Chapter 3 Adenine nucleotides in cardiac cell injury and restitution

Chapter 3

Adenine Nucleotides in Cardiac Cell Injury and Restitution

HEINZ-GERD ZIMMER

Introduction Significance of Cardiac Adenine Nucleotides for Cell Injury Possible Causes of Myocardial Cell Injury

84 85 86

Depletion of Adenine Nucleotides Accumulation of Glycolytic Products Calcium Overload Oxygen Free Radicals Nonuniformity of the Heart Atrium-Ventricle Apex-Base Subepicardium-Subendocardium Role of Endocardium in Regulating Heart Function Conduction System-Working Myocardium Left-Right Heart Pathophysiological Aspects of Myocardial Ischemia Stunning Ischemic Preconditioning Hibernation

86 89 89 90

Principles of Medical Biology, Volume 13 Cell Injury, pages 83-126. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-818-8 83

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92 93 93 95 96 97 105

106 108 109

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Catecholamines and Myocardial Metabolism Effects at the Metabolic and Molecular Biological LeVel Influence on the Pentose Phosphate Pathway A New Homeostatic Mechanism Metabolic Concepts for Treating Heart Disease Adenosine Inosine Ribose Summary

110 111 112 114 114 115 116 117 119

INTRODUCTION High-energy phosphates are essential for maintaining cellular structure, function, and metabolism. This is particularly obvious for an organ, such as the heart that does external mechanical work. The focus in this chapter will therefore be placed on this organ. The heart has to perform continuous work on a beat-to-beat basis and therefore depends on a constant supply of energy. This is used for the mechanical activity, the contraction process, for the proper function of its ionic pumps such as the Na+-K+-ATPase, and for phosphorylation processes that occur at proteins of the sarcolemma and of the sarcoplasmic reticulum (phospholamban). These are involved in Ca 2§ transport which is essential to regulate the inotropic (force and speed of contraction) and lusitropic (relaxation) activity (Tada and Katz, 1982). Furthermore, energy is needed for several biosynthetic processes, for example, for the biosynthesis of adenine nucleotides which culminates in the new production of ATP from a variety of small molecular precursor substances (Zimmer et al., 1973). It is therefore necessary that the energy-rich compounds, such as ATP and creatine phosphate are kept fairly constant during the cardiac cycle (Koretsky et al., 1983) and over a wide physiological range of work (Balaban et al., 1986). The heart is well equipped to meet this demand for energy supply because there is a multitude of small chemical factories very efficiently involved in energy production. These are the mitochondria which constitute a major portion of the cell volume (Figure 1) and in which the process of oxidative phosphorylation takes place. Based on this morphological evidence alone, one can understand that myocardial metabolism is primarily aerobic with CO 2 and H20 as the end products as opposed to glycolysis where lactic acid is the major end product. When there is a disturbance in the oxygen demand and supply relationship as occurs when coronary blood flow is reduced, then there is a rapid decline in the highenergy phosphate compounds in the heart and in other tissues. It is a characteristic metabolic feature of the myocardium that it cannot restore its adenine nucleotide pool rapidly once it has been reduced by a brief period ofischemia, although there is an abundance of mitochondria which are obviously not damaged to such a extent

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Figure 1. Electron micrograph of a myocardial cell with an intercalated disk in the middle. Mitochondria are located beside and alongside the sarcomeres. that they can no longer function properly. The postischemic mitochondria, however, cannot produce on short notice an extra amount of ATP, so that other metabolic processes have to compensate for the ischemia-induced energy deficit. These processes, however, are much slower and less efficient so that it takes some days of postischemic reperfusion until the cardiac adenine nucleotide pool is restored to its normal level (Reimer et al., 1981; Zimmer et al., 1984). For this reason, among other important new aspects, cardiac adenine nucleotide metabolism remains a major area of interesting and vivid research with special emphasis on pathways that can elevate the adenine nucleotide content by means other than oxidative phosphorylation. In fact, most of the cardioprotective strategies developed in the past and during recent years are aimed at normalizing the high energy phosphate pool with the ultimate hope of improving heart function.

SIGNIFICANCE OF CARDIAC ADENINE NUCLEOTIDES FOR CELL INJURY To assess the significance of adenine nucleotides under normal and pathophysiological conditions, it is first necessary to consider the most appropriate method to do that. Basically, there are two possibilities. The first is 31p-nuclear magnetic resonance spectroscopy with which it is possible to measure continuously the changes in creatine phosphate, ATP, and inorganic phosphate in the intact organ (Ingwall, 1982) so that each preparation can serve as its own control. Furthermore, the pH value can be assessed by this method. The other possibility is to freeze the tissue quickly, to extract the high-energy phosphate compounds, and to determine them

