimann W, Kiichengast M, Safer A. Protective effect of the new calcium antagonist anipamil against isoprenaline-induced cardionecrosis in rats. Drug Res 1988-38:215-221. 24. Todd GL, Sterns DA, Plambeck RD, Joekel CS, Eliot RS. Protective effects of slow channel calcium antagonists on noradrenaline induced myocardial necrosis. Cardiovasc Res 1986;20:645-651. 25. Dies R, Schneider G, Hahn KJ. Antihypertensive efficacy of anipamil in mild to moderate hypertension. J Cardiovasc Pharmacol 1989;13(suppl 4):S76-S78. 26. Dillon JS, Nayler WG. The Ca -antagonist and binding properties of the phenylalky- lamine, anipamil. Br J Pharmacol 1988;94:253-263. 27. Raschack M, Gries J, Heimann W, Kirchengast M, Muller CD, Szabo L, Teschendorf HJ, Unger L. Pharmakologisches Profil neuer Verapamil Derivate. In: Bender F, Flec- kenstein A, eds. Therapie und Prevention mit Ca+ +-Ant agonist en. Darmstadt, Stein- kopfVer!ag,1987. 28. Bergmeyer HU. Methods in Enzymatic Analysis. New York, Academic Press, 1963. 29. Lowry OH, Rosebrough NJ, Farr AL. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 30. Ferrari R, Cargnoni A, Ceconi C, Curello S, Belloli S, Albertmi A, Visioli O. Protective effect of a prostacyclin-mimetic on the ischemic-reperfused rabbit myocardium. J Mol Cell Cardiol 1988;20:1095-1106. 31. Brode E, Hotz D, Kern R. Internally standardized determination of anipamil in human plasma by means of high-pressure liquid chromatography. Methods Find Exp Clin Pharmacol 1985;7:427-434. 32. Ferrari R, Albertini A, Cureiio S, Ceconi C, Dilisa F, Raddiso R, Yisoli O. Myocardial recovery during postischaemic reperfusion: effects of nifedipine, calcium and magnesium. J Mol Cell Cardiol 1986;18:487~498. 33. Ruigrok TJC, van Echteld CJA, de Kruijff B, Borst C, Meijler FL. Protective effect of nifedipine in myocardial ischemia assessed by phosphorus-31 nuclear magnetic resonance. Eur Heart J 1983;4(SupplC):109-113.

~~59 Chapter 3~~

34. Ro YM, Markle DR, Goldstein SR, Speir E, Greene R, Steadman K, Aamodt R, Epstein SE, Patterson RE. Contrasting effects of verapamil and nifedipine on pH of ischémie myocardium in the dog. J Pharmacol Exp Ther 1989;248:654-660 35. Higgins AJ, Blackburn KJ. Prevention of reperfusion damage in working rat hearts by calcium antagonists and calmodulin antagonists. J Mol Cell Cardiol 1984;16:427-438. 36. Lavanchy N, Martin J, Rossi A. Effects of diltiazem on the energy metabolism of the isolated rat heart submitted to ischaemia: a 31P NMR study. J Mol Cell Cardiol 1986;18:931-941. 37. Przyklenk K, Ghafari GB, Eitzman DT, Kloner RA. The calcium channel blocking agent nifedipine improves contractile function of stunned myocardium by a direct cellular mechanism. J Mol Cell Cardiol 1989;21(suppl II):S3. 38. Gettes US, Symanski JD, Fleet WF, Johnson TA, Graebner C. The intracellular and extracellular changes associated with ischemia - effects of catecholamines in arrhyth- mogenesis. Eur Heart J 1986;7(suppl A):77-84. 39. Knopf H, Theising R, Hirche H. The effect of desipramine on ischemia - induced changes in extracellular K+, Na+, and H+ concentrations and noradrenaline release in the isolated rat heart during global ischemia. J Cardiovasc Pharmacol 1988;12(Suppl 1):S8- S14. 40. Nayler WG. Protective effects of prenylamine-induced depletion of noradrenaline in the post-ischaemic myocardium. In: Manning AS, Szendey GL, eds. Prenylamine: a Novel Approach to Myocardial Protection. New York, Raven Press, 1988:32-42. 41. Curtis MJ, Walker MJA, Yuswack T. Actions of the verapamil analogues, anipamil and ronipamil, against iscbaemia-induced arrhythmias in conscious rats. Br J Pharmacol 1986;88:355-361. 42. Gevers W. Generation of protons by metabolic processes in heart cells. J Mol Cell Cardiol 1977;9:867-874. 43. Panagiotopoulos S, Nayler WG, Kneen M, Craik D. Calcium gain in the ischaemic rat heart: the effect of anipamil. J Mol Cell Cardiol 1988; 20:V. 44. Lazdunski M, Frelin C, Vigne P. The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. J Mol Cell Cardiol 1985;17:1029-1042. 45. Brezinski ME, Darius H, Lefer AM. Cardioprotective actions of a new calcium channel blocker in acute myocardial ischemia. Drug Res 1986;36:464-466. 46. Braunwald E. Mechanisms of action of calcium-channel-blocking agents. N Engl J Med 1982^07:1618-1627. 47. Pedersen OL, Christensen CK, Ramsch KD. Comparison of acute effects of nifedipine in nonnotensive and hypertensive man. J Cardiovasc Pharmacol 1980;2:357-366. 48. Post JA, Leunissen-Bijvelt J, Ruigrok TJC, Verkleij AJ. Utrastmctural changes of sarcolemma and mitochondria in the isolated rabbit heart during ischemia and reperfusion. Biochim Biophys Acta 1985;845:119-123.

~~60 Low calcium reperfusion~~

~~LOW Ca2+ REPERFUSION AND ENHANCED SUSCEPTIBILITY OF THE POSTISCHEMIC HEART TO THE CALCIUM PARADOX.~~

~~Abstract~~

This study was designed to define the effect of postischemic low Ca perfusion on recovery of high-energy phosphates, intracellular pH, and contractile function in isolated rat hearts. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to follow creatine phosphate, adenosine tiiphosphate, intracellular inorganic phosphate and intracellular pH during control perfusion (IS minutes), total ischemia (30 minutes) and reperfusion (30 minutes). In Group I the perfusate [Ca ] was 1.3 mmol/1 throughout the experiment, whereas in Group II the perfusate [Ca2+] was reduced to 0.05 mmol/1 during the first 10 minutes of reperfusion. Hearts from Group III were not made ischemic but were subjected to 10 minutes of low Ca + perfusion followed by 20 minutes of normal Ca + perfusion. During low Ca + reperfusion (Group II) recovery of high-energy phosphates and pH were significantly better than in controls (Group I). However, after reexposure to normal Ca + metabolic recovery was largely abolished, coronary flow was suddenly impaired, and contracture developed without any rhythmic contractions. These observations indicated the occurrence of a calcium paradox rather than postponed ischemia- reperfusion damage. On the other hand, normoxic hearts (Group IH) tolerated temporary perfusion with 0.05 mmol/1 Ca very well with respect to left ventricular developed pressure, coronary flow and metabolic parameters. In conclusion, postischemic low Ca (0.05 mmol/1) perfusion may reduce reperfusion damage, but at the same time ischemia appears to enhance the susceptibility of the heart to the calcium paradox.

~~61 Chapter 4~~

~~Introduction~~

•» ygyocardial ischemia has numerous consequences, which can be explained by -^*-a reduced availability of oxygen and substrate and accumulation of metabolic waste products.1 Reperfusion after a limited period of ischemia may either initiate a direct or delayed restoration of metabolism and function or lead to a rapid deterioration, loss of cellular constituents, and accelerated cell death. ' The severity and duration of the ischemic event are generally believed to determine the eventual outcome. Several interventions preceding a temporary ischemic event have been found to limit ischemic damage and thereby enhance recovery of metabolism and function on reperfusion. " Preservation of high-energy phosphates and maintenance of ionic homeostasis during ischemia appear to be the key determinants of the protective action.3'6'7 More recently, interest has been diverted to protective interventions at the time of reperfusion, and several studies indicate that reversibility of ischemic or hypoxic damage may be affected by reperfusion conditions. ' " 12 Since Ca accumulation in myocardial cells has been shown to play a major role in reperfusion injury, limitation of uncontrolled Ca influx might enhance survival of cells after ischemia. Calcium antagonists, however effective when given before ischemia,3^'14 generally fail to protect the heart when given only during reperfusion ' ' or offer only limited protection ; this suggests a pathway for Ca + entry during reperfusion other than the slow channels.6 On the other hand, a large reduction of the extracellular [Ca + ] during reperfusion may well afford protection, although at the same time this may set the stage for the calcium paradox. The present study was performed to investigate whether a temporary reduction of the perfusate [Ca ] during the initial phase of reperfusion, before reexposure to normal Ca , can modify reperfusion injury. Phosphorus-31 nuclear magnetic resonance ( P NMR) spectroscopy offers the unique possibility of following the time course in high-energy phosphates, inorganic phosphate, and pH during ischemia and the two-step reperfusion protocol in isolated rat hearts, with a simultaneous assessment of myocardial function.

~~62 Low calcium reperfusian~~

~~Materials and Methods~~

Animal preparations Male Wistar rats weighing 325-375 g were anesthetized with diethyl ether and heparinized (250 IU i.v.). The heart was rapidly excised and cooled in ice-cold perfusate. After cannulation of the aorta, retrograde perfusion was started at a constant pressure of 100 cm H2O (10.0 kPa). The standard perfusate contained

(mmol/1) NaCl 124.0; KC14.7; MgCl21.0; CaCl213; NaHCQj 24.0; Na2HPO40.5; glucose 11.0. The perfusate was filtered (0.8 fim filters, Millipore, Bedford, Massa- chusetts) before use and saturated with 95% O2-5% CO2 resulting in a pH of 735 ±0.05 at 37 °C. For assessment of contractile function, a perfusate-filled ca- theter16'17 was inserted through the apex and connected to a Statham P23dB pressure transducer (Gould, Cleveland, Ohio). The pressure signal was recorded on a Gould Brush recorder, and the difference between end-systolic and end-diastolic pressure was taken to be the left ventricular developed pressure. Heart rate was maintained at 300 beats/min throughout the experiment by right ventricular pacing with two sodium chloride wick electrodes connected to a Grass S88 stimulator (Grass Instrument, Quincy, Massachusetts). Hearts were placed in a 20 mm NMR tube together with a capillary containing methylene diphosphonate as a spectral reference. The glass tube with the heart was then lowered into the NMR coil. The effluent was removed from a level above the heart, leaving the heart submerged in a fixed volume of perfusate. The effluent was collected in 5-minute fractions for determination of coronary flow. Myocardial temperature was maintained at 37 °C by water-jacketed perfusion lines to the heart and a continuous stream of air (37 °C) around the sample tube.

NMR measurements 31P NMR spectra were obtained at 81.0 MHz on a Bruker MSL 200 spectrometer (Bruker, Karlsruhe, FRG) equipped with a 4.7 Tesla vertical bore magnet. For each spectrum 128 free-induction decays were accumulated after 90° pulses by use of 2K data points and a 5-kHz spectral width at a repetition time of 23 s. Accumulated free induction decays were exponentially filtered, resulting in 10 Hz line broadening. After an automatic polynomial baseline correction of the spectra, quantitation of metabolites was achieved by integration of the areas under the individual peaks of interest in each spectrum. Values for creatine phosphate (CP) and ATP (/3-ATP) were expressed as a percentage of their respective preischemic values; intracellular inorganic phosphate (Pi) was expressed as a percentage of the sum of phosphate from CP, ATP, and Pi during preischemic control perfusion:

~~63 Chapter 4~~

~~(Pi / {CP + 3 ATP + Pi}pre-ischemic) x 100%.~~

CP and Pi were corrected for partial saturation; the saturation factors, 1.5 and 1.1, respectively, were determined using a 10-second recycle time. Intracellular and extracellular pH values were calculated from the chemical shift of the respective Pi peaks relative to methylene diphosphonate. A value of 0 ppm was assigned to CP.

Experimental protocol After a stabilization period of about 20 minutes, three control spectra were obtained in all hearts. The hearts were then randomly subjected to one of three protocols. In Groups I and II the hearts were made totally ischemic for 30 minutes, followed by 30 minutes of reperfusion. In control hearts (Group I) the [Ca2+] in the perfusate was 1.3 mmol/l throughout the experiment, whereas in Group II the [Ca +] during the initial 10 minutes of reperfusion was lowered to 0.05 mmol/l. For this purpose a second perfusion line was used, which allowed immediate start of reperfusion at a low [Ca2+]. During the last 20 minutes of reperfusion the hearts from Group II were perfused with the standard perfusate. The hearts from Group III were not made ischemic, but were exposed to 10 minutes of low Ca2+ perfusion, followed by 20 minutes of normal Ca perfusion.

Statistical analysis Results are presented as mean ± standard deviation (SD) of nine or 11 experiments. Analysis of variance with repeated measurements was used for determination of the effect of temporary reperfusion with low Ca2+ containing perfusate on metabolic parameters. Results obtained during ischemia, during the first 10 minutes of reperfusion and during the remainder of the reperfusion period were analyzed separately. In normoxic hearts exposed to 10 minutes of low Ca2+ (Group HI), analysis of variance with repeated measurements was carried out for comparison of normal Ca + reperfusion with the initial control perfusion. A test result of p < 0.05 was considered significant.

~~Results~~

~~Ischemia Figure 1 shows the effect of total interruption of coronary perfusion on CP, ATP, and intracellular Pi. The rapid degradation of CP and ATP was balanced by an~~

~~64 Low calcium reperfusion~~

ISCHEMIA REPERFUSION Figure 1. Time course in creatinephos- ca 0j)5[ jo i.-3mmni/t 1 phate(CP),ATPandintracellularinor- Js. J 3 mjpo"! _ _ J ganic phosphate (Pi) in isolated rat hearts during ischemia (30 minutes) and reperfusion (30 minutes) as measured by P nuclear magnetic resonance. During ischemia no significant differences were observed between control Group I (•),/« which [Ca2+] was 1.3 mmolll throughout the experiment (n = 9), and Group [I (m), in which [Ca2+] was lowered to 0.05 mmolll during the first 10 minutes of reperfusion (n = ll). Low Ca + reperfusion temporarily improved recovery ofCP (p < 0.001) and ATP (p < 0.02) and restored low levels of intracellular Pi (p < 0.001) as compared with controls. Normalization ofperfusate Ca2+ largely abolished this recovery. For further explanation, see text. Data are presented as mean ± SD.

increase in intracellular Pi. Intracellular pH dropped rapidly from 7.06±0.02 during control perfusion to 5.84 ±0.06 after 15 minutes of ischemia. Contractile activity was no longer observed after 2.5-3 minutes of ischemia.

Normal Ca2+ reperfitsion Reperfusion with the standard perfusate (Group I) resulted in a partial recovery of CP and ATP (Figure 1). Intracellular Pi decreased, but remained elevated in comparison with preischemic levels. Statistical analysis indicated that after 10 minutes of reperfusion, no further changes occurred. In addition, the P NMR

~~I 65 Chapter 4~~

20 10 -10 -20 ppm Figure 2. 31 rP nuclear magnetic resonance spectrum abtainedfrom an isolated rat heart between 25 and 30 minutes of normal Ca reperfusion after 30 minutes of ischemia (Group I). Numbered peaks are as follows: I, methylene diphosphonate; 2, perfusate inorganic phosphate (P\) at pH7.40; 3a, intracellular Pi at a normalized pH of 7.05; 3b, extracellular or intracellular P\ at pHbetween 7.00 and 5.85; 4, creatine phosphate; 5,6,8, y-, a - and ^-phosphate group of ATP, respectively; 7, nicotinamide adenine dinucleotide (NAD(H)). spectra were characterized by the presence of multiple Pi peaks (Figure 2), most likely due to differences of intracellular pH among myocardial cells. Despite this incomplete or nonhomogeneous recovery, the main Pi peak corresponded with a pH of 7.00-7.10. Figure 3 shows that coronary flow upon reperfusion was impaired, which indicated in combination with the different pH values, a partial no-reflow phenomenon. By the end of 30 minutes of reperfusion, left ventricular function had recovered to 40±18% of preischemic values.

Low Ca reperfusion Initial reperfusion with 0.05 mmol/1 Ca2+ (Group II) resulted in a significantly better recovery of CP (p < 0.001) and ATP (p < 0.02) than in control hearts (Figure 1). During low Ca + reperfusion, intracellular Pi levels returned to preischemic levels and were significantly lower than intracellular Pi levels in control hearts during the corresponding period (p < 0.001). In eight of 11 hearts intracellular pH

ISCHEMIA REPERFUSION Figure 3. Coronary flow in isolated rat hearts Ca 0.05 { Ca 1.3 mmol/l \ Ca_ JJLmmol/l j during control perfiision (C) and during reperfusion after 30 minutes of ischemia. Coronary flow during control perfusion did not differ between control Group I (•), in which [Ca ] was 1.3 10 .1-I.l-J mmolll throughout the experiment (n = 9), and Group II (m), in which [Caz+] was lowered to 0.05 mmol/l during the first 10 minutes of reperfusion (n = H). During normalization of perfusate [Caz+] after 10 minutes of low Ca2+ reperfixsion, coronary flow was significantly less than in controls (p < 0.001). Data are presented 30 50 60 Time (mini as mean ± standard deviation.

~~66 Low calcium reperfusion~~

~~z SI J / 25-30 Time (min) z 2^,3~~

~~W^MvfVvWW^if Control~~

~~20 10 0~~

Figure 4. 31P nuclear magnetic resonance spectra obtained from an isolated perfused rat heart during control perfusion, ischemia and reperfusion. During the first 10 minutes of repeifusion theperfusate [Ca ] was lowered to 0.05 mmolll (Group II). Numbered peaks are as follows:

(I)methylenediphosponate, (2) extracellular inorganic phosphate (Px), (3) intracellular Pj, (4) creatine phosphate (CP), (5),(6),(8), y-, a-,and ^-phosphate group of ATP, (7) nicottnamide adenine dinucleotide. recovered homogeneously to preischemic values, although in four hearts accurate pH determination was hampered due to low levels of intracellular Pi. In three hearts the intracellular Pi peak was split into a peak corresponding with normalized pH and a peak corresponding with low intracellular pH. In all respects metabolic recovery at the end of the 10-minute reperfusion period was significantly better in Group II than in Group I (control) hearts. However, on returning to the standard perfusate the initial metabolic recovery was largely abolished, as indicated by a second decrease in CP and ATP (Figure 1). The simultaneous increase in intracellular Pi did not parallel the fall in high-energy phosphates, indicating a loss of intracellular Pi. The metabolic state at the end of the reperfusion period was significantly worse than in control hearts (p < 0.0001). Figure 4 shows a set of 31P NMR spectra obtained from a heart from Group II during control perfusion, ischemia, and reperfusion.

~~67 Chapter 4~~

Ca 005 mmol/1 Figure 5. Functional and metabolic parameters in normoxic hearts subjected to 10 minutes oj perfusion with 0.05 mmolll CaZ+ followed by 20 minutes with 1.3 mmolll Ca2+(Group III). Creatine phosphate (CP) and ATT are given as percentage of their respective control values obtainedbefore low Ca perfusion. Intra- cellular inorganic phosphate (P\) is expressed as a percentage of the sum of phosphate from CP, ATP and Pi during control perfusion preceding low Ca + perfusion. Each point represents mean ± SD of nine experiments. LVDP: left ventricular developed pressure.

~~0 5 10 15 20 25 30 Time (rain)~~

Coronary flow during the first 10 minutes of reperfusion (Figure 3) was better (although not significantly) than in control hearts, but reexposure to normal Ca2+ caused a considerable drop in flow, indicating a sudden rise in coronary resistance. In Group II, despite metabolic recovery, no contractile activity was observed during the initial period of reperfusion due to the low extracellular [Ca2+]. Immediately after reexposure to normal Ca2+, most hearts showed severe contracture without any rhythmic contractile activity.

Normoxic low Ca perfusion For assessment of whether temporary reduction of the extracellular [Ca2+] to 0.05 mmol/1 would initiate similar adverse effects in nonischemic hearts, a separate series of experiments was performed (Group III). Figure 5 shows that in these nonischemic hearts metabolic parameters were almost unaffected by the switch to 0.05 mmol/1 Ca2+ for 10 minutes and back to 1.3 mmol/1 Ca2+ for another 20 minutes. The large changes in high-energy phosphates and Pj that occurred during Ca repletion in postischemic hearts (Group II) were not observed. Intracellular

~~68 Low calcium reperfusion pH did not change during the protocol, although during the low Ca2+ period pH could not always be accurately determined due to very low levels of intracellular Pi-~~

Left ventricular function recovered completely immediately after the returning to normal Ca +. Coronary flow returned to control values after a slight increase during the low Ca perfusion. Concomitantly, CP increased slightly and Pj decreased during low Ca perfusion, most likely due to the absence of contractile activity and the observed increased coronary flow. It is obvious that, in contrast to postischemic hearts, normoxic hearts tolerated a temporary reduction of Ca2+ to 0.05 mmol/1 very well.

~~Discussion~~

The present study showed that a reduction of the perfusate [Ca2+] to 0.05 mmol/1 during the first 10 minutes of reperfusion after 30 minutes of ischemia enabled myocardial energy metabolism to recover. However, upon reintroduction of 13 2+ mmol/1 Ca this beneficial effect was not only abolished but even became a detrimental effect. By now it is accepted that reperfusion injury is associated with uncontrolled Ca + influx during the first minutes of reperfusion. ' Therefore, it seems appropriate that the extracellular [Ca2+] be lowered during this vulnerable period. On the other hand, complete omission of Ca 4 from the perfusion fluid followed by readmission of Ca inevitably leads to the calcium paradox, characterized by contracture of the myofibrils, rapid depletion of high-energy phosphates, accumulation of Ca2+ and Pi by mitochondria, and uncoupling of oxidative phosphorylation, sarcolemmal disruption, and loss of intracellular constituents. It is generally assumed that a [Ca2+] of 0.05 mmol/1 is safe in that the calcium paradox will not occur, although this is based mainly on electron microscopic observations in rabbit myocardium at 28 °C that separation of the glycocalyx could be prevented by an extracellular [Ca2+] of 0.05 mmol/1.25 However, in normothennic rabbit hearts 0.05 mmol/1 did not completely prevent the calcium paradox when leakage of intracellular enzymes was used to define myocardial cell damage. The question of the optimal [Ca2+] early during reperfusion after ischemia is yet to be solved, but will certainly depend on the experimental conditions and the extent of ischemic damage and possibly also on the species used. This may explain why the results of previous studies, in which reperfusate [Ca 4 waging from 0.05 to 0.75 mmol/1 has been used, are not uniform (Table 1). 69 Chapter 4

~~Table 1. Summary of literature on temporary Low Ca2+ reperfusion after myocardial ischemia~~

~~2+ Authors Species Model [Ca ]low Protection (mmol/1)~~

Allen et al8 Dog Regional ischemia 0.25* + in vivo 0.15* + ChappeUetal' Rabbit, cat Hypoxic/reoxygenated 0.125 + isolated papillary muscle Ferrari et al3 Rabbit Langendorff 0.75 - perfused heart 0.15 +/-** O.OS + Follette et al10 Dog Hypothermic cardiac 0.5 + arrest in vivo <0.05 - Koomen et al27 Rat Langendorff perfused 0.1 heart Kuroda et al" Rat Working heart QJS*** + Nayler28 Rabbit Isolated mitochondria 0.05 + from Langendorff perfused heart Shine and Douglas12 Rabbit Perfused interventricular 0.75 + septum Watts etal29 Rat Langendorff 0.75 perfused heart

*In presence of diltiazem ** Protection did not last after normalization of extracellular [Ca2+] *** Optimal [Ca2+]

Our results showed that reduction of the [Ca2+] to 0.05 mmol/1 on reperfusion resulted in a significantly better recovery of myocardial energy metabolism in comparison with hearts reperfused with 1.3 mmol/1 Ca2+. The explanation for this may be threefold. 1) Resumption of mitochondrial respiration on reperfusion with a normal [Ca +] leads to the accumulation of Ca2+ and Pi in mitochondria at the expense of ATP production12'28. This is prevented by a large reduction of the [Ca ], leaving all newly produced energy available for repair processes and restoration of ionic homeostasis. 2) The absence of contractile activity may also contribute to metabolic recovery by diminishing energy expenditure. 3) Further- more, Ca + induced activation of (phospho)lipases and proteases28'30 will not occur, thereby preventing a further aggravation of tissue damage. When, after 10 minutes of low Ca2+ reperfusion, the standard perfusate was used again, a sharp drop in CP and ATP levels occurred, which was not accompanied by an equal increase of intracellular Pi, as was observed during ischemia. Together

70 Low calcium reperfusion with the concomitant contracture of the heart and the sudden increase in coronary resistance, these observations suggested the occurrence of a calcium paradox.23 The decreased total phosphate content may be explained by washout of Pi, or by deposition of calcium phosphate in mitochondria, which is NMR invisible.23 In addition, small amounts of CP and ATP may have left the cells before breakdown.31 Since in our experiments the normoxic hearts tolerated temporary perfusion with 0.05 mmol/1 Ca very well, it must be concluded that postischemic hearts are more susceptible to the calcium paradox. A possible additive effect of Ca depletion and ischemia in the isolated rat heart has been discussed in one other study32. It was demonstrated that the volume of Ca2+-free perfusate needed to evoke a calcium paradox was largely reduced when the heart was made ischemic between Ca depletion and Ca repletion. However, one may also argue that the exposure to the Ca2+-free perfusate lasted longer when the hearts were made ischemic, and therefore Ca + depletion was more complete, without any specific additive effect of the intervening ischemic period. At the same time, ischemic damage may have been less, attributable to the Ca2+ depletion before ischemia.5'27 In our experiments ischemia preceded low Ca + perfusion and, therefore, a true additive effect occurred, since these hearts (Group H) finally recovered significantly less than after ischemia (Group I) or low-Ca2+ perfusion alone (Group HI). It is unclear why ischemia enhances the susceptibility of the heart to the calcium paradox. It is possible that the effects of Ca2+ depletion24125 and ischemia33534 on sarcolemmal integrity are additive, as suggested by Jynge.32 Alternatively, this enhanced sensitivity may also be associated with intracellular Na+, which increases during both ischemia35'37 and Ca2+ depletion.38"39 Increased levels of intracellular Na+ during ischemia are supposed to be caused by failure of the sarcolemmal Na+/K+ pump due to lack of ATP35'37 or high levels of Pj.35 The cause of Na+ influx during Ca2+-free perfusion is not completely understood, but a reduced activity of the Na+/K+ pump has been reported as well as transport of Na+ ions through the Ca2+ channels.38 High levels of intracellular Na+ may eventually lead to a massive Ca2+ influx through the Na+/Ca2+ exchange mechanism 20=35-38'4u after normalization of the perfusate [Ca ] in the hearts of Group II rate. This in turn may lead to an addition of the postponed reperfusion injury and the newly introduced mild calcium paradox damage. It may be expected that Na /Ca exchange will be reactivated during resynthesis of ATP, after an inhibition during ischemia due to dephosphorylation41 and acidosis,35 but that massive exchange cannot occur unless the extracellular [Ca +] is normalized. It is conceivable that in normoxic hearts, in contrast with postischemic hearts, during 10 minutes of perfusion with 0.05 mmol/1 Ca2+, weakening of the sarcolemma and the increase in

71 Chapter 4 intracellular Na+ are not sufficient to predispose the heart to the calcium paradox 38 In conclusion, this study demonstrates that recovery of myocardial energy metabolism upon reperfusion is not solely determined by the extent of ischemic damage, but can be modulated by temporary lowering of the perfusate [Ca ] to 0.05 mmol/1. However, a previous period of ischemia may enhance the susceptibility of the heart to the calcium paradox. This may be of importance for future investigations, both experimental and clinical, dealing with protective interventions at the time of reperfusion.