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by biochemical methods (Wollenberger, 1957). This procedure implies that separate controls always have to be prepared as a baseline with which to compare the experimental data. A particular experimental model in which the behavior of high-energy phosphates has been studied extensively is the rapid decline in cardiac contractile performance during oxygen deficiency (Tennant and Wiggers, 1935). The basic metabolic feature of ischemia and anoxia is the rapid decline in tissue creatine phosphate and the relatively slow decrease in the ATP content (e.g. Feinstein, 1962). The pathway of ATP degradation is shown at the bottom of Figure 2. However, it is also known that contractile failure can occur long before tissue ATP levels fall below a critical value. On the other hand, contractile performance can be maintained despite low ATP values. Another approach has been utilized in that the phosphorylation potential [ATP]/[ADP].[Pi] was taken into consideration. This determines the free energy of ATP hydrolysis. A crucial problem in that regard is estimating the cytosolic ADP concentration. This has been solved by a calculation which uses the creatine kinase equilibrium equation (Kammermeier et al., 1982). When this approach was applied, early hypoxic contractile failure of the isolated perfused rat heart was shown to be mainly due to a reduction of the free energy change of ATP hydrolysis beyond the level of about 45 kJ/mol (Kammermeier et al., 1982). When considering this approach, one has to be aware that the cytosolic concentrations are measured throughout the entire heart. The heart, however, is not a homogeneous organ (see "Nonuniformity of the Heart' section, p. 91). It contains cardiac myocytes and also interstitial cells, endothelium, and smooth muscle cells belonging to the coronary vessels. Thus, there may be considerable compartmentation in functional and metabolic terms (Gudbjarnason et al., 1970; Schrader and Gerlach, 1976; Soboll and Bringer, 1981; Nees et al., 1985). The phosphorylation potential calculated for the entire heart, therefore, does not reflect the actual situation in the cardiac myocyte compartment which is responsible for providing the energy for contraction.

POSSIBLE CAUSES OF MYOCARDIAL CELL INJURY Depletion of Adenine Nucleotides As already mentioned, there is rapid deterioration in heart function subsequent to oxygen deficiency, such as global ischemia, in the isolated, perfused heart (Neely et al., 1973) and after regional ischemia in the heart in situ induced by ligation of a coronary artery in acute (Tennant et Wiggers, 1935) and chronic experiments (Zimmer et al. 1989). In the isolated rat heart preparation, a substantial decline in myocardial ATP and creatine phosphate occurred after the onset of anoxia before contractile failure was evident (Hearse, 1979). Several other experimental studies

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on isolated rat or rabbit heart preparations, however, have demonstrated a correlation between myocardial ATP content and cardiac function (Reibel and Rovetto, 1978; Watts et al., 1980; Nishioka and Jarmakani, 1982). Also in the in situ dog ventricle, the changes in ATP concentration ran parallel to those in mechanical function of the nonischemic part of the infarcted myocardium (Gudbjarnason et al., 1971). Furthermore, there is a close relationship between the depletion of high-energy phosphates and the development of lethal cell injury occurring in the acutely ischemic myocardium (Jennings et al., 1978). Despite the good correlation between residual ATP, on the one hand, and mechanical function and morphology, on the other, a clear cause and effect relationship has not been established (Neely and Grotyohann, 1984). Another approach has been applied in which the cardiac ATP content was manipulated experimentally in various pathophysiological conditions, and then its effect on global heart function was determined in the intact animal. An easy and very effective procedure to increase the myocardial ATP pool is intravenous administration of ribose. This pentose sugar bypasses the first and rate-limiting step in the oxidative pentose phosphate pathway, the glucose-6-phosphate dehydrogenase reaction, and elevates the available pool of 5-phosphoribosyl-1-pyrophosphate (PRPP), an essential precursor substrate for purine and pyrimidine nucleotide biosynthesis and for salvaging adenine to build up AMP and hypoxanthine to form IMP (Figure 2). Two types of experiments were done to illustrate the role of myocardial ATP in regard to heart function. An already increased heart function induced by isoproterenol, a 131-adrenergic stimulator, was shown to be further enhanced by ribose. At the same time, after five hours following application of this catecholamine, ribose given at a high dose had normalized the ATP content of the heart which had been depressed by isoproterenol (Zimmer and Ibel, 1983). Thus in a condition characterized by a stimulated heart function, normalization of ATP is accompanied by a further increase in function. From a clinical point of view, however, it is interesting to examine whether a depressed heart function can be improved by affecting the cardiac ATP pool. To examine this possibility, two models of impaired heart function were developed. In the first, depression of all hemodynamic parameters was induced in rats by severely constricting the abdominal aorta in combination with a single subcutaneous dose of isoproterenol. Twenty-four hours after this combined intervention, both the ATP level and heart function had deteriorated when only 0.9% NaC1 had been infused. When ribose was administered for 24 hours, the biosynthesis of cardiac adenine nucleotides was stimulated to such a degree that the depression in ATP and in the total adenine nucleotide pool was prevented. The normalization of the ATP pool was accompanied by an elevation of the depressed left ventricular systolic pressure and left ventricular dp/dtmax. The pressure-rate product, which was markedly diminished due to the combined intervention, had also returned to a near-normal value. Thus, ribose was in fact capable of normalizing an impaired heart function concomitantly with the restoration of the cardiac adenine nucleotide pool (Zimmer, 1983).