~~The authors would like to thank Pieter van der Meer for excellent technical assistance and Ingeborg van der Tweel for statistical advice.~~

~~References~~

1. Jennings RB, Reimer KA, Steenbergen C. Myocardial ischemia revisited. The osmolar load, membrane damage, and reperfusion. J Mol Cell Cardiol 1986; 18:769-780. 2. Hearse DJ: Reperfiision of the ischemic myocardium. J Mol Cell Cardiol 1977;9:605-616. 3. Ferrari R, Albertini A, Curello S, Ceconi C, DiLisa F, Raddino R, Visioli O. Myocardial recovery during post-ischaemic reperfusion: effects of nifedipine, calcium and magnesium. J Mol Cell Cardiol 1986;18:487-498. 4. Flaherty JT, Weisfeldt ML, Bulkley BH, Gardner TJ, Gott VL, Jacobus WE. Mechanisms of ischemic myocardial cell damage assessed by phosphorus-31 nuclear magnetic resonance. Circulation 1982;65:561-571. 5. Watts JA, MaioranoLJ, MaioranoPC. Comparison of the protective effects of verapamil, diltiazem, nifedipine, and buffer containing low calcium upon global myocardial ischemic injury. J Mol Cell Cardiol 1986;18:255-263. 6. Bourdillon PD, Poole-Wilson PA. The effects of verapamil, quiescence, and cardioplegia on calcium exchange and mechanical function in ischemic rabbit myocardium. Circ Res 1982-^0:360-368. 7. Ruigrok TJC, Van Echteld CJA, De Kruijff B, Borst C, Meijler FL. Protective effect of nifedipine in myocardial ischemia assessed by phosphorus-31 nuclear magnetic resonance. Eur Heart J 1983;4(suppl C):109-H3. 8. Allen BS, Okamoto F, Buckberg GD, Acar C, Partington M, Bugyi H, Leaf J. Studies of controlled reperfusion after ischemia. IX. Reperfusate composition: benefits of marked hypocalcemia and diltiazem on regional recovery. J Thorac Cardiovasc Surg 1986;92:564- 572. 9. Chappell SP, Lewis MJ, Henderson AH. Myocardial reoxygenation damage: can it be circumvented? Cardiovasc Res 1985;19:299-303.

~~72 Low calcium reperfusion~~

10.Follette DM, Fey K, Buckberg GD, Helly JJ, Steed DL, Foglia RP, Maloney J V. Reducing postischemic damage by temporary modification of reperfusate calcium, potassium, pH, and osmolarity. J Thorac Cardiovasc Surg 1981;82:221-238. ll.Kuroda H, Ishiguro S, Mori T. Optimal calcium concentration in the initial reperfusate for post-ischemic myocardial performance (calcium concentration during reperfiision). J Mol Cell Cardiol 1986; 18:625-633. 12.Shine Kl, Douglas AM. Low calcium reperfusion of ischémie myocardium. J Mol Cell Cardiol 1983;15:251-260. 13.Poole-Wilson PA, Harding DP, Bourdillon PD V, Tones MA. Calcium out of control. J Mol Cell Cardiol 1984;16:175-187. 14.Hugenholtz PG, Serruys PW, Fleckenstein A, Nayler W. Why Ca2+ antagonists will be most useful before or during early myocardial ischaemia and not after infarction has been established. Eur Heart J 1986;7:270-278. 15.Zimmerman ANE, Hülsmann WC. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 1966;211:646-647. 16.NeeIy JR, Liebermeister H, Battersby EJ. Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol 1967;212:804-814. 17.Neely JR, Grotyohann LW. Role of glycolytic products in damage to ischémie myocardium. Dissociation of adenosine triphosphate levels and recovery of function of reperfused ischémie hearts. Circ Res 1984;55:816-824. 18.Bailey IA, Seymour AML, Radda GK. A 31P-NMR study of the effets of reflow on the ischaemic rat heart. Biochim Biophys Acta 1981;637:l-7. 19.Yano Y, Riggs TR, Milam DF, Alexander JC. Calcium-accentuated ischémie damage during reperfusion: the time course of the reperfiision injury in the isolated working rat heart model. J Surg Res 1987;42:51-55. 20Daly MJ, Elz JS, Nayler WG. Contracture and the calcium paradox in the rat heart. Circ Res 1987;61:560-569. 21.Ganote CE, Sims MA. Physical stress-mediated enzyme release from calcium-deficient hearts. J Mol Cell Cardiol 1983;15:421-429. 22.Ruigrok TJC. The calcium paradox and the heart, in Parratt JR (ed): Control and Manipulation of Calcium Movement, New York, Raven Press, 1985, pp 341-365. 23Jluigrok TJC, Kirkels JH, Van Echteld CJA, Borst C, Meijler FL. ^*P NMR study of intraceUular pH during the calcium paradox. J Mol Cell Cardiol 1987;19:135-139. 24 .Post JA, Nievelstein PFEM, Leunissen-Bijvelt J, Verkleij AJ, Ruigrok TJC. Sarcolemmal disruption during the calcium paradox. J Mol Cell Cardiol 1985;17:265-273. 25.Crevey BJ, Langer GA, Frank JS. Role of Ca2 + in maintenance of rabbit myocardial cell membrane structural and functional integrity. J Mol Cell Cardiol 1978;10:1081-1100. 26.Capucci A, Janse MJ, Ruigrok TJC. The calcium paradox: an electrophysiological study in the isolated rabbit heart. Eur Heart J 1983;4(suppl H):13-2L 27.Koomen JM, Schevers JAM, Noordhoek J. Myocardial recovery from global ischemia and reperfusion: effects of pre- and/or post-ischemic perfusion with low-Ca +. J Mol Cell Cardiol 1983;15:383-392. 28.Nayler WG. The role of calcium in the ischémie myocardium. Am J Pathol 1981;102:262- 270. 29.Watts JA, Koch CD, LaNoue KF. Effects of Ca2+ antagonism on energy metabolism: Ca2+ and heart function after ischemia. Am J Physiol 1980;238:H909-H916

~~73 Chapter 4~~

3O.Steenbergen C, Jennings RB. Relationship between lysophospholipid accumulation and plasma membrane injury during total in vitro ischemia in dog heart. J Mol Cell Cardiol 1984,16:605-621. 31 .Boink ABTJ, Ruigrok TJC, Maas AHJ, Zimmerman ANE. Changes in high-energy phosphate compounds of isolated rat hearts during Ca2+-free perfusion and reperfusion with Ca2+. J Mol CeU Cardiol 1976;8:973-979. 32Jynge P. Protection of the ischemic myocardium. Calcium-free cardioplegic infusates and the additive effects of coronary infusion and ischemia in the induction of the calcium paradox. Thorac Cardiovasc Surgeon 1980;28:303-309. 33.Ganote CE, Humphrey SM. Effects of anoxic or oxygenated reperfusion in globally ischemic, isovolumic, perfused rat hearts. Am J Pathol 1985;120:129-145. 34JPost JA, Leunissen-Bijvelt J, Ruigrok TJC, Verkleij AJ. Ultrastructural changes of sarcolemma and mitochondria in the isolated rabbit heart during ischemia and reperfusion. Biochim Biophys Acta 1985;845:119-123. 35.Grinwald PM, Brosnahan C. Sodium imbalance as a cause of calcium overload in post-hypoxic reoxygenation injury. J Mol Cell Cardiol 1987;19:487-495. 36.Pridjian AK, Levitsky S, Krukenkamp I, Silverman NA, Feinberg H. Intracellular sodium and calcium in the postischemic myocardium. Ann Thorac Surg 1987;43:416-419. 37.Renlund DG, Gerstenblith G, Lakatta EG, Jacobus WE, Kallman CH, Weisfeldt ML. Perfusate sodium during ischemia modifies post-ischemic functional and metabolic recovery in the rabbit heart. J Mol Cell Cardiol 1984;16:795-801. 38.Tunstall J, Busselen P, Rodrigo GC, Chapman RA. Pathways for the movements of ions during calcium-free perfusion and the induction of the 'calcium paradox". J Mol Cell Cardiol 1986;18:241-254. 39.Nayler WG, Perry SE, Elz JS, Daly MJ. Calcium, sodium and the calcium paradox. Circ Res 1984;55:227-237. 40JLamers JMJ, Stinis JT, Ruigrok TJC. Biochemical properties of membranes isolated from calcium-depleted rabbit hearts. Circ Res 1984^4:217-226. 41.Haworth RA, Goknur AB, Hunter DR, Hegge JO, Berkoff HA. Inhibition of calcium influx in isolated adult rat heart cells by ATP depletion. Circ Res 1987;60:586-594.

~~74 Intracellular sodium~~

~~INTRACELLULAR SODIUM DURING ISCHEMIA AND CALCIUM-FREE PERFUSION: A 23Na NMR STUDY~~

~~Abstract~~

Accumulation of sodium-ions (Na+) in myocardial cells during both ischemia and calcium (Ca )-free perfusion has been suggested to play an important role in the damage occurring during subsequent reperfusion and calcium repletion, respectively. We have used 23Na NMR spectroscopy in combination with shift reagents to determine intracellular Na+-concentration ([Na+]i) in isolated rat hearts during either control perfusion followed by ischemia and reperfusion or during control perfusion, Ca +-free perfusion and subsequent ischemia. [Na+]j during control perfusion was found to be 10.5 ±0.6 mmol/l. During ischemia a substantial rise in [Na+]i was observed. During reperfusion [Na+]j decreased, but remained elevated when compared to control perfusion. Most surprisingly, however, no significant increase of [Na ]i was observed during Ca -free perfusion, although severe calcium paradox damage was shown to occur under the used conditions, when calcium was readmitted to the heart. The absence of a rise of [Na+]i during Ca2+-free perfusion was substantiated when during subsequent ischemia a similar rise of [Na+]i was observed as during ischemia without previous Ca +-depletion. We conclude that an increased [Na ]i during Ca -depletion is not a prerequisite for the calcium paradox to occur, but that increased [Na+]i during ischemia may influence the subsequent reperfusion damage through Na+-Ca +-exchange.

~~75 Chapters~~

~~Introduction~~

ccumulation of sodium-ions (Na+) in myocardial cells during both ischemia Aand calcium (Ca2+)-free perfusion has been suggested to play an important role in the damage occurring during subsequent reperfusion and calcium repletion, respectively. The damage occurring upon readmission of Ca2+ after a period of Ca +-free perfusion in a mammalian heart is known as the calcium paradox and is characterized by a massive influx of calcium into the cells 2, contracture of the myofibrils , release of cell constituents , and a rapid depletion of high energy phosphates . The early gain in cytosolic Ca + is generally believed to play a crucial role in the subsequent events of the calcium paradox. This gain in Ca may be due to a decreased ability to remove Ca2+ from the cytosol '6, entry of Ca2+ through the slow channels 7'8 or via Na+-Ca2+ exchange. The possible involvement of the Na+-Ca exchanger has been inferred from increased intracellular Na+ " during Ca +-free perfusion, and the protective effect of reduced or replaced extracellular Na+ 2'9>14)16. The slow Ca2+ channels have been suggested as a possible route for entry of Na+ in the absence of Ca2+ n'13'16. The rise in intracellular Na+-concentration ([Na+]j) has even been suggested to be a prerequisite for the calcium paradox 16 In postischemic reperfusion injury and posthypoxic reoxygenation injury again a rise in Ca + in myocardial cells has been shown to play an important role1 "21. Ca overload may result in decreased energy production by the mitochondria due to Ca + uptake and increased energy utilization by excessive stimulation of the contractile elements, eventually leading to contracture. Already during ischemia or hypoxia 2+ 22 23 cytosolic Ca starts to rise ' most likely caused by failure of the energy dependent sequestration of Ca2+ in the sarcoplasmic reticulum. At the same time [Na+]i increases too ' but, unlike during Ca +-free perfusion, now most probably as a consequence of impaired Na+-K+-pump activity or via Na+-H+ exchange. Upon reperfusion intracellular Ca may substantially increase due to increased membrane permeability or in exchange for Na . Reduction of extracellular Na during low-flow ischemia was found to improve functional and metabolic recovery upon reperfusion ^ and provides further evidence for the involvement of Na+-Ca2+ exchange. Although the rise in [Na+]i has been found to be a common feature of both energy and Ca +-depletion, it has been reported to be much larger during Ca2+-depletion . Unfortunately, information on myocardial [Na+]j levels has been obtained with a variety of techniques and models which do not allow for a straightforward compari-

76 Intracellular sodium son of ischemia and Ca +-free perfusion. The introduction of shift reagents in the perfusate 30 has made it possible to continuously monitor t^e [Na+)j of isolated functioning hearts with "^Na NMR spectroscopy by separating signals from intra-and extracellular Na+. We have used this approach to study and compare the extent of intracellular Na+ accumulation in isolated perfused rat hearts under conditions known to predispose the heart to severe calcium paradox damage or to produce severe ischemic injury.

~~Materials and Methods~~

Heart preparation Male Wistar rats weighing between 350 and 450 g were anesthetized with diethyl ether and heparinized (250 IU i.v.). The heart was rapidly excised and cooled in ice-cold perfusate. After cannulation of the aorta perfusion was started at a constant pressure of 100 cm H2O (10.0 kPa). The perfusion apparatus was equipped with three reservoirs and perfusion lines, which allowed immediate switching of perfusates. Left ventricular developed pressure (LVDP) was monitored by inserting a latex balloon through the left atrium in the left ventricle. The latex balloon was connected to a Statham P23dB pressure transducer and was filled with distilled water to produce an enddiastolic pressure of 10 cm H2O (1.0 kPA). Coronary flow was continuously measured using a Skalar MDL 503 flow meter. The heart rate was maintained at 300 beats/min throughout the experiment by right ventricular pacing with two KCl-wick electrodes connected to a Grass S88 stimulator. Hearts were placed in a 20 mm NMR tube together with a reference capillary containing a solution of Dy(PPP)2 "- The glass tube was lowered into the magnet and the heart was positioned in the center of a Helmholtz NMR coil. The effluent was removed from a level above the heart, leaving the heart submerged for better magnetic susceptibility matching. Myocardial temperature was maintained at 37 °C by water-jacketed perfusion lines and a continuous stream of air of 37 °C around the sample tube. if.

Solutions Stock solutions of dysprosium triethylenetetraminehexaacetate (Dy(TTHA) ") (50 mmoiyi) and dysprosium tripolyphosphate (Dy(PPP)2 ") (62.5 mmol/T) were prepared from DyCl3.6H2O, H6TTHA and NasPPP (Sigma Chemical Co). HeTTHA was dissolved by titrating with 5 moW NaOH. Subsequently, a DyCb-solution was added in small aliquots with continuous pH correction with 5 mol/1 NaOH until a Final pH of 7.4 was reached. A slight excess of TTHA6" over Dy3+ (mole ratio 1.03) was used to absolutely avoid the presence of possibly toxic free Dy3+. The

~~77 Chapter 5~~

~~Na7Dy(PPP)2.3NaCl solution was used as reference for quantitation.~~

Perfusates Three different perfusion solutions were used (Table 1). The amount of NaCI in the perfusate containing shift reagent was adjusted to ensure a constant [Na+]. In addition, the amount of CaCl2 in the perfusate containing shift reagent was increased to correct for the Ca +-affinity of the shift reagent and to maintain normal cardiac performance. The activity of Na+ and Ca2+ in the perfusates was verified using ion-selective electrodes (Radiometer, Copenhagen). The perfusates were filtered (0.8 fim Millipore) prior to use and saturated at ambient temperature with 95% O2/5% CO2 resulting in a final perfusate pH at 37 °C of 7.35 ± 0.05.

~~Table 1. Composition of perfusion fluids (mmol/1)~~

~~Standard Standard + Standard + Shift Reagent Shift Reagent without Ca2+~~

~~NaCI 124.0 6Z0 62.0~~

~~NaHCO3 24.0 24.0 24.0 KC1 4.7 4.7 4.7~~

~~MgCl2 1.0 1.0 1.0 CaCl2 13 2.6 0.0 Na3DyTTHA3NaCl 0.0 10.0 10.0 glucose 11.0 11.0 11.0~~

Experimental protocol Two experimental protocols were followed to study ischemia and reperfusion or Ca -free perfusion followed by ischemia ("calcium paradox" protocol). After a stabilization period of 20 min, a 31P spectrum was obtained in five min, followed by a Na spectrum in one min. Subsequently, a rapid switch from standard perfusate to perfusate containing shift reagent took place and minor adjustments to field homogeneity and coil tuning were completed within 5 min. In the next 20 min, 10 one min Na spectra were collected. The heart was then made globally ischemic for 30 min by cross clamping the perfusion line. During ischemia 15 Na spectra were obtained. The heart was then reperfused for 15 min with perfusate containing shift reagent, while 15 consecutive 23Na spectra were obtained. The "calcium paradox" protocol runs parallel to the ischemia protocol up to the ischemic period. Instead of this ischemic period another rapid switch took place to perfusate containing shift reagent without Ca2+. During the 30 min of Ca2+-free

78 IntraceUular sodium perfusion six Na-spectra were collected. After this period the hearts were made ischemic and in a period of 45 min 22 Na-spectra were collected. After completion of the experiments the wet weight and dry weight of the hearts were determined. The difference (= total tissue water) was assumed to consist of 54 % intracellular water 31

NMR measurements aNa and 31P NMR spectra were obtained at 52.9 and 81.0 MHz respectively on a Bruker MSL 200 spectrometer equipped with a 4.7 Tesla vertical, 150 mm room temperature bore magnet and a multinuclear 20 mm probe. Magnetic field homogeneity was optimized using either the H or Na free induction decay with comparable results. P spectra were obtained to evaluate the quality of the heart preparation. For each P spectrum 128 free induction decays were accumulated following 90° pulses with a repetition time of 2.3 s using 2K data points and a 5 kHz spectral width. For each ^Na spectrum 256 free induction decays were accumulated following 90° pulses with a repetition time of 0.25 s, using 2K data points and 5 kHz spectral width. In separate experiments a progressive saturation sequence was used, which showed that the repetition time of 0.25 s exceeded Gve times the longest Ti-relaxation time. Quantitation of the relatively small peak area corresponding to the intracellular Na resonance was achieved in the following way. Subtraction of spectra obtained during perfusion in the presence of shift reagent from spectra obtained after 30 min of ischemia reduced the large contribution from Na+ in the bathing Quid. The remaining intracellular Na+ peak could easily be quantified by integration and compared to the peak area of the reference. Quantitation of the reference signal was obtained by calibrating against known amounts of NaCl solution, positioned well within the boundaries of the Helmholtz coil.

Creatine kinase activity Parallel experiments were performed to verify that under the conditions used, the calcium paradox occurs to a similar extent in the presence and absence of shift reagent. Coronary effluent was collected during 30 min of reperfusion with standard perfusate after 30 min of Ca2+-free perfusion with or without shift reagent The effluent was analyzed for creatine kinase activity.

~~Statistics All numerical results are presented as mean ± standard deviation.~~

~~79 Chapters~~

~~Results~~

Upon switching from standard perfusate to perfusate containing shift reagent the heart showed a transient increase in LVDP and coronary flow, which soon leveled off at values not significantly different from those observed with standard perfusate as indicated in Table 2, demonstrating that the hearts tolerated the introduction of Dy(TTHA)3"well. Table 2. Left ventricular developed pressure (LVDP) and coronary flow prior and after perfusion with shift reagent.

~~ischemia/reperfusion protocol "calcium paradox" protocol LVDP flow LVDP flow (mm Hg) (ml/min) (mm Hg) (ml/min)~~

A. prior to perfusion 79±12 12.1+2.0 79+6 13.0±2.6 with shift reagent B. during perfusion 85±11 12.8+2.7 81+7 13.6+2.2 with shift reagent C. during reperfusion 8+8 10.3+4.3 with shift reagent D. during Ca2*-free 0 12.5 ±1.4 perfusion with shift reagent

Figure 1 shows a stacked plot of ^Na NMR spectra of an isolated rat heart which was perfused with perfusate containing shift reagent and subsequently made ischemic for 30 min and reperfused for 15 min. The amount of Na+ in the bathing fluid combined with vascular and interstitial Na+ gives rise to such a strong signal that its resonance peak had to be clipped to properly display the intracellular Na+ and reference signals. The intracellular signal shows up as a shoulder on the high-field site of the clipped "extracellular" peak, whereas the reference signal is well separated. The increase of the intracellular Na+-signal during ischemia and the subsequent decrease during reperfusion can be clearly seen in Figure 1. Figure 2 shows the [Na+]i during 20 min of control perfusion followed by 30 min of global normothermic ischemia and 15 min of reperfusion from 6 separate experiments. The mean [Na+]i during control perfusion was calculated from these and the "calcium paradox" experiments and was found to be 10.5 ±0.6. During ischemia a substantial rise in [Na+]i can be seen. During reperfusion [Na+]j did not decrease to preischemic values.

~~80 Intracellular sodium~~

~~0 -10 -20 ppm~~

23 ; Figure 1. Na NMR (4.7 T, 52.9 MHz) spectra of an isolated rat heart, during control peifiision withperfusate containing Dy(TTHA)3' (a), after 6 (b), 12 (c), 18 (d), 24 (e), 30 (f) min of global normothermic ischemia, and afier6minofreperfusion (g). Spectra were resolwion enhanced and each spectrum represents 256 scans obtained in 1 min.

~~40 control ischemia reperfuskxi~~

~~10 20 30 40 50 6O 7O time~~

~~Figure 2. [Na +/i (n=6) during 20 min of control perfiision, 30 min of ischemia and 15 min of reperfusion. [Na+J was calculated assuming a constant intracellular volume of 54 % of toial tissue water .~~

~~81 Chapter 5~~

~~10 0 -10 -20 ppm~~

Figure 3. 23Na. NMR spectra of an isolated rat heart during control perfusion with perfusate containing Dy(TTHAf' (a), after 5 (b), 15 (c), 30 (d) min of Ca2-free perfusion, and after 15 (e), 30 (f), 45 (g) min of global normothermic ischemia.

~~40 control calcium-free ischemia~~

~~0 10 20 3O 40 50 60 70 80 90 100~~

~~time (min)~~

~~Figure 4. [Na+]i (n = 6) during 20 min of control perfusion, 30 min of Ca%Jr-pee perfusion and 45 min cf global normothermic ischemia.~~

~~82 Intracelhdar sodium~~

Figure 3 shows a stacked plot of Na NMR spectra of an isolated rat heart which was perfused with perfusate containing shift reagent followed by 30 min of Ca2+-free perfusion and a subsequent ischemic period of 45 min. It can be clearly seen that no significant increase of the intracellular Na+-signal occurs after 5 or even 30 min of Ca +-free perfusion, whereas the rise in intracellular Na+-signal after 30 or 45 min of subsequent ischemia is obvious. Figure 4 shows the [Na+]i derived from 6 separate "calcium paradox" experiments. No significant increase in [Na+]i can be observed during Ca +-depletion whereas during subsequent ischemia [Na+]i rises to thesame extent as without a previous period of Ca -free perfusion. Reperfusion with Ca +-containing perfusate resulted in a massive release of creatine kinase: 3700±300 IU/30 min per g dry heart tissue (mean ± SD; n=6).

~~Discussion~~

Methodological considerations Na NMR in combination with paramagnetic shift reagents allows for the continuous, non destructive monitoring of intracellular Na+ levels in intact isolated hearts. These shift reagents interact weakly with extracellular Na+ and alter its resonance frequency, leaving the signal from intracellular ions virtually unchanged. As can be seen in Figures 1 and 3, the shift produced by Dy(PPP)2 " is much larger than that produced by Dy(TTHA)3". Nevertheless, we included Dy(TTHA)3~ in the Ifl "7 perfusate since we and others have found the Dy(PPP)2 " complex to be cardio- toxic and cause precipitation problems, most likely due to its high affinity for Ca and magnesium (Mg +). Furthermore, the shift of the Na+ resonance frequency induced by Dy(PPP)27" shows a strong pH dependence, whereas the Dy(TiHA) " shift has been reported constant between pH 6 and 8 , which is advantageous when studying ischemia. Dy(TTHA)3" also shows an, albeit much lower, affinity for Ca +, which could easily be corrected for by increasing the perfusate [Ca +], as evidenced by identical Ca2+ activities with and without shift reagent. Furthermore, upon switching to perfusate with shift reagent, no significant differences in LVDP and coronary flow were found. It appears reasonable to assume that Dy(TTHA) " shows a low affinity for Mg2+ too. However, it is very difficult to determine the free [Mgf+] and since no data on complexation of Mg2+ and Dy(TTHA)3" are available, no attempts were made to correct for a possibly decreased free [Mg +]. The activity of Na+ in the perfusate appeared not to be influenced by the presence of the shift reagent. The omission of Ca2+ resulted in a slightly further downfield shift for the extracellular Na+, since Ca2+ and Na+ compete for Dy(TTHA)3". The better

83 Chapters separation of intra- and extracellular Na+ signal in the absence of Ca made for easier quantitation. Attempts to increase this separation in the presence of Ca by increasing the Dy(TTHA)3" concentration failed, since the increased separation did not outweigh the concomitant increased linewidths. Once the shift reagent had entered the vascular and interstitial spaces of the heart, the resonance frequency of the intracellular Na+ signal remained unaltered throughout the entire experiment, indicating that Dy(TTHA)3" cannot cross the plasma membrane, as also has been demonstrated by Pike et al and found to be true for a variety of shift reagents and cell types 33"41. For absolute quantitation of [Na+]i, knowledge of the NMR visibility of intracellular Na+ is required. Visibility factors varying from 0.4-1.0 have been reported and appear to be related to quadrupolar coupling of the Na-nucleus to intracellular electric field gradients . However, for erythrocytes , renal cortical tubules 37 and heart38>39 an NMR visibility factor of 1 for intracellular Na+ has been firmly established. The accurate measurement of [Na+]j during control perfusion is difficult due to its low level and the incomplete separation of signal from intracellular Na+ and the large signal from Na+ in the bathing fluid. Nevertheless, our value of 15 27 38 39 10.5 ±0.6 mmol/1 is well within the range of values obtained by NMR ' > > and other methods 11-13'16-19'24-26.

[Na+]\ during ischemia and reperfusion With the great advantage of being able to monitor [Na+]i continuously, we have followed the change in [Na+]i during 30 ruin of global ischemia and confirmed the substantial increase in [Na+]i, previously reported to occur during conditions of hypoxia * '25«26>2 and ischemia 24>27, although actual extent and time course varied with different experimental conditions. We have observed a rise in [Na+]i within the first minutes of ischemia. Intracellular inorganic phosphate (Pi) levels have also been reported to increase already substantially (> 2-fold) during the very first minutes of hypoxia ' 3. The concurrent rise of [Na+] i and Pi suggests that Pi may decrease the Na+-K+-ATPase activity through product inhibition. On the other hand, Neubauer et al have reported a smaller rise in [Na+]j during 28 min of hypoxia than during an ischemic period of 10 min, which resulted in comparable ATP depletion (= 40%). From the difference in intracellular pH they suggested a role for the Na+-H+ exchange mechanism in the Na+ accumulation during ischemia. In agreement with this small rise in [Na+]i, Poole-Wilson and Tones K reported that [Na+]i only started to rise after 20 min of substrate-free hypoxic perfusion. In contrast, Fiolet et al M found an immediate rise in [Na+]j during anoxic perfusion. Detailed, high time-resolution studies on intracellular [Pi] and [Na+]i during hypoxia and ischemia are required to determine the relative contributions of the Na+-H+ exchange ;md the

~~84 Intracellular sodium~~

inhibition of the Na+-K+-ATPase to the rise in [Na+]j. The contribution of Na+-Ca + exchange to Ca2+ overload during ischemia and reperfusion has been inferred from the stimulation of post-ischemic Ca2+ uptake by low extracellular Na+ levels during reperfusion17 and from Ca2+ uptake after normoxic Na+ loading19. These results are corroborated by the beneficial effects of low extracellular Na+ during ischemia on functional and metabolic recovery during reperfusion . In contrast, Poole-Wilson and Tones could not detect an increased „ efflux of Na+ upon reoxygenation, whereas they did find an increased efflux of Na+ . ry . under conditions known to stimulate Na -Ca exchange otherwise. Although they acknowledge the Na -Ca exchange mechanism being active at the moment of reoxygenation, they invoke Na+-H+ exchange to explain a net Ca2+-influx without a net Na+-efflux. However, this hypothesis fits the ischemic heart with its large H+-gradient during the first stage of reperfusion better than the hypoxic heart Therefore, additional H+-generation upon reoxygenation by the mitochondria in ty , *}c exchange for cytosolic Ca was suggested . In our experiments we did observe a decrease of [Na+]i upon reperfusion, which leveled off after approximately 5 min and remained elevated when compared to preischemic [Na+]i. This finding can be reconciled with the occurrence of Na+-Ca + exchange and a sustained inhibition of the Na+-K+-ATPase due to lack of ATP or elevated Pj levels. Alternatively, these results may indicate heterogeneous reperfusion and heterogeneous recovery. [Na ]\ during Ca -free perfusion Most surprisingly, we have not been able to demonstrate a significant increase of [Na+]iduring30 minof Ca +-free perfusion, and certainly not to the extent observed during ischemia, although only a few minutes of Ca +-free perfusion are already ,; sufficient to cause a full-blown calcium paradox upon readmission of Ca2+. This J - finding does not support the previous suggestion , that additive detrimental effects V of ischemia and low Ca2+-reperfusion would depend on intracellular Na+. The V ' occurrence of the calcium paradox in the presence of the shift reagent Dy(TTHA) '{'•••• was confirmed by measurements of creatine kinase release. Furthermore, we feel ^ that the validity of the [Na ]i measurement is convincingly demonstrated by the rise V in [Na+]i in the ischemic period following the period of Ca2+-free perfusion. f| A reduced maximal Na+-K+-ATPase activity has been reported in sarcolemmal || vesicles, prepared from calcium-depleted hearts 45. On the other hand, in cells fi. isolated in the absence of Ca2+ an increased Na+-K+-ATPase activity has been iff demonstrated 46, which could maintain low [Na+]i in the presence of increased influx. feS Since during Ca2+-free perfusion Pi levels decrease, phosphocreatine levels increase I I 85 Chapter 5

47 and no ATP is used for contractile activity, the conditions for increased Na+-K+- ATPase activity are favourable. However, a more likely explanation for our results is that the supposed Na+-influx through the slow Ca2+-channels is effectively blocked by the presence of Mg2+. This would also explain why in so many previous studies an increased [Na+]j was found during Ca2+-depletion, since in most of those studies chelators were used to ensure the complete absence of Ca , which at the same time could substantially reduce extracellular [Mg ], or Mg + itself was omitted too. The inhibiting influence of Mg2+ on Na+-influx during Ca2+-depletion has been recognized before 12>29148. Although in our experiments the exact free [Mg +] was unknown, it appeared to be sufficient to block Na+ -influx since in preliminary experiments we have found that in the absence of both Ca + and Mg + an increase in [Na+]i does indeed occur, and of course a calcium paradox upon Ca2+-repletion. Furthermore, Busselen 49 has demonstrated that the attenuating effect of reduced extracellular Na+ during Ca +- depletion on the extent of the calcium paradox damage does not depend on the Na+-gradient across the sarcolemma, and he suggests that Na+ or other monovalent cations displace Ca from its binding sites upon Ca -free perfusion, which would predispose the myocardial cells to the calcium paradox. In conclusion, it has been demonstrated that an increased [Na ]i during Ca - depletion is not a prerequisite for the calcium paradox to occur, but an increased [Na+]j during ischemia may influence the subsequent reperfusion damage.

~~References~~

1. Zimmerman ANE, Hulsmann WC. Paradoxical influence of calcium ions on the permeability of the cell membranes of isolated rat hearts. Nature 1966;211:646-647. 2. Alto LE, Dhalla NS. Myocardial cation contents during induction of calcium paradox. Am J Physiol 1979;237:H713-H719. 3. Ganote CE, Nayler WG. Contracture and the calcium paradox. J Mol Cell Cardiol 1985;17:733-745. 4. Boink ABTJ, Ruigrok TJC, M

~~86 Intracellular sodium~~

7. Ohhara H, Kanaide H, Nakamura M. A protective effect of verapamil on the calcium paradox in the isolated perfused rat heart. J Mol Cell Cardiol 1982;14:13-20. 8. Meno H, Kanaide H, Nakamura M. Effects of diltiazem on the calcium paradox in isolated rat hearts. J Pharmacol Exp Ther 1984;228:220-224. 9. Goshima K, Wakabayashi S, Masuda A. Ionic mechanism of morphological changes of cultured myocardial cells on successive incubation in media without and with Ca2+. J Mol Cell Cardiol 1980;12:1135-1157. 10. Cheung JY, Thompson IG, Bonventre JV. Effects of extracellular calcium removal and anoxia on isolated rat myocytes. Am J Physiol 1982;243:C184-C190. 11. Chapman RA, Rodrigo GC, Tunstall J, Yates RJ, Busselen P. Calcium paradox of the heart: a role for intracellular sodium ions. Am J Physiol 1984;247:H874-H879. 12. Chapman RA, Fozzard HA, Friedlander IR, January CT. Effects of Ca^TMg2* removal on aNa', aK1, and tension in cardiac Purkinje fibers. Am J Physiol 1986;251:C920-C927. 13. Nayler WG, Perry SE, Elz JS, Daly MJ. Calcium, sodium, and the calcium paradox. Circ Res 1984;55:227-23X 14. Ruano-Arroyo G, Gerstenblith G, Lakatta EG. 'Calcium paradox5 in the heart is modulated by cell sodium during the calcium-free period. J Mol Cell Cardiol 1984;16:783-793. 15. Wittenberg BA, Gupta RK. NMR studies of intracellular scdhun ions in mammalian cardiac myocytes. J Biol Chem 1985;260:2031-2034. 16. Guarnieri T. Decrease hi the transmembrane sodium activity gradient hi ferret papillary muscle as a prerequisite to the calcium paradox. J Clin Invest 1988;81:1938-1944. 17. Grinwald PM. Calcium uptake during post-ischemic reperfusion in the isolated rat heart* influence of extracellular sodium. J Mol Cell Cardiol 1982-14:359-3.55. 18. Poole-Wilson PA, Harding DP, Bourdillon PDV, Tones MA. Caicium out of controL J Mol Cell Cardiol 1984-16:175-187. 19. Grinwald PM, Brosnahan C. Sodium imbalance as a cause of calciiun overload hi post-hypoxic reoxygenation injury. J Mol Cell Cardiol 1987;19:487-495 20. Pridjian AK, Levitsky S, Krukenkamp I, Silverman NA, Feinberg H. Intracellular sodium and calcium in the postischemic myocardium. Ann Thorac Surg 1987;43:416-419. 21. Nayler WG, Panagiotopoulos S, Elz JS, Daly MJ. Calcium-mediated damage during post- ischaemic reperfusion. J Mol Cell Cardiol 1988;2Q(Suppl n):41-54. 22. Shen AC, Jennings RB. Kinetics of calcium accumulation in acute myocardial ischemic injury. Am J Pathol 1972;67:441-452. 23. Steenbergen C, Murphy E, Levy L, London RE. Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Core Res 1987;60:700-707. 24. Regan TJ, Broisman L, Haider B, Eaddy C, Oldewurtel HA. Dissociation of myocardial sodium and potassium alterations in mild versus severe ischemia. Am J Physiol 1980;238:H575-H580. 25. Poole-Wilson PA, Tones MA. Sodium exchange during hypoxia and on reoxygenation hi the isolated rabbit heart. J Mol Cell Cardiol 1988;20(Suppl n):15-22. 26. Fiolet JWT, Baartscheer A, Schumacher CA, Coronel R, ter Welle, HF. The change of the free energy of ATP hydrolysis during global ischemia and anoxia in the rat heart. J Mol Cell Cardiol 1984;16:1023-1036. 27. Neubauer S, Balschi JA, Springer CS, Smith TW, Ingwall JS. Intracellular Na+accumulation hi hypoxic vs. ischemic rat heart: evidence for Na+-H+exchange. Circulation 1987;76(SupplIV):IV56.

~~87 Chapters~~

28. Renlund DG, Gerstenblith G, Lakatta EG, Jacobus WE, Kallman CH, Weisfeldt ML. Perfusate sodium during ischemia modifies post-ischemic functional and metabolic recovery in the rabbit heart. J Mol Cell Cardiol 1984,16:795-801. 29. Chapman RA, Tunstall J. The calcium paradox of the heart. Prog Biophys Molec Biol 1987;50:67-96. 30. Pike MM, Frazer JC, Dedrick DF, Ingwall JS, Allen PD, Springer CS, Smith TW. ^Na and 39K nuclear magnetic resonance studies of perfused rat hearts. Discrimination of intra-and extracellular ions using a shift reagent. Biophys J 1985;48:159-173. 31. GonzalezF, Harris C, Bassingthwaighte JB. Volumes of distribution of intact rabbit hearts. Physiologist 1980;23:78. 32. Chu SC, Pike MM, Fossel ET, Smith TW, Balschi JA, Springer CS. Aqueous shift reagents for high-resolution cationic nuclear magnetic resonance. III. Dy(TTHA)3", Tm(TTHA)3", 7 and Tm(PPP)2 ~. J Magn Res 1984;56:33-47. 33. Gupta RK, Gupta P. Direct observation of resolved resonances from intra- and extracellular sodium-23 ions in NMR studies of intact cells and tissues using dysprosium(III)tripolyphosphate as paramagnetic shift reagent. J Magn Reson 1982;47:344-350. 34. Balschi JA, Cirillo VP, Springer CS. Direct high-resolution nuclear magnetic resonance of cation transport in vivo. Na+ transport in yeast cells. Biophys J 1982;38:323-326. 35. Civan MM, Degani H, Margalit Y, Shporer M. Observations of aNa in frog skin by NMR. Am J Physiol 1983;245:C213-C219. 36. Ogino T, Shulman GI, Avison MJ, Gullans SR, den Hollander JA, Shulman RG. ^Na and ^K NMR studies of ion transport in human erythrocytes. Proc Natl Acad Sci USA 1985;82:1099-1103. 37. Gullans SR, Avison MJ, Ogino T, Giebisch G, Shulman RG. NMR measurements of intracellular sodium in the rabbit proximal tubule. Am J Physiol 1985;249:F160-F168. 38. Fossel ET, Hoefeler H. Observation of intracellular potassium and sodium in the heart by NMR: a major fraction of potassium is "invisible". Magn Reson Med 1986;3:534-540. 39. Burstein D, Fossel ET. Nuclear magnetic resonance studies of intracellular ions in perfused frog heart. Am J Physiol 1987;252:H1138-H1146. 40. Naritomi H, Kanashiro M, Sasaki M, Kuribayashi Y, Sawada T. In vivo measurements of intra- and extracellular Na+ and water in the brain and muscle by nuclear magnetic resonance spectroscopy with shift reagent. Biophys J 1987^2:611-616. 41. Blum H, Schnall MD, Chance B, Buzby GP. Intracellular sodium flux and high-energy phosphorus metabolites in ischemie skeletal muscle. Am J Physiol 1988;255:C377-C384. 42. Berendsen HJC, Edzes HT. The observation and general interpretation of sodium magnetic resonance in biological material. Ann NY Acad Sci 1973^04:459-485. 43. Allen DG, Morris PG, Orchard CH, Pirolo JS. A nuclear magnetic resonance study of metabolism in the ferret heart during hypoxia and inhibition of glycolysis. J Physiol 1985;361:185-204. 44.Kirkels JH, Ruigrok TJC, van Echteld CJA, Meijler FL. Low Ca2+ reperfusion and enhanced susceptibility of the postischemic heart to the calcium paradox. Circ Res 1989;64:1158-1164. 45. Lamers JMJ, Stinis JT, Ruigrok TJC. Biochemical properties of membranes isolated from calcium-depleted rabbit hearts. Circ Res 1984;54:217-226. 46. Haworth RA, Hunter DR, Berkoff HA. Mechanism of Ca2+-resistance in adult heart cells isolated with trypsin plus Ca2+. J Mol Cell Cardiol 1982;14:523-530.

~~88 Intracellular sodium~~

V7. Ruigrok TJC, Kirkels JH, van Echteld CJA, Borst C, Meijler FL. 31P NMR study of intracellular pH during the calcium paradox. J Mol Cell Cardiol 1987;19:135-139. 48. Bhqjani IH, Chapman RA. The relationship between intracellular sodium activity (a*Na) and extracellular sodium activity (a°Na) in isolated ferret ventricular muscle, in the presence and absence of divalent cations. J Physiol 1987;390:61P. 49. Buasebn P. Effects of sodium on the calcium paradox in rat hearts. Pflugers Arch 1987;408:458-464.

~~89 Intracellular magnesium~~

~~INTRACELLULAR MAGNESIUM DURING MYOCARDIAL ISCHEMIA AND REPERFUSION: POSSIBLE CONSEQUENCES FOR POSTISCHEMIC RECOVERY.~~

~~Abstract~~

Magnesium (Mg ) is an important regulator of cell energy metabolism, since only MgATP can serve as asubstrate for ATP utilizing processes. We used 31P NMR spectroscopy to determine the complexation of ATP with Mg + and intracellular free Mg + (Mgf) in isolated rat hearts during control perfusion, ischemia and reperfusion. Atomic absorption spectrophotometry was used to determine preischemic and postischemic tissue Mg and release of Mg into the coronary effluent during reperfusion. Mgf increased from 0.60 mmol/1 during control perfusion to > 6.5 mmol/1 after 15 min of ischemia, while we estimated that at that time 6.7 mmol/1 Mg + had been liberated from ATP. Less than 2 % of cellular Mg2+ was released to the effluent during reperfusion after 30 min of ischemia. From spectra obtained during reperfusion the fraction of ATP that was bound to Mg was calculated to be = 96% (compared to 94 % during control perfusion), indicating that intracellular Mg2"1" did not limit the metabolic use of the newly produced ATP. Mgf remained elevated during reperfusion (0.85 mmol/1). We conclude that intracellular Mg2+ deficiency due to leakage of Mg2+ to the extracellular space does not play a role in the poor postischemir recovery in this isolated rat heart model. Nevertheless, high Mg + prior to ischemia or during reperfusion may well be protective, due to interactions of Mg + with the sarcolemma or intracellular sites, affecting Ca2+, K+ and Na+ distribution and fluxes.

~~91 Chapter 6~~

~~Introduction~~

-* yragnesium (Mg2+) is an essential cofactor in many ATP producing and -*•* -"-utilizing enzymatic processes 1>2, such as the Na+/K+ pump activity, myofibrillar contraction, oxidative phosphorylation and several steps of glycolysis ' ' . Another important feature of Mg2+ is that it can compete with Ca2+ \ both at the sarcolemma (depending upon the species) and at several intracellular sites such as the troponin-C binding site 7 and mitochondrial membranes . Magnesium therefore has been called "nature's physiologic calcium blocker" . Although not all effects of magnesium are equally well understood, it has long been known from epidemiological studies for a review see 10 that magnesium deficiency may be related to a host of diseases, including cardiac arrhythmias, hypertension and stroke. In recently published animal experiments acute or chronic Mg + deficiency was found to reduce tolerance to myocardial ischemia in isolated rat 111*7 1*4 o t hearts ' , and to result in larger infarcts in dogs . In addition, high Mg during postischemic reperfusion proved sometimes beneficial in an isolated rabbit ' or rat heart model and high Mg prior to ischemia or during cardioplegia improved postischemic recovery 6>16; for a review see \ In a few clinical trials patients admitted to hospital with a myocardial infarction were given MgCl2 or MgSO4 infusions with 1*7 1 ft favourable results, as determined by infarct size, arrhythmias or mortality rate ' . However, it is still unclear what role Mg2+ may play in the limitation of ischemic myocardial damage or recovery during reperfusion. The present study was performed (1) to gain insight in the possible role of Mg in a model of ischemia, which is known to cause (at least to some extent) irreversible metabolic and functional damage, and (2) to establish whether postischemic recovery is limited by intracellular Mg2"*" deficiency due to loss of Mg2+ to the extracellular space. 31P NMR spectroscopy enabled us to calculate the fraction of ATP complexed to Mg2"1", the intracellular free Mg2+ concentration (Mgf), high- energy phosphates and intracellular pH under normal and ischemic conditions, as well as during reperfusion. Atomic absorption spectrophotometry was used to assay total Mg + in tissue and coronary effluent.

~~I Materials and Methods \ £ }• | Animal model | Male Wistar rats, weighing 300-350 g were anesthetized with diethylether and~~

92 Intracellular magnesium heparinized. The heart was rapidly excised and cooled in ice-cold perfusate. After cannulation of the aorta, perfusion was started by the Langendorff technique at a constant pressure of 9.81 kPa. The perfusate had the following composition

(mmol/1): NaCl, 124.0; KC1,4.7; MgCl2,1.0; NaHCO3) 24.0; Na2HPO4,0.5; CaCl2, 1.3; glucose, 11.0, and was equilibrated with 95% O2 and 5% CO2 (pH 7.4 at 37 °C).The heart was stimulated at 300 beats/min throughout the experiment by two NaCl-wick electrodes. A fluid-filled open catheter was inserted through the apex and connected to a Statham P23dB pressure transducer to measure left ventricular developed pressure . The hearts were observed during 15 min of control perfusion and 30 min of total ischemia, followed by 30 min of reperfusion.

NMR-studies In the first series of experiments hearts (n=8) were placed in a 20 mm NMR tube with a capillary containing methylene diphosphonate for spectral reference. The glass tube with the submerged heart was then lowered into the NMR magnet Coronary flow was measured by collecting the effluent from the hearts in 5 min fractions. Myocardial temperature was carefully maintained at37 °Cby using a water jacketed perfusion line and heat-controlled air around the sample tube inside the magnet. 31P NMR spectra were obtained on a Bruker MSL 200 spectrometer equipped with a 4.7 Tesla vertical bore magnet. No field frequency lock was used. Consecutive five minute spectra were obtained from 128 accumulated free induction decays after 90° pulses repeated at 2.3 s intervals. The data were accumulated using 2K datapoints and 5 kHz spectral width. Exponential multiplication resulting in 10 Hz line broadening was applied and polynomial baseline correction was performed on the spectra. Quantitation of metabolites was achieved by intregrating the individual peaks of interest in each spectrum. The /J-phosphate peak of ATP was used to determine ATP levels. Intracellular and perfusate pH were calculated from the chemical shift of the respective inorganic phosphate (Pj) peaks relative to creatine phosphate (CP). Data on ATP and CP were expressed as percent of baseline levels; intracellular Pi was expressed as percent of the total amount of phosphate from CP, ATP and intracellular Pi during preischemic control perfusion:

~~Pi/{CP + 3ATP + Pi}preischemicx 100%.~~

Saturation factors for CP and Pi (1.5 and 1.1, respectively) were determined in a separate experiment with an interpulse time of 10 s. The effect of binding of Mg2+ to ATP on the chemical shift of the a- and

~~93 Chapter 6~~

/J-phosphate peaks of ATP was previously established from solutions containing either (mmol/1): KC1 (150), ATP (5) and EDTA (1), or KC1 (100), ATP (5), EDTA (1) and MgCh (25). In order to mimic ischemic conditions in the heart, the NMR measurements were repeated at different pH values by titration of the solutions with 0.1 M HCl. The difference between the chemical shift of ATPa and (pa-fi) obtained from P NMR spectra of the isolated rat hearts could then be used to calculate the fraction (<£) of free ATP by the following equation 20'21:

~~M ATP [ATP]free _ 6af - daf 8~~

(ATPl ~~ A OATP A ,,MgATP ' J [ATPJiotal Oa-P -Oa-fi tn MsATP 2+ ATP where <5a-^ represents the da-p at excess Mg and &a-p in the absence of Mg2+, both at the pH determined from the shift of the Pi peak. Next, using a dissociation constant (KD) for the MgATP complex of 0.038 mmol/120, free Mg2+ (Mgf) could be calculated according to:

~~M8A r 1 = KD ' V -l) [2]~~

~~KDMgATP was corrected for the intracellular pH (pHi) by multiplication with a factor (f) 2222:~~

~~6 5 Hi I + i0 - -P f i + io6-5-7-2~~

3 In order to improve the accuracy of the determination of da -fi fromth e *P NMR spectra, two additional methods of data processing were used. First, to enhance the signal to noise ratio, exponential multiplication resulting in 30 Hz line broadening was used, and secondly, to enhance spectral resolution, Gaussian multiplication was used.

Magnesium assay In a separate series of experiments isolated rat hearts (n=5) were perfuse' under similar experimental circumstances for 15 minutes, followed by 30 min of total ischemia and 5 min of reperfusion. During reperfusion the coronary effluent was collected in 1 min fractions for determination of Mg2+. The hearts were then perfused with physiologic saline for 30 s to remove the Mg2+-containing perfusate in the vascular compartment. After dismounting the heart from the perfusion

94 Intracellular magnesium cannula, it was cut open and gently blotted with a tissue, weighed and dried in a stove at 70 °C for 24 hours. After determination of dry weight the heart was extracted in 6 N HNO3 for 7 days, and tissue Mg2+ was determined from this extract. A control series of hearts was analyzed for total tissue Mg + after 15 min of perfusion without ischemia. Mg2+ was determined by atomic absorption spectrophotometry (A.A.S.). All effluent samples were measured sixfold. The difference in [Mg2+] between coronary effluent during reperfusion and during control perfusion was evaluated for statistical significance by Student's t-test.

~~Maximal Mg + release In order to determine to what extent Mg + could be released from severely~~

Figure 1. Time course in intracellu- ischemia reperfusion lar levels of creatine phosphate (CP), ATP, and inorganic phosphate (Pi), as measured with P NMR in isolated perfused rat hearts. CP and ATP are expressed as a percent of their respective preischemic values. Pi is expressed as a percent of the amount of phosphate groups from CP, ATP and Pi during preischemic control perfusion: P-J{CP

~~+ 3ATP + Pi}preischemic* 100 %. Each point represents the mean ± SD of 8 experiments.~~

95 Chapter 6 damaged myocardial cells, hearts (n=4) were subjected to 5 min of Ca-2 + -free perfusion followed by 5 min of normal Ca perfusion (L3 mM), which gives rise to the calcium paradox . During readmission of calcium the coronary effluent was collected in 1 min fractions for determination of Mg +.

~~Results~~

High-energy phosphates, cardiac Junction and coronary flow Figure 1 shows the data on CP, ATP and Pi during ischemia and reperfusion. The rapid degradation of CP and ATP during ischemia was balanced by an equal increase in Pi. During reperfusion CP recovered to 47±5 % and ATP to 25±9 % of preischemic values. Pi remained elevated but the sum of phosphate from ATP, CP and Pj was no longer equal to the preischemic sum, indicating loss of phosphate or accumulation of NMR-invisible (calcium)phosphate in mitochondria. Left ventricular developed pressure recovered to 40±18 % and coronary flow to 84± 17 % of preischemic values.

~~Intracellular Mg2+~~

~~The experimentally determined relationship between pH and da-fi of ATP both 2+ 2+ at excess Mg and without Mg is shown in Fig-2. The actual da-fi as determined~~

6.8 7.2 7.4 pH Figure 2. Chemical shift difference (daf) ofATPaandATPfi as a function of nil determined from 3XP NMR spectra of an ATP-containing solution with excess Mg2* (daJT^1*: x—x) or without £+ ATP

~~96 Intracellular magnesium~~

W WV VWV" Vw* 20 10 -10 ppm -20 Figure 3. PNMR spectra obtained from an isolated perfused rat heart during controlperfusion (a), from 0 - 5 min of ischemia (b), from 5-10 min of ischemia (c), and from 10 -15 min of ischemia (d). Each spectrum consists of 128 accumulated free induction decays after 90°pulses with a repetition time of 2.3 s. Signal to noise was improved by exponential multiplication resulting in 10 Hz line broadening. Numbered peaks include: 1. methylene diphosphonate (external reference); 2. extracellular inorganic phosphate; 3. intracellular inorganic phosphate;

4. creatinephosphate; 5. ATPY; 6. ATPa; 7. NAD(H);8.ATPp. The vertical lines indicate the consistency of the chemical shift difference between ATPa andATPp (aa-P ). from a P NMR spectrum of a rat heart consists of the weighted average of contributions from free ATP and MgATP, due to rapid exchange. The intracellular pH necessary to perform the calculations was derived from the same spectrum. Fig. 3 shows consecutive spectra obtained from an isolated rat heart during control perfusion (a), from 0-5 min of ischemia (b), from 5-10 min of ischemia (c) and from 10-15 min of ischemia (d). In subsequent spectra the signal to noise ratio of ATPa and ATP^ was not sufficient to determine da-P- The vertical lines indicate the consistency oida-fi during the first 15 min of ischemia, despite large differences in the amount of ATP. Table 1 summarizes the data directly derived from the NMR spectra or calculated from these. The rapid drop of ATP during the first 15 min of ischemia was accompanied by a decrease in the fraction of free ATP from 6.1 ±0.5 % to less than 2 %. From the % ATP and the fraction of ATPfce an estimation is

~~97 Chapter 6~~

~~Table 1. Data directly obtained or calculated from 31 P NMR spectra during control perfusion and ischemia.~~

~~pH ATP Mg2+liberated j^MgATP Mgf % from ATP (mmol/1) (mmol/1) (mmol/1)~~

control 7.06+0.02 100 0.061+0.005 0.039 0.60+0.05 0- 5 minisch. 6.58+0.16 93±11 0.043+0.009i 0.49 0.063 1.40±0.29 5-10 min isch. 6.06±0.04 55±H 0.030±0.011 4.06 0.111 350+132 10-15 minisch. 5.86+0.05 27±8 <0.02 6.74 0.158 >6J>

~~Mean+SD of 8 experiments; pH derived from the chemical shift of inorganic phosphate relative to cretine phosphate; ATP: % preichemic value;~~

MsATP KD : dissociation constant of MgATP, corrected for pH. obtained of the amount of Mg + liberated from ATP, assuming a preischemic cytosolic [ATP] of 10 mmol/119. By 15 min of ischemia about 6.74 mmol/1 Mg2+ had been liberated (Table 1), and the calculated intracellular free [Mg +] had risen accordingly from 0.60 mmol/1 during control perfusion to > 6.50 mmol/1. Although the accuracy of these values decreases with decreasing ATP levels, as will be discussed later, they indicate that Mg + liberated from ATP during ischemia

appears largely as Mgf. During reperfusion, the da-fi detennined from the P NMR spectra was slightly but consistently lower than during control perfusion. Conse- quently, the estimated fraction of free ATP is somewhat smaller and Mgf corre- spondingly elevated (Table 2). The calculations are based on the assumption that,

~~Table 2. Data calculated from 3IP NMR spectra during reperfusion after 30 minutes of ischemia.~~

~~Mgr(mmolA)~~

0- 5 min reperfusion < 0.020 >1.90 5-10 min reperfusion < 0.020 > 1.90 10-15 min reperfusion 0.036±0.023 1.04+0.66 15-20 min reperfusion 0.044±0.014 0.85+0.27 20-25 min reperfusion 0.040+0.010 0.94+0.23 25-30 min reperfusion 0.044+0.005 0.85+0.10

~~Mean+SD of 8 experiments;~~

~~98 Intracellular magnesium~~

~~Table 3. Mg2+in tissue samples and coronary effluent~~

~~fimollg dry weight~~

~~Preischemic tissue Mg2+(n=8) 39.72 ± 0.41~~

~~Postischemic tissue Mg2+(n=8) 37.51 ± 1.55~~

~~Postischemic Mg2+loss* (n=5) 0.73 ± 0.22~~

~~Calcium paradox related Mg2+ loss" (n=4) 25.47 ± 2.02~~

•Accumulated Mg2+ loss over 5 minutes of reperfusion Mean ± SD although metabolic recovery of myocardial cells may be heterogeneous ^ cells that regenerated ATP also restored a normal intracellular pH. Since abnormal values for Mgf during reperfusion may have important consequences, we subsequently analyzed the data in a different way. Spectra from corresponding 5 minute periods in the different experiments were added, after careful matching of the reference resonance positions. Thus, the improved signal to noise ratio allowed for an over-all measurement of the fraction of MgATP and intracellular free Mg2+ (not shown in

Table 2). The da-fi determined from these spectra confirmed that during the first three 5-min periods of reperfusion more than 98% and during the next three periods 96% of ATP was complexed to Mg2+. Therefore, at the end of 30 min of reperfusion, when ATP had recovered to 25 % of preischemic levels, 6.99 mmol/1 Mg2+ of the original 9.39 mmol/1 complexed to ATP were no longer bound, whereas the Mgf was calculated to be only 0.85 mmol/1.

Mg + in coronary effluent and heart tissue During postischemic reperfusion the [Mg2+] of the coronary effluent was higher than during preischemic control perfusion in the first and sometimes the second minute fraction. The cumulative Mg2"*" loss is given in Table 3. The comparison of 0.73,umol/g dry weight with the preischemic tissue Mg2+ of 39.72 ^mol/g dry weight implies that less than 2 % of total heart Mg2+ was released during reperfusion after 30 min of ischemia. Accordingly, tissue Mg2+ was slightly but not significantly lower after reperfusion compared to preischemic values. For comparison, cumulative Mg2+ loss to the coronary effluent during the calcium paradox was calculated to be about 64 % of total heart Mg2+.

~~99 Chapter 6~~

~~Discussion~~

Although intracellular free magnesium (Mgr) is an important regulator of many cell functions, values for Mgf under normal or abnormal circumstances are still a matter of debate. Four different methods are available to determine Mgf3-25*26: indirect calculations based on equilibrium reactions, NMR measurements of molecules that chelate Mg such as ATP and citrate, ion selective microelectrodes, and nul! point measurements using metallochromic dyes. All recent NMR studies 26>27 present values for Mgf of less than 1 mmol/l under normal circumstances ? in contrast to most studies using other techniques, which usually report higher values 3,28,29 The use of 31P NMR to calculate Mgf is attractive, since it is non-destructive, can be repeated in time, and does not necessitate the use of intracellular complexing agents which may bind Ca and H in addition to Mg . At the same time, however, there are some inherent problems and inaccuracies, which may limit the 31 application of P NMR. In order to obtain reliable values for da-p, the signal to noise ratio of ATPa and ATP/j must be sufficiently high, which in the present study was no longer true after 15 min of ischemia. An implicit assumption in the use of 3 J P NMR to study Mgf is that the chemical shifts of ATPa and ATP£ in tissue and in calibration solutions are influenced in a similar way by the presence of Mg . This has been proven to be true for erythrocytes and is likely to be valid for other cell types as well30. Due to the low KDMgATP most of the ATP is saturated with

Mg ' , and small changes in the observed 6a-§ give rise to rather large changes 2+ in the calculated fraction of Mg bound to ATP. Finally, the value for KDMSATP 31 is crucial for the eventual calculation of Mgf. Gupta et al. determined KDMSATP in three different ways and suggested a value of about 0.050 mmol/l at 25 °C We used the value of 0.038 mmol/l determined by the same group of investigators in frog skeletal muscle at 35 °C and pH 7.2 7a. Our value for Mgf of 0.60 mmol/l during control perfusion is well in agreement with values of 0.5 or 0.6 mmol/l obtained by others using 31P NMR in isolated hearts 1S>21>31. 19F NMR with intracellular Quorinated magnesium chelators yielded values of 0.25 mmol/l in erythrocytes and 0.85 mmol/l in isolated rat hearts 27. It has been reported that postischemic myocardium loses Mg2+ s^2, favouring mitochondrial calcium accumulation, which could be reduced by a high extracellular [Mg2+] during reperfusion 8'14. A high extracellular [Mg2+] during reperftision was also found to improve postischemic metabolic recovery . Chronic Mg + deficiency may affect Na+ fluxes, giving rise to elevated intracellular [Na+] 1>n. At a later stage this may lead to an increased [Ca2+] due to Na+/Ca2+ exchange. These ionic

100 Intracellular magnesium disturbances may become critical during ischemia and reperfusion as suggested by the reduced tolerance to ischemia in Mg deficient rat hearts ' and larger infarct sizes in LAD ligated hearts in Mg + deficient dogs . Our experiments, causing largely irreversible metabolic and functional damage due to ischemia and reperfusion, did neither reveal a major Mg release to the coronary effluent, nor a reduction in tissue Mg content. Nevertheless, a large intracellular liberation of Mg2+ from ATP occurred during ischemia (6.74 mmol/1 after 15 min) with a large or even equal increase in Mgf. However, after 15 min of

ischemia, when the observed 5a-§ was close or equal to 6a-p ^ at the relevant pH, the formulas [1] and [2] can no longer be expected to yield reliable results. Therefore, it can only be inferred that ATP was completely complexed to Mg and thai Mgf was much higher than the residual [ATP]. Consequently, it seems reasonable to assume that during ischemia Mg + liberated from ATP appeared as Mgf. Other groups also reported an equal15 or a large but incomplete 27 appearance of Mg2+ from ATP as Mgf. However, the former study suffered from the same inherent inaccuracies as our study, and the latter necessitated the use of complexing agents, which could also bind Ca2+ and H+; in addition, preischemic contractility of the isolated hearts was depressed. Therefore, the exact extent of elevation of Mgf during ischemia is still a matter of debate. In case the increase in Mgf did not follow the liberation of Mg + from ATP, our observations indicate that Mg2+ is (temporarily) bound to other intracellular sites, such as inorganic phosphate , rather than lost to the extracellular space. This may especially apply to the situation during reperfusionlerfi , when mitochondria may actively take up Mg in an energy- linked way.34 From the individual and accumulated P NMR spectra during reperfusion we estimated that the small amount of newly produced ATP ( ~ 25 % of preischemic levels) was sufficiently complexed to Mg + ( > 98 % and 96 % during early and late reperfusion, respectively). This excludes postischemic intracellu'ar Mg + deficiency as a limiting factor for metabolic recovery in this model, as has been suggested ' . On the other hand, the = 50 % increase of Mgf during reperfusion as compared to the control situation, which is in agreement with observations by Murphy et al. , may well have several effects, including a reduction of cardiac function ' . The difficulty for Mg2+ to leave the cell may be explained in different ways. Only about 20 to 60 % of total muscle Mg2+ appeared to be diffusable, whereas the other 40 to 80 % is tightly bound to intracellular structures, such as sarcoplasmic reticulum, mitochondria 29 , nucleic acids and cell membranes 35. We observed that during the calcium paradox about 64 % of total Mg2+ is released. Normal sarco-

101 Chapter 6 lemma is highly impermeable to Mg2+ 7, but specific ionophores may accelerate transport processes . Although membrane permeability may increase due to ischemia and reperfusion 16>33, the actual transport rate is influenced by the concentration and electrical gradients between the intracellular and extracellular compartment. Although knowledge on the regulation of Mgf is very limited, the electrochemical equilibrium for Mgf would suggest that active extrusion of Mg is responsible for the low levels of Mgf under normal conditions 27. Therefore, it may not be surprising that Mg + is retained in ischemic-reperfused cells. Accord- ingly, by elevating the perfusate [Mg ] during the initial phase of reperfusion to 15 mmol/1 one may well cause an increase in intracellular Mg2+ with protective effects ' ' . Others ' could only protect isolated rat hearts when a high extracellular [Mgf+] was present prior to ischemia. The negative inotropic effect of Mg seemed essential, since rabbit hearts, which are not sensitive to this effect ofMg +, were not protected .

Conclusions The question whether Mg + plays a role during ischemia, myocardial infarction and reperfusion still has to be answered. During ischemia Mgf increased possibly proportionally to the degradation of ATP (i.e. from 0.60 to > 6.5 mmol/I), and during reperfusion Mgf remained elevated at 0.85 mmol/1, which may have several effects including depressed cardiac function. Intracellular Mg + deficiency due to leakage of Mg to the extracellular space does not play a role in the poor postischemic recovery in our isolated rat heart model Nevertheless, high Mg + prior to ischemia or during reperfusion may well exert protective effects in animal and man, due to interactions of Mg with the sarcolemma or intracellular sites, such as the mitochondria and myoEbrils, affecting Ca2+, K+ and Na+ distribution and fluxes.

~~The authors wish to thank Mr. P. van der Meer and Mrs. C. Kasbergen for their excellent technical assistance and Professor F.L. Meijler for stimulating discussions.~~

~~102 Intracellular magnesium~~

~~References~~

1. Altura BM, Altura BT. New perspectives on the role of magnesium in the pathophysiology of the cardiovascular system. II. Experimental aspects. Magnesium 1985;4:245-271. 2 + 2. Lawson J WR, Veech RL. Effects of pH and free Mg on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions J Biol Chem 1979;254:6528-6537. 3. Garfinkel L, Altschuld RA, Garfinkel D. Magnesium in cardiac energy metabolism. J Mol Cell Cardiol 1986;18:1003-1013. 4. Opie LH. The Heart, Physiology, Metabolism, Pharmacology, Therapy. London, Grune & Stratton 1984: p 47. 5. Polimeni PI, Page E. Magnesium in heart muscle. Circ Res 1973;33:367-374. 6. Bersohn MM, Shine KI, Sterman WD. Effect of increased magnesium on recovery from ischemia in rat and rabbit hearts. Am J Physiol 1982;242:H89-H93. 7. Shine KI. Myocardial effects of magnesium. Am J Physiol 1979;237:H413-H423. 8. Ferrari R, Ceconi C, Curello S, Cargnoni A, Condorelli E, Belloli S, Albertini A, Visioli O. Metabolic changes during post-ischaemic reperfusion. J Mol Cell Cardiol 1988;20(Suppl II):119-133. 9. Iseri LT, French JH. Magnesium: nature's physiologic calcium blocker. Am Heart J 1984;108:188-193. 10. Altura BM, Altura BT. New perspectives on the role of magnesium in the pathophysiology of the cardiovascular system. I. Clinical aspects. Magnesium 1985;4:226-244. 11. Borchgrevink PC, Jynge P. Acquired magnesium deficiency and myocardial tolerance to ischemia. J Am Coll Nutr 1987;6:355-363. 12. Borchgrevink PC, 0ksendal AN, Jynge P. Acute extracellular magnesium deficiency and myocardial tolerance to ischemia. J Am Coll Nutr 1987;6:125-130. 13. Chang C, Varghese PJ, Downey J, Bloom S. Magnesium deficiency and myocardial infarct size in the dog. J Am Coll Cardiol 1985;5:280-289. 14. Ferrari R, Albertini A, Curello S, Ceconi C, Di Lisa F, Raddino R, Visioli O. Myocardial recovery during post-ischaemic reperfusion: effects of nifedipine, calcium, and magnesium. J Mol Cell Cardiol 1986;18:487-498. 15. Borchgrevink PC, Schie Bergan A, Bakoy OE, Jynge P. Magnesium and reperfusion of ischemic rat heart as assessed by 31P-NMR. Am J Physiol 1989;256:H195-H204. 16. Hearse DJ, Stewart DA, Braimbridge MV. Myocardial protection during ischemic cardiac arrest. The importance of magnesium in cardioplegic infusates. J Thorac Cardio- vasc Surg 1978;75:877-885. 17. Rasmussen HS, Norregard P, Lindeneg O, McNair P, Backer V, BalslevS. Intravenous magnesium in acute myocardial infarction. The Lancet 1986;847S:234-236. 18. Smith LF, Heagerty, Bing RF, Barnett DB. Intravenous infusion of magnesium sulphate after acute myocardial infarction, effects on arrhythmias and mortality. Int J Cardiol 1986;12:175-180. 19. Neely JR, Liebermeister H, Morgan HE. Effect of pressure development on membrane transport of glucose in isolated rat heart. Am J Physiol 1967^12:815-822. 20. Gupta RK, Moore RD.31 P NMR studies of intracellular free Mg2 + in intact frog skeletal muscle. J Biol Chem 1980^55:3987-3993.

~~103 Chapter 6~~

21. Garflnkei L, Garfinkel D. Calculation of free-Mg2"1" concentration in adenosine 5'-triphosphate containing solutions in vitro and in vivo. Biochemistry 1984;23:3547-355Z 22. Bock JL, Wenz B, Gupta RK Changes in intracellular Mg adenosine triphosphate and ionized Mgf+ during blood storage: detection by P nuclear magnetic resonance spectroscpy. Blood 1985;65:1526-1530. 23. Zimmerman ANE, Hiilsmann WC. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 1966;211:646-647. 24. Kirkels JH, Ruigrok TJC, van Echteld CJ A, Meijler FL. Protective effect of pretreatment with the calcium antagonist anipamil on the ischemic-reperfused rat myocardium: a phosphorus-31 nuclear magnetic resonance study. J Am Coll Cardiol 1988;ll:1087-1093. 25. Corkoy BE, Duszynski J, Rich TL, Matschinsky B, Williamson JR. Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria. J Biol Chem 1986;261:2567-2574. 26. Levy LA, Murphy E, Raju B, London RE. Measurement of cytosolic free magnesium ion concentration by 19F NMR. Biochemistry 1988;27:4041-4048. 27. Murphy E, Steenbergen C, Levy L, Raju B, London R. Cytosolic free magnesium levels in ischemic rat heart. J Biol Chem 1989;264:5622-5627. 28. Hess P, Metzger P, Weingart R. Free magnesium in sheep, ferret and frog striated muscle at rest measured with ion-selective micro-electrodes. J Physiol 1982^33:173-188. 29. Paradise NF, Beeler GW, Visscher MB. Magnesium net fluxes and distribution in rabbit myocardium in irreversible contracture. Am J Physiol 1978;234:C115-C121. 30. Gupta RK. NMR measurement of intracellular free magnesium ions in living cells. In: NMR in living systems, edited by T Axenrod, G CeccarellL D Reidel Publishing Company 1986: pp 335-345. 31. Gupta RK, Gupta P, Yushok WD, Rose ZB. On the noninvasive measurement of intracellular free magnesium by P NMR spectroscopy. Physiol Chem Phys Med NMR 1983;15:265-280. 32. Shen AC, Jennings RB. Myocardial calcium and magnesium in acute ischemic injury. Am J Pathol 1972;67:417-440. 33. Jennings RB, Reimer KA, Steenbergen C Myocardial ischemia revisited. The osmolar load, membrane damage, and reperfusion. J Mol Cell Cardiol 1986;19:769-780. 34. Brierley G, Murer E, Bachman E. Studies on ion transport. V. Restoration of the adenosine triphosphate-supported accumulation of Ca + in aged heart mitochondria. J Biol Chem 1964;239:2706-2712. 35. Mildvan AS. Role of magnesium and other divalent cations in ATP-utilizing enzymes. Magnesium 1987;6:28-33. 36. Watts JA, Koch CD, LaNoue KF. Effects of Ca2+ antagonism on energy metabolism: Ca2+ and heart function after ischemia. Am J Physiol 1980;238:H909-H916.

~~104 31P NMR during ischemia and reperfusion in vivo~~

~~IN VIVO 31P NMR STUDY OF ACUTE REGIONAL MYOCARDIAL ISCHEMIA AND REPERFUSION IN THE RABBIT: PRESENTATION OF AN ANIMAL MODEL AND PRELIMINARY RESULTS.~~

~~tets~~

~~105 Chapter 7~~

~~Introduction~~

CJ ince the introduction in the late 1970's of 3' P NMR spectroscopy as a tool to ^ study myocardial metabolism, numerous studies have appeared on the isolated heart of rats, guinea pigs or rabbits l'9. Both physiological and pathological conditions have been studied in vitro, including metabolic changes during ischemia followed by reperfusion. After the first reports on in vivo NMR spectroscopyx only a limited number of NMR studies have addressed the issue of metabolic changes during myocardial ischemia in vivo e"8' , and only part of them also included reperfusion data. Although these studies demonstrate the feasibility of the experimental approach, many important questions remain to be answered. Since reperfusion of previously ischemic myocardial tissue is becoming an increasingly important intervention in clinical cardiology " , there is a strong need for additional basic knowledge from animal experiments, in order to gain insight in the (ir)reversibility of ischemic damage and to improve functional and metabolic recovery on reperfusion. NMR speclroscopy seems ideally suited for this purpose, as it is non-destructive, can be repeated on the same object, and does not interfere with simultaneous determinations of heart function ' ' . P NMR provides information on intracellular high-energy phosphates, inorganic phosphate and pH; these parameters are known to change dramatically during ischemia and reperfusion. We developed an in vivo open thorax model of reversible regional ischemia in rabbits for several reasons. Most biochemical knowledge of the heart is obtained from isolated hearts of rodents, whereas the in vivo NMR studies almost exclusively use (for practical reasons) larger animals, which may have considerable collateral circulation (dogs) or exhibit differences in metabolism. To bridge the gap between perfused heart studies and in vivo dog or pig studies, we chose the rabbit as the smallest animal in which infarct size can be expected to match the surface coil dimensions that are needed for a reasonable temporal resolution of the NMR data. In this model it is possible to continuously and selectively study metabolic changes in regionally ischemic myocardium, from the onset of ischemia until the end of reperfusion. In this study we will present the animal model, including its advantages and problems, and the changes in myocardial metabolism that cau be observed during ischemia. Additionally, preliminary results will be presented on the relationship between the duration of the ischemic period and postischemic recovery as well as on the possible effect of AICA-riboside, a precursor of ATP and anti-granulocyte agent30

~~106 31P NMR during ischemia and reperfusion in vivo~~

~~Materials and Methods~~

Animal model New Zealand White rabbits, weighing 1.3 to 1.8 kg were premedicated with Vetranquil i.m. (4 mg/kg acepromazine and 2 mg/kg chlorbutanol) and methadon 1.5 mg/kg i.m. and anesthetized with etomidate 2 mg/kg i.v. An endotracheal tube was inserted and connected to an Amsterdam Infant Ventilator MK 3 (Hoek Loos, Schiedam). Since this is a continuous flow system, a 2.5 m long tubing could be used to keep the respirator far enough away from the NMR magnet, without any problems of respiratory dead space. Artificial ventilation was started with a mixture of 11/min O2 and 2 1/min N2O, with 1 % halothane, with a respiration rate of 25 breaths/min. The left carotid artery was then cannulated for measurement of blood pressure and heart rate, and the right jugular vein was cannulated when needed for administration of AICA-riboside or saline. The thorax was opened and part of the sternum and left ribs were removed to create an opening of about 3 cm in diameter. The pericardium was opened to expose the heart, and the coronary arteries were identified. A loose ligature (6-0) was placed around the first (or second) anterolateral branch of the left coronary artery , which was guided through a short piece of polyethylene tubing to create a reversible occluder. By briefly occluding the artery, the ischemic area was identified as a darker, blueish area in the anterior-api-

Fig. 1. Schematic representation of the opened thorax of the rabbit (a), with theNMR surface coil placed over the previously identified area of regional ischemia after ligation of the first or second anterolateral branch of the left coronary artery. The heart is shown in more detail (b). LAD.ieft anterior descending artery; LOc left circumflex artery; AL: first and second anterolateral bran- ches, with the sites of coronary artery ligation.

107 Chapter 7 cal region of the left ventricle. The surface coil was attached with cyanoacrylic glue to the epicardium over the previously demarcated area (Fig.l). The animal was then carefully positioned on an aluminium cradle, and the surface coil was connected by semi-flexible leads to the electrical circuit with the tuning and matching capacitors. The body temperature of the rabbit was maintained by a temperature controlled (37 DC) warm water pad. Finally, the cradle with the animal was entered into the vertical bore of the NMR magnet.

NMR methods 31P NMR spectra were obtained on a Bruker MSL 200 spectrometer equipped with a 4.7 T wide bore (150 mm) vertical magnet A single tuned (3IP), single turn 9 mm diameter surface coil was used for excitation and data acquisition. Shimming of the magnetic field was achieved by optimizing the H signal from H2O, resulting in typical line widths of 60 Hz. Although the surface coil was not tuned at the *H resonance frequency, the signal from H2O was sufficient for optimization. P NMR spectra were obtained from 128 accumulated free induction decays after pseudo 90° pulses of 10 fis. The data were accumulated using 2 K datapoints and 5,000 Hz spectral width. Data acquisition was triggered to the artificial ventilator and the blood pressure signal by means of a home-built combined gating device. Scan repetition time was set at 2.3 s and the first systolic rise in blood pressure after the onset of expiration was used to trigger the spectrometer, resulting in an effective scan repetition time of « 2.5 s. The spectra were analyzed after deconvolution and exponential multiplication resulting in 20 Hz line broadening. Intracellular pH was calculated from the chemical shift of the inorganic phosphate peak relative to creatine phosphate (CP). Peak heights of CP and ATP were normalized to 100 % during preocclusion, and corrected for contributions from nonischemic tissue, as will be explained later.

Experimental protocol During an equilibration period of about 15 min three control spectra were obtained. While the animal remained inside the magnet the coronary artery was then occluded for 10,15,20,25,35,40,45 or 60 min by attaching a 60 g weight to the ligature, which had been extended to the bottom of the magnet. In a model system the 6-0 ligature attached to this weight was found to completely occlude a silicon rubber tubing of about the same size as the rabbit coronary arteries, when an intraluminal pressure of 75 to 100 mm Hg was applied. It appeared essential that the coronary occluder was handled as gently as possible, to prevent vascular wall damage to occur, which might hamper subsequent reperfusion. Reperfusion was

108 31P NMR during ischemia and reperfusion in vivo achieved by releasing the coronary occluder. Metabolic recovery was followed for 30 to 45 min of reperfusion. 31P NMR spectra were made successively during the entire protocol. At the end of the experiment the animal was removed from the magnet, and after reocclusion of the coronary artery methylene blue was injected intravenously. The absence of dye clearly demarcated the myocardial infarction and the position of the surface coil relative to the infarct area could be confirmed. When appropriate, administration of AICA-riboside (1 mg/kg per min) was started at an infusion rate of 6 ml/hr, after the control period of 15 min. Infusion was continued during the remainder of the experiment. Controls were administered physiological saline only. Coronary artery occlusion was started 15 min after the onset of infusion and maintained for 60 min. Metabolic recovery upon release of the occluder was followed for 30 min.

~~Results and Discussion~~

Blood pressure, heart rate and arrhythmias Systolic and diastolic blood pressure measured during preocclusion amounted to 63±9 and 45±11 mmHg, respectively, and heart rate was found to be 235±30 beats/min (mean±SD). During coronary artery occlusion and reperfusion these values remained constant within 10 % of their baseline values. However, of the 27 experiments performed, 4 ended in ventricular fibrillation during the ischemic period. These animals turned out to have a very large infarction, as determined retrospectively with methylene blue. No arrhythmias were observed upon reperfusion in the other experiments.

Preocclusion31 P NMR spectra A spectrum obtained under fully relaxed conditions is shown in Fig.2. The spectrum contains the following signals arising from the myocardium: (1) phosphomonoesters (PME); (4) inorganic phosphate (Pj); (5) phosphodiesters (PDE); (6) creatine phosphate (CP); (7,8,10) y-, a-, and^-ATP; (9) NAD(H). The spectrum may be contaminated by blood constituents, such as 2,3 diphosphoglycerate (2,3 DPG) (2,3) and PDE. The CP/ATP ratio in this spectrum was estimated to be 2.5 (both for peak areas and peak heights), well within the range reported by others in different species, when these are corrected for partial saturation effectsfor rcview 1X6 32. The pH calculated from the chemical shift of Pi was 7.04. Chapter 7

~~8 7 i 10~~

~~1>9) ^ V ^fyv^/W~~

~~10 0 -10 -20 ppm~~

Fig.2. 3lP NMR spectrum obtained with a 9 mm diameter surface coil prior to coronary artery occlusion from the left ventricularanteriorwallofan open chestedrabbit The spectrum is based on the accumulation of 128 free induction decays afterpseudo 90 "pulses. The scan repetition time of 15 s allowed for full relaxation of the resonances. Peak assignment is as follows: (1) phosphomonoesters (PME); (2) 3-phosphate of blood 2,3 diphosphogfycerate (2,3 DPG); (3) 2-phosphate of 2,3 DPG; (4) inorganic phosphate (Pi); (5) unresolved phosphodiesters; (6) creatine phosphate (CP); (7,8,10) y-,a-, and ^-phosphate of ATP; (9) nicotinamide adenine dinucleotide (NAD(H)). Zeroppm is assigned to CP. Peaks (I) and (2) may overlap, mainly depending on the pH dependent shift of (2). Peaks (4) and (9) cannot be assigned with absolute certainty, due to insufficient spectral resolution or signal intensity. Intracellular pH, calculated from the chemical shift of the Pi resonance, would amount to 7.04.

Metabolic changes during ischemia Examples of spectra obtained during ischemia and reperfiision are shown in Fig.3-7. It should be noted that the experimental approach described above assumes the selective measurement of an area within the left ventricular free wall during normal perfusion, regional ischemia, and subsequent reperfusion. Since the sensitive volume of an NMR surface coil is roughly equal to a (half) sphere with the radius of the coil, exact positioning of the coil is essential. Due to variations in coronary anatomy it was not always possible to produce an infarction of sufficient size to match the sensitive volume of the surface coil, giving rise to spectra originating from both ischemic and non-ischemic tissue, as will be discussed below. Figures 3-7, obtained from experiments with increasing periods of ischemia up to 60 min, show that during ischemia creatine phosphate (CP) decreased rapidly, accompanied by a large rise in intracellular inorganic phosphate (Pi). ATP de- 31P NMR during ischemia and reperfusion in vivo creased much more slowly than CP. The upfield shift of the Pi peak indicated a progressive acidification of the ischemic area.

Intracellular pH Table 1 shows the changes in intracellular pH observed during the protocol in typical experiments with increasing duration of the ischemic period. In preocclusion spectra the intracellular Pj signal was sometimes obscured by the 2,3 diphosphoglycerate (2,3 DPG) signal from erythrocytes, making it impossible to assess the pH. In other spectra obtained from the same heart, Pi could be readily distinguished from the peak from 2-phosphate of 2,3 DPG. On average, intracellular pH under normal perfusion conditions amounted to 7.04 ±0.05, a value that is also observed in isolated perfused hearts ' , or in vivo ' . Others have reported in vivo values of 7.35 2°l22, but Brindle et al34 elegantly demonstrated by using spectral editing techniques that this value represents the blood pH, rather than intracellular pH.

Fig.3-7. Illustrations of serial 31P NMR spectra obtained from regionally ischemic rabbit hearts in vivo during preocclusion and increasing periods from 10 to 60min of coron- 25-30" ary artery occlusion, followed by 30 to 45 min of reperfusion. Repre- sentative sets of spectra are shown. reperfusion Peak assignment is as shown in (min) Fig.2, although not all peaks are resolved in all spectra. Residual signal from CP after 10 min of coronary artery occlusion is supposed to orig- inate from nonischemic tissue. This is used as a correction factor to estimate the decrease in ATP during ischemia and to estimate recovery of CP and ATP on reperfusion. For further explanation and interpreta- ischemia tion of results, see text. (min)

~~Wi/VV W/AJVI preocclusion~~

~~10 -10 -20 ppm 111 Chapter 7~~

~~Fig. 4, additional information. The first two ischemic spectra were ob-~~

• *"\. "\ -*.^' \„.,. 25-30 1 tained from a smaller number of I scans than the other spectra. Scaling reperfusion nas been adapted to allow for direct (min) . r. ... • visual comparison, resulting in an 0-5 1 increase in noise level

~~' \ 20-25~~

~~20 /V^\AJV^A^1O-15~~

~~ischemia 5-10 (min)~~

~~0-2.5~~

~~0-1.25"~~

~~^.- v -1 -v \ ^, preocdusion~~

10 -10 -20 ppm After 20 min of ischemia intracellular pH stabilized at 5.9 - 6.0. This value is also close to that observed in isolated rat hearts after total ischemia, and in vivo after coronary artery ligation in rabbits , cats and dogs .

High-energy phosphates Both in isolated globally ischemic hearts and in the present experiments, in which the surface coil picked up signal from the ischemic area only, it appeared that CP was depleted rapidly and completely during ischemia (Kg. 5,6). This is caused by the fact that the rabbit heart essentially lacks collaterals 28>36. If, on the other hand, CP was still observed after 10 min of ischemia (as for example in Rg. 3,4, /), it can

112 31P NMR during ischemia and reperfusion in vivo be concluded that nonischemic tissue contributed to this spectrum; this was indeed always confirmed by methylene blue injection at the end of the experiment. The amount of residual CP in the ischemic spectra can be used as a correction factor to estimate the decrease in ATP in the ischemic area and to estimate recovery of CP and ATP on reperfusion. This was done to obtain Fig.8, which shows the sharp decrease in CP (8a) and the gradual decrease in ATP (8b) during ischemia (drawn lines), as accumulated from experiments with increasing duration of the ischemic period. Although contributions from nonischemic tissue may hamper quantitation of metabolites, they may also be helpful in that they offer a reference (the CP resonance) to measure the chemical shift of Pi to calculate the pH during ischemia. Other studies using dogs ' also show that CP may not become completely depleted during ischemia. However, in this species this is likely to be due to the

~~reperfusion~~

~~ischemia (min)~~

~~preocclusion~~

~~E» r „ _, ~ ,, 10 ° "10 -20 Fig. 5. (Legends see Fig. 3) ppm~~

~~113 Chapter 7~~

Fig. d and 7. Tune series of 31P NMR spectra obtained from region- 10-15" 1 ally ischemic rabbit hearts in vivo , . during preocclusion, 60min ofco- reper fusion or . ' (rain) nonary artery occlusion, and subsequent release of the occluder. In both experiments the spectrum obtained immediately after release of the occluder shows little or no recovery ofCP and ATP, but in Fig.6 the P,peak remained elevated, indicating "no reperfitsion", whereas in Fig.7 the Pipeak decreased, indicating "no recovery". The decrease in Pi ischemia in Fig.7 may be due to wash-out or (min) depostition of NMR-invisible Ca3(PO4)2 in mitochondria-

~~preocclusion~~

10 -10 -20 ppm presence of a collateral circulation, whereas in rabbits the absence of an adequate collateral circulation usually caused a complete depletion of CP. The persistent signal from CP in cats 20>^2 may either be due to collaterals, or to the feet that the surface coils that were used simply had too large a diameter compared to the infarct size. The rapidity of CP breakdown (Fig. 8s1) is illustrated in Fig. 4, in which spectra obtained during the first 75 seconds and during the first 2.5 min of ischemia are shown. The final level of CP was already reached after the first one to two minutes of ischemia. The peaks arising from the phosphate groups of ATP were less well defined than the CP peak, and they showed rather large variations between successive spectra. However, taking all limitations into consideration, we estimated that ATP decreased gradually during ischemia to about 20 % of preocclusion values after 30 - 35 min of ischemia (Fig. 8*). After 40 min of ischemia (/8-)ATP was no longer observed (with the exception of contributions from non-ischemic tissue). The time course of metabolic changes is in agreement with results by Camacho

~~114 P NMR during ischemia and reperfusion in vivo~~

~~25-30 1 reperfusion 0.5 «T°~~

~~/ '• \ 55-60 [ v/' • , n 30-35 ischemia /If,/ i M/ v rA/C/ *iA \* 1 | \ preocclusion~~

10 0 -10 -20 ppm fig. 7. (Legends see Fig. 6) et al x , who suggested that the early decline in contractility of the ischemic myocardium may be caused by an increase in inorganic phosphate or a decrease in pH, rather than by loss of ATP.

Metabolic recovery after reperfusion The first issue to be raised is whether it is possible to discriminate between non-reperfused tissue (due to permanent obstruction as a result of damage to the vascular tissue or thrombotic obstruction) and non-recovering tissue upon successful reperfusion. The answer is shown in Fig.6, in which after release of the occluder the Pj peak remained elevated ("no reperfusion") as compared to Fig.7, in which the Pj peak decreased ("no recovery"), while in both spectra recovery of OP and ATP immediately after release of the occluder was very small or absent The decrease in Pi in Fig.7 may be due to wash-out or deposition of NMR-invisible Ca3(PO4)2 in mitochondria. In all experiments, except for the experiment of Fig.6, where no reperfusion took place, intracellular pH recovered to normal during reperfusion (Table 1). CP also recovered completely in all experiments up to 45 min of ischemia (Fig. 8a, dotted line), but after 60 min of ischemia CP did not always recover completely. As mentioned before, recovery of ATP was more difficult to evaluate. Going from 10 to 60 min of ischemia (Fig. 8b, dotted line), it appeared that recovery of ATP gradually diminished with increasing duration of the ischemic period. After 60 min

115 Chapter 7 of ischemia recovery appeared even virtually absent Importantly, irrespective of the duration of ischemia, recovery after 10 min of reperfusion was not different from 30 or 45 min of reperfusion. This is in agreement with results by Rehr et al19 who did not observe mebabolic improvement between one and five hours of reperfusion following 60 min of LAD ligation in dogs. Since ATP degradation products (adenosine, inosine and hypoxanthine) may leave the cell, restoration of ATP on reperfusion will largely depend on de-novo synthesis , and therefore final biochemical and functional recovery may take days to weeks 38>39. The open thorax model certainly does not allow for such long observations. Recovery of CP during reperfusion indicated resumption of oxidative phosphorylation in mitochondria. However, the discrepancy we and others 17"19 found between restoration of low levels of Pi and recovery of CP on the one hand, and the lack of recovery of ATP on the other hand may have important consequences. Even if absolute concentrations cannot be derived from in vivo spectra, the CP/ATP and Pi/ATP ratios will be elevated in this situation. On the basis of these ratios it may then be possible to distinguish between normal, ischemic and reperfused viable tissue. Reperfused irreversibly damaged myocardium was reported to be charac-

Fig 8. Time course of changes in creatine phosphate (CP) (a) and ATP(b) duringregionalischemia in vivo, indicated by the drawn lines. The dotted lines indicate recoveiy of CP and ATP after release of the coronary artery occluder after different periods of ischemia. Recovery after each ischemic period is represented by one experiment. Conse- quently, the changes in CPandATP (mean and SJ>.) are based on a decreasing number of experiments, from eight at 10 min of ischemia, to one at 60 min of ischemia.

~~20253O354O-S5 50556O time (mm)~~

~~116 31P NMR during ischemia and repeifusion in vivo~~

Fig 9. Illustration of serial J131P1 NMR spectra obtained from a re- 25-30 1 gionalfy ischemic rabbit heart in reperfusion vivo during preocclusion, during in- (min) fusion of AICA-riboside (1 mg/kg per min), and during 60 min of coronary artery occlusion followed by 30 min of reperfusion. During infusion of AICA-riboside no differences in the P spectra were observed, compared to the control situation. ischemia (min) On reperfusion recovery of CP was complete, and ATP recovered partially.

~~u .:&? -V V AICA-riboside~~

~~preocclusion~~

10 -10 -20 ppm terized by persistent depletion of both CP and ATP, and elevation of Pi in comparison with preischemic levels "a. Accordingly, the first human 31P NMR infarct study 40, performed at 5-9 days after onset reported a significantly reduced CP/Pi ratio and increased Pj/ATP ratio in patients treated with drugs or coronary angioplasty for anterior myocarial infarction. Although it may not be possible to predict the eventual extent of metabolic recovery, this approach holds promise for future clinical applications. Unlike our results, Bottomley et al 14 observed a reversal of changes in 31P spectra after permanent LAD occlusion in a number of dogs, and they showed that this was caused by the presence of collaterals. They also found in dogs without collaterals that between six and 15 hours of coronary artery occlusion, pH in the infarcted area was neutralized, although Pi remained elevated for several days.

Effects of AICA-riboside AICA-riboside, a naturally occurring intermediate in purine biosynthesis , was first studied as a precursor of purine de novo synthesis . However, in addition to the lack of purine precursors is postischemic hearts, the enzyme activity to convert Chapter 7

~~Table 1. Intracellular pH during regional ischemia and reperfusion~~

~~ischemic time (min) exp. pre- 0-5 5-10 10-15 15-20 20-25 25-30 30-35 35-40 40-45 55-60 repert nr. occl.~~

9 7.04 6.71 6.20 7.10 10 7.01 6.87 635 6.27 7.01 6 7.01 6.91 6.60 6.41 6.27 * 11 7.00 6.91 6.56 6.17 5.93 7.10 24 7.06 6.85 635 6.10 5.91 * 14 6.95 6.90 651 6.41 6J23 5.94 7.03 17 7.10 6.74 6.47 6.17 5.93 5.93 5.93 5.96 7.06 22 7.10 6.97 6.55 631 6.08 6.08 6.06 6.08 6.08 7.09 23 7.09 6.90 6.53 6.10 5.93 5.90 5.94 5.89 5.93 553 7.02 28 7.01 6.80 6.60 6.35 6.21 6.02 5.% 5.94 5.90 5.95 5.90 7.08

~~7.04 6.86 6.47 6.25 6.06 5.97 5.97 5.97 5.97 5.94 5.90 7.06 ± ± ± ± 0.05 0.08 0.13 0.12 0.15 0.07 0.06 0.08 0.04~~

* No reperfusion data available.

AICA-riboside to adenine nucleotides may not be sufficient . Later it was suggested that AICA-riboside may enhance adenosine release from energy deprived cells 4z by inhibiting the conversion of adenosine to inosine or from AMP to IMP 30. Adenosine, in addition to its known vasodilator action, which may enhance collateral flow, may decrease granulocyte accumulation and inhibit superaxide radical formation by granulocytes during myocardial ischemia • .This mechanism was held responsible for the prevention of the no-reflow phenomenon and arrhythmias in dog hearts, subjected to one hour of ischemia . Prevention of microvascular obstruction and improved recovery on reperfusion are suggested to occur onry when AICA-riboside is present before and during ischemia . The failure of AICA-riboside to protect ischemic isolated hearts 43, and the failure of AICA- riboside to enhance adenine nucleotides when given during reperfusion only , would emphasize the limited role of AICA-riboside as a precursor of purine resynthesis, in favour of its role as an anti-granulocyte agent.

In a small number of experiments (5), coronary artery occlusion and reperfusion 31P NMR during ischemia and reperfusion in vivo were performed in the presence of AICA-riboside, 1 mg/kg per m:n i.v., started 15 min before coronary artery occlusion. In none of the experiments > .hanges in blood pressure or heart rate were observed upon starting the infusion of AICA-riboside, nor did any changes in the 31P NMR spectra occur. The lack of hemodynamic and biochemical effects of AICA-riboside on non-ischemic tissue is in agreement with previous studies ^ ' . Preliminary qualitative evaluation of tne experiments revealed no differences in the development of acidosis and the depletion of CP and ATP during 60 min of regional ischemia, as compared to controls. Fig.9 illustrates an experiment in which biochemical recovery during reperfusion in the presence of AICA-riboside was considerably better than in control animals. However, due to technical problems (e.g. no reperfusion at all after release of the occluder), not all experiments could be evaluated and therefore it is too early to draw any conclusions with regard to possible protective effects of AICA-riboside in this animal model. Since the rabbit heart essentially lacks collaterals, it may not be the best model to study AICA-riboside, as part of its action may depend on enhancing collateral blood flow to the ischemic region. For the same reason, the ischemic period of 60 min may be too long to allow for any (acute) protective effects. Therefore, further studies are necessary, including a shorter period of ischemia (45 min).

~~Conclusions and Future Expectations~~

We have demonstrated that it is feasible to study regional myocardial ischemia and reperfusion in an in vivo rabbit model by means of surface coil 31P NMR spectroscopy. We consider the rabbit the smallest animal in which regional ischemia can be selectively studied with this technique. Changes in high-energy phosphates, intracelhilar inorganic phosphate and pH during ischemia are reported, and metabolic recovery after increasing periods of ischemia is demonstrated, with the absence of immediate and complete recovery when ischemia exceeded 10 to 15 minutes. Preliminary results with AICA-riboside show the suitability of the experimental model to study the effects of protective drugs or interventions. 31 32 40 4447 As P NMR spectroscopy of the human heart is becoming feasible > > ) unique information will emerge on energy metabolism in the normal and diseased heart 4&. Based on the CP/ATP and Pj/ATP ratios it may be possible to distinguish between normal, ischemic, and reperfused viable tissue. In addition to 31P studies, *H NMR spectroscopy may become important44'49 as it may reveal elevated levels

119 Chapter 7 of lipids, lactate and alanine after coronary occlusion 5OlS1. By making use of knowledge from well-controlled animal experiments, NMR spectroscopy may be expected to contribute to future cardiovascular diagnosis and the evaluation of treatment in patients.

~~Technical assistance by Pieter van der Meer and Cor Lekkerkerk as well as biotechical assistance by Carina Kasbergen are gratefully acknowledged.~~

~~References~~

1. Clarke K, O'Connor AJ, Willis RJ. Temporal relation between energy metabolism and myocardial function during ischemia and reperfusion. Am J Physiol 1987;253:H412- H421. 2. Hollis DP, Nunnally RL, Jacobus WE, Taylor GJ. Detection of regional ischemia in perfused beating hearts by phosphorus nuclear magnetic resonance. Biochem Biophys Res Commun 1977;75:1086-1091. 3. Ingwall JS. Phosphorus nuclear magnetic resonance spectroscopy of cardiac and skeletal muscles. Am J Physiol 1982;242:H729-744. 4. Jacobus WE, Pores IH, Lucas SK, Weisfeldt ML, Haherty JT. Intracellular acidosis and contractility in the normal and ischaemic heart as examined by P NMR. J Mol Cell Cardiol 1982;14(suppl 3):13-20. 5. Lavanchy N, Martin J, Rossi A. Graded global ischaemia and reperfusion of die isolated perfused rat heart: characterisation by P NMR spectroscopy of the extent of energy metabolism damage. Cardiovasc Res 1984;18:573-582. 6. Matthews PM, Williams SR, Seymour AM, Schwartz A, Dube G, Gadian DG, Radda GK. A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain, and the ionophore RO2-2985 (X537A) in the isolated perfused rat heart. Biochim Biophys Acta 1982;720:163-171. 7. Nunnally RL, Bottomley PA. Assessment of pharmacological treatment of myocardial infarction by phosphorus-31 NMR widi surface coils. Science 1981;211:177-180. 8. Pieper GM, Todd GL, Wu ST, Salhany JM, Clayton FC, Eliot RS. Attenuation of myocardial acidosis by propranolol during ischaemic arrest and reperfusion: evidence with 31P nuclear magnetic resonance. Cardiovasc Res 1980;14:646-653. 9. Salhany JM, Pieper GM, Wu ST, Todd GL, Clayton FC, Eliot RS. 31P Nuclear magnetic resonance measurements of cardiac pH in perfused guinea-pig hearts. J Mol Cell Cardiol 1979;ll:601-610. 10. Grove TH, Ackerman JJH, Radda GK, Bore PJ. Analysis of rat heart in vivo by phosphorus nuclear magnetic resonance. Proc Nad Acad Sri USA 1980;77:299-302. 11. Koretsky AP, Wang S, Murphy-Boesch J, Klein MP, James TL, Weiner MW. 31P NMR spectroscopy of rat organs, in situ, using chronically implanted radiofrequency coils. Proc Natl Acad Sci USA 1983;80:7491-7495.

120 12. Neurohr KJ, Gollin G, Barrett EJ, Shulman RG. In vivo 31P-NMR studies of myocardial high energy phosphate metabolism during anoxia and recovery. FEBS Letters 1983;l59:207-210. 13. Kantor HL, Briggs RW, Balaban RS. In vivo 31P nuclear magnetic resonance measurements in canine heart using a catheter-coil. Circ Res 1984;55:261-266. 14. Bottomley PA, Smith LS, Brazzamano S, Hedlund LW, Redington RW, Herfkens RJ. The fate of inorganic phosphate and pH in regional myocardial ischemia and infarction: a noninvasive 31P NMR study. Magn Res Med 1987;5:129-142. 15. Bittl JA, Balschi JA, Ingwall JS. Contractile failure and high-energy phosphate turnover during hypoxia: 31P NMR surface coil studies in living rat. Circ Res 1987;60:871-878. 16. Camacho SA, Lanzer P, Toy BJ, Gober J, Valenza M, Botvinick EH, Weiner MW. In vivo alterations of high-energy phosphates and intracellular pH during reversible ischemia in pigs: a 31P magnetic resonance spectroscopy study. Am Heart J 1988;116:701-708. 17. Guth BD, Martin JF, Heusch G, Ross J. Regional myocardial blood flow, function and metabolism using phophorus-31 nuclear magnetic resonance spectroscopy during ischemia and reperfusion in dogs. J Am Coll Cardiol 1987;10:673-681. 18. Malloy CR, Matthews PM, Smith MB, Radda GK. Influence of propranolol on acidosis and high energy phosphates in ischaemic myocardium of the rabbit. Cardiovasc Res 1986;2O:71O-72O. 19. Rehr RB, Tatum JL, Hirsch JI, Wetstein L, Clarke G. Effective separation of normal, acutely ischemic, and reperfused myocardium with P-31 MR spectroscopy. Radiology 1988;168:81-89. 20. Stein PD, Goldstein S, Sabbah HN, Liu Z, Helpern JA, Ewing JR, Lakier JB, Chopp M, LaPenna WF, Welch KMA. In vivo evaluation of intracellular pH and high-energy phosphate metabolites during regional myocardial ischemia in cats using P nuclear magnetic resonance. Magn Res Med 1986;3:262-269. 21. Werner MW. NMR spectroscopy for clinical medicine. Animal models and clinical examples. Ann N Y Acad Sci 1987; 508:287-297. 22. Wendland MF, White RD, Derugin N, Finkbeiner WE, McNamara MT, Moseley ME, Upton MJ, Higgins CB. Characterization of high-energy phosphate compounds during reperfusion of the irreversibly injured myocardium using 31P MRS. Magn Res Med 1988;7:172-183. 23. Braunwald E. Myocardial reperfusion, limitation of infarct size, reduction of left ventricular dysfunction, and improved survival. Should the paradigm be expanded? Circula- tion 1989;79:441-444. 24. Ferrari R, Niccoli L, Visioli O, Harris F. Myocardial metabolism during intracoronary thrombolysis. Two illustrative cases. Int J Cardiol 1987; 15:293-299. 25. Kiibler W, Schuler G, Schwartz F. Intervention in acute myocardial infarction. Am J Cardiol 1987;60:2A-5A. 26. O'Neill WW. Impact of different reperfusion modalities on ventricular function after acute myocardial infarction. Am J Cardiol 1988;61:45G-53G. 27. Portman MA, James S, Heineman FW, Balaban RS. Simultaneous monitoring of coronary blood flow and 31P NMR detected myocardial metabolites. Magn Res Med 1988;7:243-247. 28. Schaper W, Gorge G, Winkler B, Schaper J. The collateral circulation of the heart. Progr Cardiovasc Dis 1988;31:57-77.

~~121 Chapter 7~~

29. Swain JL, Hines JJ, Sabina RL, Holmes EW. Accelerated repletion of ATP and GTP pools in postischemic canine myocardium using a precursor of purine de novo synthesis. Ore Res 1982;51:102-105. 30. Engler R. Consequences of activation and adenosine-mediated inhibition of granulocytes during myocardial ischemia. Fed Proc 1987;46:2407-2412. 31. Day SB, Johnson JA. The distribution of the coronary arteries of the rabbit Anat Rec 1958;132:633-643. 32. Schaefer S, Gober J, Valenza M, Karczmar GS, Matson GB, Camacho SA, Botvinick EH, Massie B, Weber MW. Nuclear magnetic resonance imaging-guided phosphorus-31 spectroscopy of the human heart J Am Coll Cardiol 1988;12:1449-1455. 33. Kirkels JH, Ruigrok TJC, van Echteld CJA, Meijler FL. Protective effect of pretreatment with the calcium antagonist anipamil on the ischemic-reperfused rat myocardium: a phosphorus-31 nuclear magnetic resonance study. J Am Coll Cardiol 1988;ll:1087-1093. 34. Brindle KM, Rajagopalan B, Bolas NM, Radda GK. Editing of 31P NMR spectra of heart in vivo. J Magn Res 1987;74:356-365. 35. Katz LA, Swain JA, Portman MA, Balaban RS. Intracellular pH and inorganic phosphate content of heart in vivo: a 31P NMR study. Am J Physiol 1988;255:H1S9-196. 36. Maxwell MP, Hearse DJ, Yellon DM. Species variations in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc Res 1987^1:737-746. 37. Ip JH, Levin RI. Myocardial preservation during ischemia and reperfusion. Am Heart J 1988;115:1094-1104. 38. Ellis SG, Henschke CI, Sandor T, Wynne J, Braunwald E, Kloner RA. Time course of functional and biochemical recovery of myocardium salvaged by reperfusion. J Am Coll Cardiol 1983;l:1047-1055. 39. Patel B, Kloner RA, Przyklenk K, Braunwald E. Postischemic myocardial "stunning*: a clinically relevant phenomenon. Ann Int Med 1988;108:626-628. 40. Bottomley PA, Herfkens RJ, Smith LS, Bashore TM. Altered phosphate metabolism in myocardial infarction: P-31 MR spectroscopy. Radiology 1987;165:703-707. 41. Glower DD, Spratt JA, Newton JR, Wolfe JA, Rankin JS, Swain JL. Dissociation between early recovery of regional function and purine nucleotide content in postischaemic myocardium in the conscious dog. Cardiovasc Res 1987;2L328-336. 42. Forman MB, Puett DW, Virmani R. Endothelial and myocardial injury during ischemia and reperfusion: pathogenesis and therapeutic implications. J Am Coll Cardiol 1989;13:450-459. 43. Ambrosio G, Jacobus WE, Mitchell MC, Iitt MR, Becker LC. Effects of ATP precursors on ATP and free ADP content and functional recovery of postischemic hearts. Am J Physiol 1989;256:H560-H566. 44. Bottomley PA. Human in vivo NMR spectroscopy in diagnostic medicine: clinical tool or research probe? Radiology 1989;170:l-15. 45. Conway MA, Bristow JD, Blackledge MJ, Rajagopalan B, Radda GK Cardiac metabolism during exercise measured by magnetic resonance spectroscopy. The Lancet 1988;2(8612):692. 46. Matson GB, Twieg DB, Karczmar GS, Lawry TJ, Gober JR, Valenza M, Boska MD, Weiner MW. Application of image-guided surface coil P-31 MR spectroscopy to human liver, heart, and kidney. Radiology 1988;169:541-547.

~~122 31P NMR during ischemia and reperfusion in vivo~~

47. Oberhaensli RD, Galloway GJ, Hilton-Jones D, Bore PJ, Styles P, Rajagopalan B, Taylor DJ, Radda GK. The study of human organs by phosphorus-31 topical magnetic resonance spectroscopy. Brit J Radiol 1987;60:367-373. 48. Rajagopalan B, Blackledge MJ, McKenna WJ, Bolas N, Radda GK. Measurement of phosphocreatine to ATP ratio in normal and diseased human heart by P magnetic resonance spectroscopy using the rotating frame-depth selection technique. Ann N Y Acad Sci 1987;508:321-332. 49. BaVtoy M, Langer BG, Glick RP, Venkatasubramanian PN, Wilbur AC, Spigos DG. In vivo H-l spectroscopy in humans at 1.5 T. Radiology 1988;167:839-844. 50. Evanochko WT, Reeves RC, Sakai TT, Canby RC, Pohost GM. Proton NMR spectroscopy in myocardial ischemic insult. Magn Res Med 1987;5:23-31. 51. Richards TL, Terrier F, Sievers RE, Lipton MJ, Moseley ME, Higgiiis CB. Lactate accumulation in ischemic- and anoxic-isolated rat hearts assessed by H-l spectroscopy. Invest Radiol 1987;22:638-641.

~~123 31PNMR of human donor heart preservation~~

~~HUMAN DONOR HEART PRESERVATION STUDIED WITH 31P NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY.~~

~~125 Chapter 8~~

~~Introduction~~

-protection of the myocardium from ischemic damage by chemical cardioplegia •* was first reported in 1955 by Melrose et al. \ Since then many studies have addressed the issue of optimal protection for review see ^ by means of hypothermia ' , changing the ionic composition of the cardioplegic solution with respect to K+, Mg2+, Ca2+ and Na+ 2'6"12 and the possible effect of additives like ATP13>M, n i^ n IR creatine phosphate ' " , calcium antagonists , scavengers of oxygen free radicals l ' and substrates like lactate 21, phosphoenolpyruvate14 or aspartate 72. However, with the advent of heart transplantations, the solutions designed for relatively short periods of cardioplegia during cardiopulmonary bypass for cardiac surgery, did no longer meet the needs for prolonged preservation of donor hearts. The lower temperature of storage (0 - 4 °C as opposed to 15 - 20 °C) may also require a different ionic composition of the cardioplegic solution. The commonly used upper limit of about four hours from the onset of cardioplegia used now ' ' ' , poses severe restrictions on the transport and use of donor hearts. Furthermore, until now the quality of a donor heart could only be judged from the clinical condition and drug treatment of the donor, since rapid, noninvasive NMR-methods to assess the metabolic condition of donor organs have only become available during recent years. The metabolic state of the heart prior to implantation may be an important factor in determining the ability of the transplanted heart to resume adequate pump function following a technically successful transplantation. Conditions related to brain death of the donor may influence the metabolic condition of the heart, such as autonomic dysregulations 7A, endocrine changes ^^ and administration of dobutamine or vasopressin . Therefore, better graft preservation may be obtained when heart donors have been administered triiodotbyronine (T3)w or cortisol and insulin M. 31P NMR is ideallysuitable to determine the energy content of the donor heart and has already been applied successfully in the assess- 2930 ment of viability prior to kidney transplantation . In the Geld of organ preservation it is generally believed that a relationship exists between the development of irreversible injury and the reduction in adenine nucleotides 2'6. Until now, most studies have been performed on "healthy" animals, and the applicability to the human donor heart may be limited due to species differences and the influence of brain death 24"27. Therefore, this study was performed (1) to monitor energy metabolism with 31P NMR in human donor hearts up to approximately 12 hours of cold cardioplegia and (2) to compare the effects of two commonly used cardioplegic solutions, the "extracellular" SLThomas' Hos- pital no.2 solution, and the "intracellular" Bretschneider's HTP solution. 126 31P NMR of human donor heart preservation

~~Materials and Methods~~

Human hearts meeting the criteria for heart valve donation but not for whole heart transplantation were taken out under standard conditions for whole heart transplantation. Cardioplegia was induced either by St.Thomas' Hospital no.2 solution or the strongly buffered, calcium-free Bretschneider's HTP solution. The hearts were wrapped in plastic bags containing Ringer's lactate or the cardioplegic solution and were stored at 0 °C.

NMR technique 31P NMR spectra were obtained on a Philips Gyroscan 1.5 T whole body NMR instrument, equipped with a 60 mm diameter, P/ H switchable surface coil. In order to assess the position of the heart relative to the surface coil, *H spin echo multislice images (TE = 50 ms, TR = 820 ms) were obtained. When necessary, the heart was repositioned with the left ventricular free wall over the surface coil (Fig. 1). The tissue volume contributing to the NMR spectra is roughly equal to a half sphere with the radius of the coil (about 50 ml, taking into account the distance from the heart to the surface coil).

Figure 1. Ex vivo human donor heart as depicted by magnetic resonance imaging. The arrow points at a small reference container with methylene diphosphonate dissolved in water, placed in the center of the 6 cm diameter surface coil (not visible), used to obtain the 31PNMR spectra. The heart was subsequently repositioned with the left ventricular anterior wall exactly over the surface coil. After shimming of the magnetic Geld by optimizing the 'II signal from H20,31P spectra were obtained from 64 accumulated scans using a 5 s interpulse delay and either an adiabatic rapid half passage pulse or a hard pseudo 90° pulse. Spectra were

127 Chapter 8 analyzed after deconvolution and a combined Gaussian multiplication to enhance spectral resolution and exponential multiplication to enhance the signal to noise ratio. The first *P NMR spectra were obtained between two and three hours after the onset of cardioplegia, and from then every two to four hours up to approximately 12 hours. The time needed to obtain the first 31P NMR spectrum (including the necessary preparations) was about 30 min. Care was taken to maintain the temperature of the heart at 0 °C during the NMR measurements by placing the bag containing the submerged heart in a small plastic box surrounded by crushed ice. Between the measurements this box was stored outside the magnet in a well-isolated container with crushed ice. The position of the plastic box relative to the surface coil was marked, in order to facilitate exact repositioning of the box and thereby obtain subsequent spectra from the same volume within the heart. Approximately twelve hours after the onset of cardioplegia, when the last 31P NMR spectrum had been obtained, the hearts were sent on for dissection of the valves.

~~Results~~

Fig. 2a shows a typical time series of 31P NMR spectra of a human donor heart with St.Thomas' Hospital cardioplegia. The first spectrum, obtained after 2 h 30 min, shows resonance peaks from (1) phosphomonoesters (PME); (2) inorganic phosphate (Pi); (3) phosphodiesters (PDE); (4) crearine phosphate (CP); (5,6,7) y-, a-, and /J-phosphate from ATP. The pH calculated from the chemical shift of the Pi resonance was 6.91. Since the cardioplegic solutions mentioned above do not contain inorganic phosphate this value most likely represents intracellular pH. At 6 h 20 min after the onset of cardioplegia CP had virtually disappeared with a concomitant rise in Pi. The pH had decreased to 6.46, while ATP levels remained relatively constant After 12 h 40 min these changes were even more pronounced with a complete disappearance of CP and a pH of 6.04. On the other hand, the deterioration in a heart with Bretschneider's HTP cardioplegia (Fig. 2?) measured at three, five, seven and twelve hours after the onset of cardioplegia, was impressively less. CP stores were better preserved and the decline in pH was only moderate (from 7.25 to 6.98). Fig. 3 summarizes the data on CP (3a), ATP (3") and pH (3C) from three hearts with St-Thomas' Hospital solution, and two hearts with Bretschneider's HTP cardioplegia. Values for CP and ATP (peak heights) are normalised to 100 % in the

~~128 31P NMR of human donor heart preservation~~

Q Figure 2. 31P NMR spectra obtained from ex vivo human donor hearts at different time points after the onset of StThomas' Hospital no.2 cardioplegia (a) or Bret- Schneider's HTP cardioplegia (b). Peak as- Sr*s**SV* Wsr vT \^/trJ \+^ffiu~y/*J*>*v— signment is as follows: (1) phospho-monoesters (PME); (2) inor- 6h20 ganic phosphate (Pi); (3) phosphodiesters (PDE); (4) creatine phosphate (CP); (5,6J) y-, a-, and ^-phosphate oj ATP. 2h30

~~'.0 0 -10 -20 ppm~~

~~10 -10 ppm~~

first spectrum obtained for each heart. However, these values do not represent the same moment in time after the onset of cardioplegia, nor do they necessarily represent the same extent of depletion of CP and ATP stores as compared to the it (unknown) metabolic condition immediately after the onset of cardioplegia. Des- pite these restrictions, in all aspects the metabolic quality of the hearts appears to be better preserved with Bretschneider's HTP solution than with StThomas' Hospital solution. I?

~~129 Chapters~~

Figure 3. 31P AQMH dato on creatine phosphate (CP) (a), ATT (b), and pH (c) obtained from ex vivo human donor hearts during long 2 term hypothermic cardioplegic stor- U age. Three hearts had StThomas' Hospital no.2 cardioplegia (—), and two hearts had Bretschneider's HTP cardioplegia ( ). Values for CP and ATP are normalized to 100 % in the first spectrum obtained for each heart For further interpretation see Results section.

~~2 3 4 5 6 7 S 9 10 11 12 13 14 15~~

~~Discussion~~

Although the present study is based on a limited number of observations, it is the Grst demonstration of the potential of NMR spectroscopy to assess the metabolic state of explanted human donor hearts, and the time course of changes during prolonged cold cardioplegic storage. The preliminary results would suggest that during long term cold cardioplegia with Bretschneider's HTP solution high-energy

~~130 31PNMR of human donor heart preservation~~

Table l. Compostion of cardioplegic solutions (mmol/l) phosphates are better preserved and pH is better maintained than HTP STS with St.Thomas' Hospital no.2 Sodium 15.0 120.0 solution. Potassium 8.0 16.0 Cardioplegia starts with a Calcium - 1.2 rapid induction of cardiac arrest Magnesium 8.0 16.0 by means of perfusing the heart Chloride 54.0 160.4 with a solution with a special ionic Bicarbonate - 10.0 2 6 Histidine(HCI) 195.0 - composition ' . In StThomas' Tryptophan 2.0 - Hospital no.2 solution a high Mannitol 20.0 - [K+] (Table 1) is very effective in Osmolarity 287 285-300 achieving this, and Mg is elev- (mOsmol/kg) pHat37°C 6.9 * ated to counteract possible det- + pHat20°C 7.2 7.8 rimental effects of high K and pHatO°C 7.2 * because it is cardioplegic as well 8. On the other hand, in the HTP = Bretschneider's HTP solution ra = SLThomas'HosPitalno.2so.ution Bretschneider s HTP solution, Data are based on reports in literaturewa3W S cardioplegia is induced by a very •no data available low [Na+] and the absence of Ca +, and the solution is highly buffered by histidine. Based on their different ionic composition (Table 1), these solutions have been called "extracellular" and "intracellular", respectively 2. The common part in different types of cardioplegia is hypothermia with the objective of reducing myocardial metabolism and preserving high-energy phosphates for rewew ** 2A31. In the setting of coronary bypass surgery with relatively short periods of cardioplegia, differences in clinical outcome after application of different cardioplegic solutions have been reported 7, and these may be even more striking for long term preservation 6'12. The discussion about the desirability of the presence of Ca2+ is still open. On the one hand, complete absence of Ca2+ may predispose the heart to the calcium paradox 2 which is only prevented by the simultaneous hypothermia 33. On the other hand, a high [Ca +] may stimulate energy demanding processes during ischemia and facilitate Ca2+ overload during reperfusion, which may also be detrimental34. Accordingly, the formulation of the StThomas' Hospital solution has been changed over the last years, from 2.2 mmol/l Ca2+ to 1.2 mmol/l in the no.2 solution , with better results 9. A very recent report 36 suggested that the optimal [Ca2+] for long term hypothermic storage with St.Thomas' Hospital cardioplegia of rat hearts might be 0.5 - 0.6 mmol/l, although the differences between the various [Ca2+] were rather small. A modification of this solution 6, containing

131 Chapter S only 0.1 mmol/1 Ca2+ (and also 30 mmol/1 glucose) was found to offer better energy preservation in isolated rabbit hearts, and again this was ascribed to the lower [Ca2+]. The beneficial effects of the addition of calcium antagonists to the cardioplegic solution support the importance of a limited availability of Ca during cardioplegia, in addition to better preservation of high-engergy phosphates. On the other hand, not all studies using calcium antagonists as an adjunct to cardioplegia are equally positive for a review see 37. The temperature may be of importance, in that calcium antagonists mainly offer additional protection during normothermic, but not during hypothermic cardioplegia 37>38. Finally, the beneficial effect of CP and ATP added to the cardioplegic solution may also in part be related to their Ca2+-binding capacity13'

IntracelluIarpH during storage The most striking difference between the two cardioplegic solutions used, was the intracellular pH, which remained almost 7.0 up to 12 hours of storage in Bretschneider's HTP solution, to be attributed to the large amount of histidine. Histidine may enter the cells and thereby increase the intracellular buffering capacity, or the high extracellular histidine concentration provides an efficient proton-sink and facilitates proton efflux. On the other hand, the StThomas' Hospital no.2 solution offers only a very limited buffering capacity with 10 mmol/1 bicarbonate, which is unlikely to do more than stabilize the pH of the cardioplegic solution before infusion . However, the importance of the pH during cardioplegic preservation remains to be clearly defined .

ATP and CP during preservation This study is based upon the assumption that tissue levels of ATP and CP during heart preservation have some relevance to subsequent viability. Although this seems reasonable, there are both reports in favour 1 'l ™' and against *" this contention. Rosenkranz et al. suggested that myocardial preservation can be better expressed in terms of mitochondrial capacity of oxidative phosphorylation rather than in terms of (static) levels of ATP. An attempt by Takami et aL 12 to improve 24 hour preservation in isolated dog hearts by continuous hypothennic perfusion revealed that ATP, CP and lactate remained at control levels, but functional recovery was not better than in hearts simply stored in CoUin's M solution at 2 °C, and coronary vascular resistance even increased significantly. The latter finding may be related to interstitial edema, and may indicate poor viability of the graft. The intermittent administration of an

132 31PNMR of human donor heart preservation oxygenated cardioplegic solution in isolated rat hearts U was also found to maintain normal ATP levels till the end of a five hour arrest period at 20 °C, and again this was not related to a better functional recovery after five hours of reperfusion. On the other hand, Ledingham et al.35 reported a clear cut improvement in biochemical and functional recovery after three or four hours of hypothennic arrest in working rat hearts, when the cardioplegic solution was oxygenated. Despite the lack of (immediate) functional improvement reported by some authors , the use of oxygenated cardioplegic solutions is still advocated, especially for long term preservation 35>44. Exogenous ATP and CP added to cardioplegic solutions at their optimal dosage offered considerable protection during hypothermic13'15 or normothermic14'* '17 cardioplegia, the combination ATP and CP being most effective 13. The question is, however, whether these (charged) compounds can cross the intact cell membrane to replenish intracellular high-energy phosphate stores. Vasodilating (ATP) and antiarrhythmic (ATP and CP) actions may also be involved ' • ' , in addition to protective effects on the glycocalyx and cell membrane integrity (CP) .

Alternative protective measures. Extended preservation of hearts for transplantation, may necessitate the application of additional protective measures, such as free radical scavengers. Allopuri- nol and oxypurinol enhanced the protection afforded by St/Thomas' Hospital cardioplegic solution in a rat heart model19, and superoxide dismutase combined with catalase as additives to continuous hypothermic perfusion of sheep hearts improved myocardial preservation up to eight hours . Wicomb et al. , using continuous hypothennic (4 -10 °C) perfusion of baboon hearts, were able to prevent biochemical and functional deterioration up to 48 hours, when contaminating iron (Fe ), essential in the production of hydroxyl radicals, was largely removed from the perfusion fluid. However, continuous perfusion itself may not be harmless, as it may cause an increase in coronary vascular resistance and edema . The com- promise approach of combined hypothermic storage and intermittent normothermic reperfusion improved biochemical and functional protection in ex vivo dog hearts up to nine hours . Finally, modification of reperfusion conditions after ischemia or cardioplegia 42 46 49 may determine the eventual extent of recovery > ' ) and this may influence any possible relationship between the ex vivo metabolic condition of the heart and later performance of the heart.

~~133 Chapter 8~~

~~Conclusions and Future Expectations~~

Although the exact importance of improved preservation of CP and ATP stores during human heart preservation remains to be established, we tend to interpret the better preservation of high-energy phosphates with Bretschneider's HTP solution as a beneficial effect The same applies to the maintenance of a near normal intracellular pH during long term storage. Nevertheless, the final answer can only be given when improved long term cardioplegia allows for an additional 30 min time delay before human heart transplantation, necessary to obtain a 31P NMR spectrum. Only then the possible relevance of the metabolic state of the human heart prior to implantation can be assessed, and the influence on immediate and long term survival may be elucidated. If the decision whether or not to use a donor heart were to be based on the metabolic condition of the heart as assessed with P NMR, an additional time delay would ensue, since the preparations of the acceptor would depend on the verdict from the NMR department Therefore, considerable effort will be needed to improve long term preservation of donor hearts.

~~References~~

1. Melrose DG, Dreyer B, Bentall HH, Baker JB. Elective cardiac arrest; preliminary communication. Lancet 1955;ii:21-22. 2. Hearse DJ, Braimbridge MV, Jynge P. Protection of the ischemic myocardium, Cardio- plegia. New York, 1981, Raven Press. 3. Silvennan NA, Levitsky S. Intraoperative myocardial protection in the contest of coronary revascularization. Progr Cardiovasc Dis 1987;29:413-428. 4. Baumgartner WA, Williams GM, Fraser CD, Cameron DE, Gardner TJ, Burdick JF, Augustine S, Gaul PD, Reitz BA. Cardiopulmonary bypass with profound hypothermia. An optimal preservation method for multiorgan procurement Transplantation 1989;47:123-127. 5. Nayler WG. Protection of the myocardium against posuschemicreperfusion damage: the combined effect of hypothermia and nifedipine. J Thorac Cardiovasc Surg 1982;84:897- 905. 6. English TA, Foreman J, Gadian DG, Pegg DE, Wheeldon D, Williams SR. Three solutions for preservation of the rabbit heart at 0 °C. A comparison with phosphorus-31 nuclear magnetic resonance spectroscopy. J Thorac Cardiovasc Surg 1988;96:54-61. 7. Gallandat Huet RCG, Karliczek GF, Homan van der Heide JN, Brenken U, Mooi B, van der Broeke JJW, Jenkins I, de Geus AF. Clinical effect of Bretschneider-HTK and St Thomas cardioplegia on hemodynamic perfomance after bypass measured using an automatic datalogging database system. Thorac Cardiovasc Surgeon 1988^36:151-156.

~~134 31P NMR of human donor heart preservation~~

8. Hearse DJ, Stewart DA, Braimbridge MV. Myocardial protection during ischemic cardiac arrest. The importance of magnesium in cardioplegic infusates. J Thorac Cardio- vasc Surg 1978;75:877-885. 9. Ledingham SJM, Braimbridge MV, Hearse DJ. The St.Thomas' Hospital cardioplegic solution. A comparison of the efficacy of two formulations. J Thorac Cardiovasc Surg 1987;93:240-246. 10. Pernot AC, Ingwall JS, Menasche P, Grousset C, Bercot M, Piwnica A, Fossel ET. Evaluation of high-energy phosphate metabolism during cardioplegic arrest and reperfusion: a phosphorus-31 nuclear magnetic resonance study. Circulation 1983;67:1296- 1303. ll.Stinner B, Krohn E, Gebhard MM, Bretschneider HJ Intracellular pH, Na+- and K+-activities at the onset of St.Thomas' cardioplegia: a study with ionselective microelectrodes. Thorac Cardiovasc Surgeon 1988;36:247-253. 12. Takami H, Matsuda H, Hirose H, Kaneko M, Ohtani M, Tagawa K, Kawashima Y. Myocardial energy metabolism hi preserved heart: comparison of simple storage and hypothermic perfusion. J Heart Transplant 1988;7:205-212. 13. Robinson LA, Braimbridge MV, Hearse DJ. Enhanced myocardial protection with high-energy phosphates in St.Thomas' Hospital cardioplegic solution. J Thorac Cardio- vasc Surg 1987;93:415-427. 14. Thelin S, Hultman J, Ronquist G, Hansson HE. Enhanced protection of rat hearts during ischemia by phosphoenolpyruvate and ATP in cardioplegia. Thorac Cardiovasc Surgeon 1986-34:104-109. 15. Semenovski ML, Shumakow VI, Sharov VG, Mogilevsky GM, Asmolovsky AV, Makho- tina LA, Saks VA. Protection of ischemic myocardium by exogenous phosphocreatine II. Clinical, ultrastructural, and biochemical evaluations. J Thorac Cardiovasc Surg 1987;94:762-769. 16. Sharov, VG, Saks VA, Kupriyanov W, Lakomkin VL, Kapelko VL, Steinschneider Y, Javadov SA. Protection of ischemic myocardium by exogenous phosphocreatine I. Mor- phologic and phosphorus 31-nuclear magnetic resonance studies. J Thorac- Cardiovasc Surgl987;94:749-761. 17. Thelin S, Hultman J, Ronquist G, Juhlin C, Hansson HE, Lindren PG. Improved myocardial protection by creatine phosphate in cardioplegic solution. An in vivo study in the pig during normothennic ischemia. Thorac Cardiovasc Surgeon 1987;35:137-142. 18. Ip JH, Levett JM, Kadowaki MH, Karp RB. Preservation of myocardial high energy phosphates during cardioplegic arrest with nifedipine. J Surg Res 1988;44:216-223. 19. Chambers DJ, Braimbridge MV, Hearse DJ. Free radicals and cardioplegia: allopurinol and oxypurinol reduce myocardial injury following ischemic arrest. Ann Thorac Surg 1987;44:291-297. 20. Gharagozloo F, Melendez FJ, Hein RA, Shemin RJ, DiSesa VJ, Cohn LH. The effect of superonde dismutase and catalase on the extended preservation of the ex vivo heart for transplantation. J Thorac Cardiovasc Surg 1988;95:1008-1013. 21. Teoh KH, Mickle DAG, Weisel RD, Madonik MM, Ivanow J, Harding RD, Romaschin AD, Mullen JC. Improving myocardial metabolic and functional recovery after cardioplegic arrest. J Thorac Cardiovasc Surg 1988;95:788-798.

~~135 Chapter 8~~

22. Choong YS, Gavin JB. L-Aspartate improves the functional and metabolic recovery of arrested hearts after prolonged hypothennic storage. J Mol Cell Cardiol 1989;21(suppl II):S.157. 23. Beeman SK, Shuman TA, Perna AM, Atkinson JB, Hammon JW, Bender HW, Merrill WH. Intermittent reperfusion extends myocardial preservation for transplantation. Ann Thorac Surg 1987;43:484-489. 24. Cooper DKC, Novitzky D, Wicomb WN. Pathophysiology of brain death in the experimental animal: extracranial aspects. Hemodynamic and electrocardiographic responses. Transpl Proc 1988;20(suppl 7):25-28. 25. Novitzky D, Cooper DKC, Wicomb WN. Pathophysiology of brain death in the experimental animal: extracranial aspects. Endocrine changes and metabolic responses. Transpl Proc 1988;20(Suppl 7):33-38. 26. Novitzky D, Cooper DKC. Pathophysiology of brain death in the experimental animal: extracranial aspects. Results of hormonal therapy in human brain-dead potential organ donors. Transpl Proc 1988;20(Suppl 7)^9-62. 27. Wicomb WN, Cooper DKC, Novitzky D. Effects of storage and preservation. Added effects of organ (heart) storage after brain death in the experimental animaL Transpl Proc 1988;20(Suppl 7)39-43. 28. Montero JA, Mallol J, Alvarez F, Benito P, Concha M, Blanco A. Biochemical hypothy- roidism and myocardial damage in organ donors; are they related? Transpl Proc 1988;20:746-748. 29.Bretan PN, Baldwin N, Novick AC, Ng TC, Majors A, Stowe N, Streem S, Steinmuller D, Go R, Meaney T. Preliminary clinical experience with pretransplant assessment of renal viability by phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Transpl Proc 1988;20:852-853. 30. Pomer S, Hull WE, Rohl L. Assessment of renal viability for transplantation by high field 31P-NMR. Transpl Proc 1988;20:899-901. 31. Ip JH, Levin RI. Myocardial preservation during ischemia and reperfusion. Am Heart J 1988;115:1094-1104. 32. Zimmerman ANE, Hulsmann WC. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 1966^11:646-647. 33. Ruigrok TJC, de Moes D, Borst C. Bretschneider's histidine-buffered cardioplegic solution and the calcium paradox. J Thorac Cardiovasc Surg 1983;86:412-417. 34. Poole-Wilson PA, Harding DP, Bourdillon PDV, Tones MA. Calcium out of control. J Mol Cell Cardiol 1984;16:175-187. 35. Ledingham SJM, Braimbridge MV, Hearse DJ. Improved myocardial protection by oxygenation of St.Thomas' Hospital cardioplegic solutions. J Thorac Cardiovasc Surg 1988;95:1O3-11L 36. Takahashi A, Chambers DJ, Braimbridge MV, Hearse DJ. Long term preservation of the heart: the optimal concentration of calcium in the St.Thomas' Hospital cardioplegic solution. J Mol Cell Cardiol 1989;21(suppl H):S.121. 37.de Jong JW. Cardioplegia and calcium antagonist: a review. Ann Thorac Surg 1986;42:593-598. 38. Yamamoto F, Manning AS, Braimbridge MV, Hearse DJ. Nifedipine and cardioplegia: rat heart studies with the St.Thomas' cardioplegic solution. Cardiovasc Res 1983;17:719.

~~136 31P NMR of human donor heart preservation~~

39. Whitman GJR, Roth RA, Kieval RS, Harken AH. Evaluation of myo. ardial preservation using 31P-NMR. J Surg Res 1985;38:154-161. 40. Vary TC, Angelakos ET, Schaffer SW. Relationship between adenine nucleotide metabolism and irreversible ischémie tissue damage in isolated perfused rat heart. Circ Res 1979;45:218-225. 41. Ambrosio G, Jacobus WE, Bergman CA, Weisman HF, Becker LC. Preserved high energy phosphate metabolic reserve in globally " stunned " hearts despite reduction of basal ATP content and contractility. J Mol Cell Cardiol 1987; 19:953-964. 42. Rosenkranz ER, Okamoto F, Buckberg GD, Vinten-Johansen J, Allen BS, Leaf J, Bugyi H, Young H, Barnard RJ. Studies of controlled reperfusion after ischemia II. Biochemi- cal studies: failure of tissue adenosine triphosphate levels to predict recovery of contractile function after controlled reperfusion. J Thorac Cardiovasc Surg 1986;92:488-501. 43. Watts JA, Maiorano LJ, Maiorano PC. Protection by verapamil of globally ischémie rat hearts: energy preservation, a partial explanation. J Mol Cell Cardiol 1985; 17:797-804. 44. de Wit L, Coetzee A, Kotze J, Lochner A. Oxygen requirements of the isolated rat heart during hypothermie cardioplegia. J Thorac Cardiovasc Surg 1988;95:310-320. 45. Wicomb WN, CooperDKC.NovitzkyD.Loss of myocardial viability following hypothermie perfusion storage from contaminating trace elements in the perfusate. Transplanta- tion 1987;43:23-29. 46. Allen BS, Okamoto F, Buckberg GD, Acar C, Partington M, Bugyi H, Leaf J. Studies of controlled reperfusion after ischemia IX. Reperfusate composition: benefits of marked hypocalcemia and diltiazem on regional recovery. J Thorac Cardiovasc Surg 1986;92:564- 572. 47. Follette DM, Fey K, Buckberg GD, Helly JJ, Steed DL, Foglia RP, Maloney J V. Reducing postischemic damage by temporary modification of reperfusate calcium, potassium, pH, and osmolarity. J Thorac Cardiovasc Surg 1981;82:221-238. 48.Kirkels JH, Ruigrok TJC, van Echteld CJA, Meijler FL. Low Ca2+ reperfusion and enhanced susceptibility of the postischemic heart to the calcium paradox. Circ Res 1989;64:1158-1164. 49. Lazar HL, Wei J, Dirbas FM, Haasler GB, Spotnitz HM. Controlled reperfusion following regional ischemia. Ann Thorac Surg 1987;44:350-355.

~~137 General Discussion~~

~~GENERAL DISCUSSION Chapter 9~~

rrrhe work presented in this thesis deals with energy metabolism and ionic homeostasis in different models of myocardial ischemia and reperfusion. By making useof 31P and Na NMR spectroscopy it was possible to continuously study metabolic and ionic changes occurring during ischemia and reperfusion in isolated rat hearts and regionally ischemic rabbit hearts in vivo, and also in ex vivo human donor hearts during hypothermic storage.

~~Metabolic Changes during Ischemia and Reperfusion~~

Ischemia In both animal models normothermic ischemia caused a depletion of high-energy phosphates (creatine phosphate and ATP), associated with acidosis of the cytosol and accumulation of inorganic phosphate (Pi). The time course in metabolic deterioration was rather similar, with a depletion of creatine phosphate (CP) within 5 to 10 minutes, and a depletion of ATP within 20 (rat) to 40 (rabbit) minutes, with an almost equal acidification of the cytosol, from 7.0 - 7.1 under normoxic conditions to 5.9 - 6.0 after 20 min of ischemia. The difference in the rate of depletion of ATP may be partly explained by the higher metabolic rate in rat hearts. It should be noted that the absence of NMR visible ATP does not necessarily mean that it is absolutely absent . As only nuclei in mobile molecules give rise to a narrow NMR peak, bound ATP (e.g. to myofibrils or ATP-ases such as the Na+/K+ pump) will not show up. This also applies to Pi during reperfusion, when its NMR signal may decrease either due to wash-out and resynthesis of CP and ATP, or due to deposition of immobile Ca3(PO4)2 in mitochondria. Furthermore, the low sensitivity of the technique does not allow for small signals to be discriminated bom background noise. The rapid changes during ischemia preclude long term signal averaging, which could improve the signal to noise ratio of the spectra. Since peak areas in an NMR spectrum are proportional to the amount of spins contributing to that spectrum, magnetic field inhomogeneity and variations in magnetic susceptibility (large line-widths) has a detrimental effect on the signal to noise ratio. Especially in in vivo spectra this may hamper adequate quantitation of low levels of metabolites.

Reperfusion Reperfusion after limited periods of ischemia (approximately 20 min in the isolated rat heart and 45 min in the rabbit hearts) initiated a rapid and complete replenishment of CP stores, whereas recovery of ATP was very limited. In the in General Discussion vivo model of regional ischemia, a relationship was shown between the duration of the ischemic period and the extent of recovery after reperfusion. Inorganic phosphate levels decreased upon reperfusion, although not always to baseline values. Resumption of oxidative phosphorylation in mitochondria leads to a partial restoration of ATP stores, limited by low levels of adenine nucleotides, due to wash-out of ATP degradation products. On the other hand, a small pool of ATP with a high turnover rate, may very well be compatible with normal cell functions. Theoretically, there are three possible explanations for depressed postischemic cardiac function: reduced energy production in the mitochondria, reduced energy transfer via the creatine kinase reaction, and reduced energy consumption at the myoGbrillar site. Saturation transfer NMR experiments showed that the unidirectional flux through the creatine kinase reaction was reduced during postischemic reperfusion, although it was calculated to be still approximately 8 times higher than the ATP synthesis rate, based on oxygen consumption measurements 2. This would indicate that cytosolic energy transfer does not limit postischemic cardiac performance. 31P NMR saturation transfer measurements also indicated that mitochondrial uncoupling is not the cause of depressed postischemic mechanical function , suggesting that ATP utilization at the myofibrillar site might be inefficient. However, the discussion on possible causes for postischemic dysfunction is still open.

~~Animal Models~~

The in vivo model of regional ischemia was developed to extend our knowledge from isolated perfused hearts to a model that is closer to clinical reality. The similarities between metabolic changes in ischemic-reperfused isolated rat hearts and in vivo rabbit hearts once more stress the importance of the isolated perfused heart model as a basis for metabolic studies. Only when specific blood components, such as granulocytes, or an intact circulation with all normal feed-back mechanisms are involved in the aim of the study, the isolated perfused heart cannot be expected to yield valuable information.

~~141 Chapter 9~~

~~Protective Interventions~~

Hypothermia in human donor hearts Protection of the ischemic heart can be achieved by slowing down metabolic deterioration, which is clearly shown in the study on ex vivo donor hearts (Chapter 8). During hypothermic storage after cardioplegia, CP was detectable up to six to 12 hours, while ATP stores were maintained or only slightly decreased. Differences in buffering capacity and ionic composition of the preservation fluids (StThomas' Hospital no.2 or Bretschneider's HTP solution) were found to have a large influence on the development of acidosis and the depletion of high-energy phosphates, in favour of Bretschneider's HTP solution. It is yet to be demonstrated that these differences correlate with a better function and survival of the transplanted heart. Future studies will be needed to further improve long term preservation of donor hearts, and possibly to assess the metabolic condition of the ex vivo donor heart prior to transplantation. This may shed a light on the influence of e.g. brain death and drug treatment of the donor on the metabolic condition of the heart. Hopefully, this type of information will eventually help to extend the preservation time of donor hearts in order to expand the applicability of heart transplantation, and to prevent failure of successfully transplanted hearts.

Calcium antagonists (anipamil) In isolated rat hearts improved metabolic and functional postischemic recovery was achieved by pretreating the animals for five days with the new calcium antagonist anipamil (Chapters 2 and 3). Unlike the majority of studies using calcium antagonists, we did not find a negative inotropic effect during preischemic control perfusion. Accordingly, there was no reduction in metabolic deterioration of the hearts during ischemia. The only effect during ischemia appeared to be an attenuation of acidosis. Since ischemic acidosis may be related to glycogen degradation and catecholamine release, these were also studied (Chapter 3). However, glycogen and noradrenaline stores were not changed by five days of pretreatment with anipamil, and the degradation or release during ischemia and reperfusion was not diminished. Furthermore, pretreatment with anipamil did not change the blood pressure and heart rate in the intact animals during the pretreatment period. Although reduction of myocardial energy utilization is an important aspect of clinical treatment of myocardial ischemia and infarction, protection in the absence of a negative inotropic effect before ischemia (and therefore in the absence of an energy sparing effect during ischemia) is also very interesting. Since commonly

142 General Discussion known mechanisms of myocardial protection fail to explain the effects of anipamil, alternative modes of action have to be considered. Due to the highly lipophilic properties of the drug and the long pretreatment period, stabilization of cell membranes and prevention of Ca +-induced conformational changes of the sarcolemma are proposed as protective mechanisms.

~~Ionic Changes, low Ca Reperfusion, and the Calcium Paradox.~~

Calcium during ischemia and reperfusion While cytosolic Ca increases during ischemia, due to redistribution of cellular Ca2+, there is a large influx of Ca2+ during reperfusion, with numerous detrimental effects (see Chapter 1). The view of a pivotal role of Ca2+ in reperfusion-induced cell damage, led to studies of interventions at the time of reflow in order to improve metabolic and functional recovery after ischemia. Calcium antagonists, given only during reperfusion, failed to protect against Ca overload, eliminating the slow channels as an important route for Ca2+ entry. More likely routes are the Na+/Ca2+ exchanger or increased membrane permeability for Ca +, although the importance of Na+/Ca2+ exchange was recently questioned 4. However, in both cases a reduction of the availability of Ca2+ in the extracellular compartment during reperfusion might limit Ca influx.

Low Ca reperfusion The effects of temporary lowering of the extracellular [Ca ] to 0.05 mmol/1 early during reperfusion are presented in Chapter 4. Recovery of high-energy phosphates was significantly improved during the period of low Ca2+ reperfusion, indicating that interventions at the time of reperfusion can modify postischemic recovery and reperfusion damage can be circumvented. At the same time, this suggests that the concept of expressing the extent of ischemic damage in terms of (ir)reversibility may no longer be valid.

Postischemic susceptibility to the calcium paradox The next step was to return to a normal extracellular [Ca2+] to see whether the improvement in metabolic recovery would be a lasting effect Surprisingly, metabolic recovery was totally abolished and the hearts showed all signs of a calcium paradox. The calcium paradox is known to occur when a heart is reperfused with a

~~143 Chapter 9~~

Ca + containing solution after a short period of perfusion in the absence of Ca2+. However, temporary perfusion of normoxic hearts with 0.05 mmol/1 Ca2+ was not found to have any lasting detrimental effects. Therefore, it must be concluded that the postischemic heart is more susceptible to the calcium paradox. Future investigations should be directed at finding an optimal [Ca2+], low enough to prevent reperfusion-induced Ca + overload, and high enough to prevent the calcium paradox. The mechanism of enhanced susceptibility of the postischemic heart to the calcium paradox is yet to be established, but it might be related to intracellular Na+, which has been reported to increase during both ischemia and calcium-free perfusion. If these effects would be additive, increased intracellular Na+ could be exchanged for Ca + during normalization of the extracellular [Ca ], with fetal consequences.

NaNMR during ischemia and calcium-free perfusion Na NMR spectroscopy with the use of shift reagents to separate the signal from intracellular and extracellular Na+ (Chapter 5) confirmed the large increase in intracellular Na+ during 30 min of ischemia in isolated rat hearts. However, Ca2+-free perfusion was not associated with an increase in intracellular Na+, quite opposite to what has been reported in literature. Consequently, the assumed causal relationship between intracellular Na+ accumulation and the occurrence of the calcium paradox has to be reconsidered. It also appears unlikely that the increased susceptibility of the postischemic heart to the calcium paradox (Chapter 4) is related to additive effects on intracellular Na+.

Intracellular Mg2+ Energy metabolism and ionic homeostasis during ischemia and reperfusion are interrelated, as is commonly known for e.g. the Na+/K+ ATPase. Less well known is the importance of Mg2+ as an essential cofactor in all enzymatic processes involved in the production and utilization of ATP. More than 90% of ATP is complexed to Mg2+. Intracellular tree MgT+ (Mgf) is considered to be an important regulator of myoBbrillar contraction and ion pumps. It has been suggested that loss of Mg during postischemic reperfusion may limit the utilization of newly formed ATP. 31P NMR spectroscopy offers a unique possibility to calculate Mgf from the

chemical shift difference between ATPa and ATP/3. By applying this technique to the ischemic isolated rat heart (Chapter 6), it was found that Mgf rises considerably as Mg2+ is liberated from ATP. During reperfusion after 30 min of ischemia there was no significant loss of Mg + to the coronary effluent, and ATP was still

144 Uenerm uiscusswn complexed to Mg2+ for more than 90 %, indicating that lack of Mg2+ in this model is not a likely cause of impaired ATP utilization. On the other hand, Mgf remained elevated during reperfusion. In view of the important regulatory role of Mgf, this may contribute to an explanation of postischemic depressed cardiac function.

~~References~~

1. Humphrey SM, Burns S, Garlicfc P. ATP and PCr in postischemic hearts: NMR vs chemical analysis. J Mol Cell Cardiol 1988;20(Suppl V):S78. 2. Neubauer S, Hamman BL, Perry SB, BittI JA, Ingwall JS. Velocity of the creatine kinase reaction decreases in postischemic myocardium: a P-NMR magnetization transfer study of the isolated ferret heart. Circ Res 1988;63:1-15. 3. Sako EY, Kingsley-Hickman PB, From AHL, Foker JE, Ugurbil K. ATP synthesis kinetics and mitochondrial function in the postischemic myocardium as studied by 31P NMR. J Biol Chem 1988;263:10600-10607. 4. Poole-Wilson PA, Tones MA. Sodium exchange during hypoxia and on reoxygenation in the isolated rabbit heart. J Mol Cell Cardiol 1988;20(Suppl D):15-22.

~~145 Summary~~

~~SUMMARY~~

~~147 Chapter 10~~

Tn this study several aspects of myocardial energy metabolism and ionic homeostasis during ischemia and reperfusion were investigated in isolated perfused rat hearts, regionally ischemic rabbit hearts, and ex vivo human donor hearts during long term hypothermic cardioplegia. Phosphorus-31 nuclear magnetic resonance ( P NMR) spectroscopy was used as a powerful tool to non-destructively follow the time course in changes in intracellular high-energy phosphates, (creatine phosphate and ATP), inorganic phosphate, and pH. In addition, changes in intracellular free magnesium were followed during ischemia and reperfusion. Sodium-23 ( Na) NMR spectroscopy was used to study intracellular sodium during ischemia and reperfusion and during calcium-free perfusion.

Chapter 1 provides a general introduction and survey of the literature on metabolic and ionic changes occurring during ischemia and reperfusion, as well as on the possible role of oxygen derived free radicals, and the transition from reversible to irreversible cell damage. A summary of possible protective interventions before and during ischemia is given, and possible protective interventions at the time of reperfusion are summarized. NMR spectroscopy is concisely introduced, and the aim of the thesis is presented.

Chapter 2 presents data on the protective effects of the new calcium antagonist anipamil. Pretreatment of rats for five days significantly improved metabolic and functional recovery of the isolated hearts during postischemic reperfusion. In contrast with most animal studies using calcium antagonists, protection occurred in the absence of a negative inotropic effect during preischemic control perfusion, and without an effect on high-energy phosphate depletion during ischemia. Only intracellular acidosis during ischemia was attenuated.

Chapter 3 deals with further investigations of the possible protective mechanisms of anipamil. Since ischemic acidosis may be related to glycogen metabolism and catecholamine release, these were also measured. It was found that five days of treatment of rats with anipamil did not alter their blood pressure and heart rate. By using a different technique to study function of the isolated hearts, the lack of a negative inotropic effect during preischemic control perfusion (and the absence of an energy sparing effect during ischemia) was confirmed. Glycogen and noradrenaline stores were not altered by anipamil pretreatment, and the degradation and release due to ischemia and reperfusion were not different from controls. Since commonly known mechanism of myocardial protection fail to explain protection by pretreatment with anipamil, as observed in these experiments, alternative modes

~~148 Summary of action have to be considered. Some of these alternatives are discussed.~~

Chapter 4 presents data on experiments, designed to prevent postischemic reperfusion damage by temporary lowering of the extracellular Ca concentration to 0.05 mmol/1. During 10 min of low Ca + reperfusion metabolic recovery was significantly improved as compared to controls, but on normalization of the Ca concentration metabolic recovery was largely abolished. The combination of findings would suggest that a (partial) calcium paradox occurred, initiated by the period of low calcium reperfusion. On the other hand, normoxic hearts tolerated 10 min of perfusion with 0.05 mmol/1 Ca very well. These experiments demonstrate that recovery on postischemic reperfusion is not solely determined by the extent of ischemic damage, but may be modified by interventions at the time of reperfusion. At the same time, they demonstrate that postischemic susceptibility to the calcium paradox is enhanced.

Chapter 5 presents Na NMR experiments with the use of shift reagents to study changes in intracellular sodium during ischemia (and reperfusion) and calcium-free perfusion. As both conditions have been reported to be accompanied by an increase in intracellular sodium, this might lead to explanation of the increased postischemic susceptibility to the calcium paradox (Chapter 4). During 30 min of total ischemia a large increase in intracellular sodium was indeed found. However, calcium-free perfusion did not appear to be associated with an increase in intracellular sodium, quite contrary to what has been reported in literature. Based on these experiments, the causal role of intracellular sodium in the occurrence of the calcium paradox is questioned. Enhanced susceptibility of the postischemic heart to the calcium paradox through additive effects on intracellular sodium is also less likely.

Chapter 6 deals with intracellular magnesium during ischemia and reperfusion. ATP can only be used when complexed to Mg2+, and intracellular free magnesium (Mgf) is considered to be an important regulator of cell functions, such as active ion transport and contraction. Loss of Mg2+ during postischemic reperfusion has been suggested as a limiting factor for the use of newly produced ATP. By making use of

the Mg -dependent changes in chemical shift difference between ATPa and ATPjS, as measured from a 31P NMR spectrum, the fraction of MgATP and Mgf could be calculated (94 % and 0.60 mmol/1, respectively, under normal conditions). During ischemia, a large increase in Mgf was observed, parallel to the estimated liberation of Mg + from ATP. During reperfusion the newly synthesized ATP (» 25 % of preischemic values) was sufficiently complexed to Mg2+ (96 %), while

~~149 Chapter 10~~

atomic absorption spectrophotoraetric determinations of Mg2"1" in myocardial tissue and coronary effluent indicated that less than 2 % of cellular Mg2+ was released. Mgf remained elevated during reperfusion (0.85 mmol/1). It is concluded that intracellular Mg + deficiency due to leakage of Mg to the extracellular space does not play a role in the poor postischemic recovery in this isolated rat heart model. Nevertheless, high Mg + prior to ischemia or during reperfusion may well be protective, due to interactions of Mg with the sarcolemma or intracellular sites, affecting Ca +, K+, and Na+ distribution and fluxes.

Chapter 7 is dedicated to a new experimental model: in vivo regional myocardial ischemia and reperfusion in the rabbit, studied with P NMR surface coil spectroscopy. Intracellular high-energy phosphates, inorganic phosphate, and pH were continuously followed under normal conditions, increasing periods of regional ischemia, and reperfusion. Changes occurring during unprotected ischemia and reperfusion are demonstrated, and a relation between the duration of the ischemic period and the extent of metabolic recovery is shown. Preliminary results obtained from rabbits which were administered AICA-riboside before and during ischemia are presented. However, it is too early to evaluate possible protection by AICA-riboside.

Chapter 8 presents P NMR data obtained from ex vivo human donor hearts during long term hypothermic cardioplegia with either StThomas' Hospital no.2 solution or Bretschneider's HTP solution. The metabolic condition of donor hearts was studied up to approximately 12 hours after the onset of cardioplegia. Acidosis and the decrease in high-energy phosphates were much more pronounced with St.Thomas' Hospital cardioplegia. Assuming that these factors are relevant for the resumption of adequate pump function after (successful) transplantation, Bret- schneider's HTP solution might be preferable. This type of information may contribute to improvement of long term preservation of (human) donor hearts. When the clinically accepted period of preservation (now 4 hours) will have been extended to allow for the extra time delay, 31P NMR may offer a non-invasive assessment of the metabolic condition of an ex vivo donor heart prior to transplantation.

~~150 Samenvatting~~

~~SAMENVATTING~~

~~151 Chapter 10~~

irn dit proefschrift worden verschillende aspekten belicht van de veranderingen in de energie- en ionenhuishouding van het hart tijdens ischémie en reperfusie. Daarvoor werd gebruik gemaakt van geïsoleerde, doorstroomde ratteharten, van harten van konijnen onder narcose waarbij regionale ischémie (infarct) en reperfusie werden bestudeerd, en tenslotte van menselijke donorharten tijdens het langdurig bewaren van deze harten bij lage temperatuur. Fosfor-31 kernspin resonantie spectroscopie (31P NMR) werd gebruikt om op non-destruktJeve wijze het tijdsverloop in intracellulaire energierijke fosfaten (creatine fosfaat en ATP), anorganisch fosfaat en pH te bestuderen. Bovendien werden de veranderingen in intracellulair ongebonden magnesium bestudeerd tijdens ischémie en reperfusie. Natrium-23 (^Na) NMR spectroscopie werd gebruikt om de veranderingen in intracellulair natrium te bestuderen tijdens ischémie en reperfusie, alsmede tijdens perfusie van het geïsoleerde hart in afwezigheid van calcium.

Hoofdstuk 1 is bedoeld als algemene inleiding en overzicht van de literatuur betreffende de veranderingen in metabolisme en ionenhuishouding tijdens ischémie en reperfusie; bovendien wordt de mogelijke rol van vrije (zuurstof) radicalen en de overgang naar onherstelbare celbeschadiging kort belicht Vervol- gens wordt een kort overzicht gegeven van beschermende maatregelen vóór en tijdens ischémie en van de recent ontdekte mogelijkheden voor bescherming tijdens reperfusie. Tenslotte wordt de gebruikte NMR spectroscopie techniek in het kort uitgelegd, en wordt het doel van de studie geformuleerd.

In Hoofdstuk 2 wordt het beschermende effekt van de nieuwe calcium antagonist anipamil beschreven. Voorbehandeling van ratten gedurende vijf dagen ver- beterde het metabole en funktionele herstel van de geïsoleerde harten tijdens reperfusie na dertig minuten totale ischémie. In tegenstelling tot de meeste andere dierexperimentele studies die calcium antagonisten bestuderen, vond deze bescherming plaats in afwezigheid van een negatief effekt op de contractiekracht vóór ischémie en zonder effekt op de uitputting van de energierijke fosfaten tijdens ischémie. Alleen de intracellulaire verzuring tijdens ischémie was minder uitgesproken in de voorbehandelde harten.

Hoofdstuk 3 behandelt verder onderzoek naar het mogelijke werkingsmecha- nisme van anipamil. Daar de intracellulaire verzuring tijdens ischémie (voor een deel) bepaald wordt door de afbraak van glycogeen en de vrijmaking van catecholamines, werden deze gemeten naast de NMR parameters. Bovendien werden de bloeddruk en hartfirequentie van de ratten tijdens de vijf dagen voorbehandeling

152 Samenvatting met anipamil gemeten, waarbij bleek dat anipamil hierop geen invloed had. In de geïsoleerde harten bleek wederom, met gebruikmaking van een andere techniek, dat de contractiekracht vóór ischémie niet verminderd was door voorbehandeling met anipamil en het ontbreken van een "energie-sparend" effekt tijdens ischémie werd bevestigd. De intracellulaire voorraden van glycogeen en noradrenaline waren niet veranderd door de voorbehandeling met anipamil en ook de afbraak respektievelijk vrijmaking tijdens ischémie en reperfusie bleken niet te verschillen van controle dieren. Daar de bescherming door anipamil, zoais in deze experimenten beschreven, niet verklaard kan worden op basis van algemeen aanvaarde principes van bescherming van het ischemische hart, moeten alternatieve werkingsmecha- nismen worden overwogen. Enkele van deze alternatieven worden besproken.

In Hoofdstuk 4 worden experimenten gepresenteerd, opgezet om post-ischemische reperfusie beschadiging te voorkomen door tijdelijk de extracellulaire Ca concentratie te verlagen tot 0,05 mmol/1. Tijdens tien minuten reperfusie met deze lage Ca + concentratie was er een duidelijk beter metabool herstel vergeleken met controles, maar na het normaliseren van de Ca concentratie tot 1,3 mmol/1 werd dit herstel vrijwel geheel teniet gedaan. Deze bevindingen suggereren dat er een (partiële) calcium paradox optrad, geïnitieerd door de periode van reperfusie met lage Ca concentratie. Anderzijds ondervonden harten die niet ischemisch waren geweest geen blijvende schade van tien minuten doorstroming met 0,05 mmol/1 Ca2+. Deze studie toont aan dat het herstel tijdens post-ischemische reperfusie niet alleen bepaald wordt door de mate van ischemische beschadiging, maar dat ook de specifieke omstandigheden tijdens reperfusie van invloed kunnen zijn. Tevens toont deze studie aan dat het post-ischemische hart gevoeliger is voor de calcium paradox beschadiging.

In Hoofdstuk 5 worden Na NMR experimenten beschreven, waarin "shift reagentia" zijn gebruikt om de signalen van intra- en extraceüulair natrium te scheiden. De veranderingen in intracellulair natrium tijdens ischémie (en reperfusie) en tijdens calcium-vrije perfusie werden bestudeerd. Daar van beide omstandigheden wordt aangenomen dat zij gepaard gaan met een stijging van de intracellulaire Na+ concentratie, zou deze studie kunnen bijdragen tot een verkla- ring voor de verhoogde gevoeligheid van het post-ischemische hart voor de calcium paradox beschadiging (Hoofdstuk 4). Tijdens dertig minuten totale ischémie werd inderdaad een sterke toename in intracellulair natrium gevonden. In tegenstelling tot wat beschreven is in de literatuur, bleek calcium-vrije perfusie echter niet gepaard te gaan met een toename in intracellulair natrium. Op basis van deze

153 Chapter 10 experimenten moet een oorzakelijke rol van intracellulair natrium in het ontstaan van de calcium paradox beschadiging betwijfeld worden. Het is ook onwaarschijnlijk dat de verhoogde gevoeligheid van het post-ischemische hart voor de calcium paradox optreedt op basis van additieve effekten met betrekking tot intracellulair natrium.

Hoofdstuk 6 gaat over intracellulair magnesium (Mg +) tijden? ischémie en reperfusie. ATP kan alleen gebruikt worden als het gecomplexeerd is met Mg en de intracellulaire concentratie van ongebonden Mg2+ wordt verondersteld een belangrijke regulator te zijn van allerlei celfunkties, zoals aktief ionentransport en contractie van de hartspiercel. Verlies van Mg2+ tijdens post-ischemische reperfusie is beschreven en verondersteld werd dat dit een beperkende faktor zou kunnen zijn voor het benutten van ATP dat weer aangemaakt wordt tijdens reperfusie. Door gebruik te maken van de Mg2+-afhankelijke veranderingen in chemische verschuiving van ATPa en ATP/j, gemeten aan het P NMR spectrum, konden de fraktie MgATP/ATPtotaai en de concentratie van ongebonden Mg2**" berekend worden (94 % resp. 0,60 mmol/1 onder normale omstandigheden). Tijdens ischémie werd een forse toename in ongebonden Mg waargenomen, parallel aan de geschatte vrijmaking van Mg van ATP. Tijdens reperfusie bleek dat het nieuw gevormde ATP (ongeveer 25 % van de normale voorraad) in voldoende mate was gecomplexeerd met Mg2"1" (96 %). Atomaire absorptie spectrofotometrie van hartspierweefsel en coronair effluent toonde tegelijkertijd aan dat minder dan 2 % van het cellulaire Mg uitgewassen werd. De concentratie ongebonden Mg + bleef verhoogd tijdens reperfusie (0,85 mmol/1). Geconcludeerd wordt dat een intracellulair Mg2+ tekort door weglekken van Mg2+ naar de extracellulaire ruimte geen rol speelt in het slechte post-ischemische herstel in dit geïsoleerde rattehart model. Desondanks zou een hoge Mg concentratie vóór ischémie of tijdens reperfusie bescherming kunnen bieden, door interakties van Mg^+ met de celmem- braan of intracellulaire bindingsplaatsen, die de verdeling en het transport van Ca2+, K+, en Na+ beïnvloeden.

Hoofdstuk 7 is gewijd aan aan nieuw experimenteel model: in vivo regionale ischémie en reperfusie in het konijn, bestudeerd met 31P NMR spectroscopie met behulp van oppervlakte spoelen. Intracellulaire energierijke fosfaten, anorganisch fosfaat en pH werden continu gevolgd onder nonaaie omstandigheden, tijdens toenemende periodes van regionale ischémie (infarct) en tijdens reperfusie. Ver- anderingen die optreden tijdens onbeschermde ischémie worden beschreven, en een relatie tussen de duur van de ischemische periode en de mate van metabool

154 Samenvatting herstel tijdens de daaropvolgende reperfusie wordt getoond. De eerste resultaten worden gepresenteerd van konijnen die AICA-riboside kregen toegediend vóór en tijdens ischémie. Het is echter nog te vroeg om de mogelijke bescherming door AICA-riboside te beoordelen.

In Hoofdstuk 8 worden resultaten beschreven die verkregen zijn met behulp van P NMR spectroscopie aan ex vivo humane donorharten tijdens langdurige koude cardioplegie met St.Thomas' Hospital no.2 oplossing ofwel met Bretschneiders HTP oplossing. De metabole conditie van deze donorharten werd vervolgd tot ongeveer 12 uur na begin van de cardioplegie. Cellulaire verzuring en afname in energierijke fosfaten waren het meest uitgesproken in harten met St.Thomas' Hospital no.2 cardioplegie. Vanuit de veronderstelling dat deze f aktoren van belang zijn voor het hervatten van een adequate pompfunktie na (geslaagde) transplantatie, lijkt Bretschneiders HTP cardioplegie de voorkeur te genieten. Dit soort informatie kan een bijdrage leveren aan verbetering van het langdurig in goede conditie houden van (humane) donor harten. Wanneer de momenteel in de kliniek gehanteerde maximale periode van vier uur voldoende kan worden verlengd, kan lP NMR een bijdrage leveren in het non-invasief vaststellen van de metabole conditie van het (ex vivo) donor hart vóór transplantatie.

~~155 Dankwoord~~

Aan het eind van dit proefschrift wil ik graag een aantal mensen bedanken die niet alleen onmisbare bijdragen hebben geleverd aan de tot stand koming van het proefschrift, maar bovendien de afgelopen jaren mijn dagelijkse omgeving hebben gevormd, die zich zowel in de arbeidssfeer als in de sociale sfeer vaak tot in de late uren uitstrekte.

Mijn promotor profldr. F.L. Meijler dank ik dat hij mij via het ICIN in staat heeft gesteld het werk te verrichten waarop dit proefschrift is gebaseerd, waarbij ik zijn steun en originele suggesties onder het motto "the sky is the limit" als zeer waardevol heb ervaren. Anderzijds dank ik hem voor de vrijheid die mij gegeven werd en het feit dat ik af en toe voor mijn beurt mocht spreken. Mijn promotor prof.dr. T. J.C. Ruigrok, beste Tom, jou dank ik voor de prettige begeleiding en samenwerking in het dagelijkse werk, waarbij ik ruim heb kunnen proGteren van je (internationale) kontakten, je ervaring in het fundamentele cardiologische onderzoek en je precieze en gedegen steun bij het schrijven van wetenschappelijke verhandelingen. Co-promotor dr. C. J. A. van Echteld, beste Cees, jou ben ik veel dank verschuldigd ik voor wat ik nu weet van NMR, voor de bijna dagelijkse discussies die de brug vormden tussen onze disciplines, en voor het enthousiasme waarmee je mij telkens meenam op nieuwe wegen in het onderzoek. Van jou heb ik ook geleerd dat werk niet vervelend móét zijn. Pieter van der Meer, jouw steun en inzet bij de technische kant van het NMR werk met al zijn tegenslagen en problemen heb ik zeer gewaardeerd, evenals je relativerende inbreng in menig gesprek. Prof.dr. C. Borst, beste Kees, jou dank ik voor het vertrouwen datje in me hebt gesteld en je kritische suggesties vooral in de beginperiode van het onderzoek. Carina Kasbergen, zonder jouw voortreffelijke biotechnische inbreng zou met name het in vivo werk nooit zijn geworden wat het nu is. Ik dank je ook voor de prettige manier waarop je mij dat hebt leren inzien. Dirk de Moes, jou dank ik voor je biotechnische bijdrage in de beginfase van het onderzoek en je steun bij mijn eerste stappen in de wereld van het Langendorff hart. Ton Hoogenberk, jou dank ik voor je hulp bij het maken van grafieken en later bij het maken van computer-diagrammen en dia's.

157 Voor de statistische verwerking van de gegevens ben ik Ingeborg van der Tweel dank verschuldigd, evenals voor de boeiende gesprekken die wij hadden over dit onderwerp. Cor Lekkerkerk, hoewel je het team pas recent hebt versterkt wil ik je bedanken voor je inzet op (electro)technisch gebied, waardoor niet alleen nieuwe ontwikkel- ingen gerealiseerd kunnen worden, maar ook het "oude spul" kan overleven. Mijn kamergenote Dorien Dekker, jou wil ik bedanken voor de prettige sfeer en het eindeloze geduld waarmee je mijn verhalen hebt aangehoord; ik hoop dat ze je van pas zullen komen bij je eigen onderzoek. Je hulp in de laatste moeilijke weken van het voorbereiden van het manuscript heb ik zeer gewaardeerd. Jan Vermeulen, destijds student, de samenwerking met jou in het eerste (frustrerende) jaar van het onderzoek heeft mij niet alleen geholpen maar ook veel plezierige herinneringen opgeleverd. De studenten Marcel Eygelshoven en Michiel van Nieuwenhoven dank ik voor hun geleverde respektievelijk verwachte bijdragen aan het natrium NMR werk. Joop Schreur, jou dank ik voor de prettige samenwerking, ook toen je nog als student bij ons was en ik hoop dat je veel van het werk dat nog in een pril stadium verkeert zult kunnen afronden. George Jambroes, Jeroen Kodde en de hartchirurgen van het AZU bedank ik voor hun onmisbare hulp en bijdragen in het humane donorhart onderzoek. Ik hoop dat het manuscript spoedig een publiceerbare vorm zal krijgen, Ruud Verdaasdonk, jou dank ik voor je hulp bij de moeizame kennismaking met computers en programma's, die mij, na vele uren ergernis en teleurstelling, uiteindelijk toch ook geholpen hebben. De medewerkers van de mechanische werkplaats wil ik bedanken voor de perfectie waarmee ze de ideeën van een onderzoeker weten te realiseren in een technisch ontwerp. Alinda Diepeveen, jou wil ik bedanken voor je secretariële ondersteuning. Ingrid Janssen dank ik voor de tekeningen. De internisten en interne assistenten van Ziekenhuis De lichtenberg in Amers- foort dank ik voor hun steun bij mijn klinische come-back. Tenslotte wil ik jou bedanken, Rian, dat je naast je eigen werk altijd begrip hebt gehad voor de tegenslagen, de successen en de inzet die het onderzoek voor mij met zich mee bracht, terwijl je gelukkig nooit mijn humeur hebt geaccepteerd.

~~158 Curriculum Vitae~~

De schrijver van dit proefschrift werd op 11 juli 1958 geboren te Hoensbroek. In 1976, na het behalen van het eindexamen Gymnasium/} (cum laude) aan het StJanscollege te Hoensbroek, werd begonnen aan de studie Biologie aan de Rijksuniversiteit Utrecht In 1977 werd van studierichting gewisseld en startte hij de studie Geneeskunde. In 1980 werd het kandidaats examen behaald. Van 1980 tot 1982 was bij diverse malen werkzaam als student-assistent bij de vakgroep anatomie. In 1981-1982, tijdens de wachttijd voor de co-assistentschappen verricht- te hij onderzoek op de afdeling cardiologie van het Academisch Ziekenhuis Utrecht (begeleiders: Prof-dr. T. van der Werf en Pro£dr. C. Borst). In 1983 werd het doctoraal examen behaald (cum laude), waarna een aanvang werd gemaakt met de co-assistentschappen in het Elizabeth en het Maria Ziekenhuis te Tilburg. Na behalen van het artsexamen in november 1984 begon hij in januari 198S met het onderzoek waaruit dit proefschrift is voortgekomen. Het onderzoek werd verricht in het Laboratorium voor Experimentele Cardiologie van het Academisch Zieken- huis Utrecht (hoofd: Pro£dr. G Borst), in dienst van het Interuniversitair Cardio- logisch Instituut Nederland (vanaf 1988 tevens Koninklijke Nederlandse Akademie van Wetenschappen). Vanaf augustus 1989 werkt hij als arts-assistent interne geneeskunde in Ziekenhuis de Lichtenberg te Amersfoort (opleider, dr. ïLCh. Hart) als vooropleiding voor de specialisatie cardiologie.