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HEINZ-GERD ZIMMER Glycogen

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Figure 2. Schematic representation of the oxidative pentose phosphate pathway (center) its connections to glycolysis (right-hand side) via the transaldolase and transketolase reactions (arrows), to purine and pyrimidine nucleotide synthesis, and the pathways of degradation of ATP in the heart (broken arrows). G-l-P, Glucose-l-phosphate;G-6-P, Glucose-6-phosphate;F-6-P, Fructose-6-phosphate; F-l, 6-phosphate, Fructose-I, 6-biphosphate; GAP, Glyceraldehyde-3-phosphate;6-PGL, 6-Phosphogluconolactone; 6-PG, 6-Phospho'gluconate; Ru-5-P, Ribulose-5-phosphate; R-5-P, Ribose-5-phosphate; PRPP, 5-Phosphoribosyl-l-pyrophosphate; NADP§ Nicotinamide Adenine Dinucleotide Phosphate; IMP, Inosine monophosphate; AMP, Adenosine monophosphate; ADP, Adenosine diphosphate; ATP, Adenosine triphosphate; OMP, Orotidine monophosphate; UMP, Uridine monopho~phate; UDP, Uridine diphosphate; UTP, Uridine triphosphate; GSH, Reduced glutathione; GSSG, Oxidized glutathione; G-6-PD, Glucose-6-phosphatedehydrogenase; 6-PGD, 6-Phosphogluconate dehydrogenase; GP, Glutathione peroxidase;GR, Gutathione reductase; SOD, Superoxide dismutase; XD, Xanthinedehydrogenase;XO, Xanthineoxidase. ..

A marked impairment of heart function can also be induced by experimental myocardial infarction brought about by ligation of the descending branch of the left coronary artery (LAD) in rats. This intervention leads to a progressive deterioration in all hemodynamic parameters. A characteristic feature is the elevation of the left ventricular end-diastolic pressure. When ribose was infused for two and four days, this increase was markedly attenuated, and this correlated with an elevated ATP

Cardiac Adenine Nucleotide Metabolism

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content in the noninfarcted myocardium (Zimmer et al., 1989). These two results demonstrate that there is a close relationship between the ribose-manipulated ATP content and global heart function in certain in vivo pathophysiological conditions.

Accumulation of Giycolytic Products In 1935 the role of metabolic products, such as lactate in the development of ischemic damage, had already been recognized (Tennant, 1935). In the isolated rat heart, good correlation between residual ATP and heart function was found only when high levels of lactate had accumulated in the presence of 2.5 mM Ca 2§in the perfusion medium (Neely and Grotyohann, 1984). Depletion of glycogen and removal of lactate prior to the ischemic period resulted in much better recovery of ventricular function. There was a negative correlation between tissue levels of lactate during ischemia and recovery of function during the reperfusion period. When lactate was added to the perfusion medium prior to ischemia, ventricular function during the postischemic reperfusion period deteriorated linearly with added perfusate lactate. Taken together, these results indicated that ventricular function during reperfusion is inversely related to tissue lactate during ischemia. Obviously, there was no correlation between residual ATP and functional recovery. Therefore, high levels of lactate during ischemia are associated with accelerated cellular injury. The mechanism underlying the harmful effects of lactate accumulation is not known yet. Tissue damage in this condition could be mediated by changes in intracellular pH. In fact, in the isolated interventricular septum of the rabbit heart, the onset of acidosis preceded a decline in mechanical function during total ischemia (Cobbe and Poole-Wilson, 1980). It has been estimated that about 40 to 50% of the depression in left ventricular developed pressure of the isolated, perfused rat heart during the early phases of ischemia may be due to the effects of intracellular acidosis (Jacobus et al., 1982). Not only lactate and the concomitant pH-decline has to be considered for early ischemic failure of the heart, but also inorganic phosphate. When creatine phosphate and ATP are broken down, phosphate ions are accumulated and retained. Increased phosphate levels may cause calcium to be sequestered and trapped in the sarcoplasmic reticulum and possibly in the mitochondria so that Ca 2§ ions are not available to participate in the process of excitation-contraction coupling. As a consequence, early "pump" failure of the ischemic heart may occur (Kiibler and Katz, 1977).

Calcium Overload The idea that Ca 2§ overload may be involved in the processes leading to cell injury has been developed by Fleckenstein (1983) in studies related to I]-adrenergic overstimulation of the heart. Catecholamines with 13-adrenergic activity increase cardiac contractility by enhancing Ca 2+ -dependent utilization of high-energy phos-

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phates in the contractile apparatus. With high doses of isoproterenol, Ca 2§ uptake and the breakdown of high-energy phosphates become so excessive that depletion of creatine phosphate and ATP occurs and focal myocardial cell lesions develop (Rona et al., 1959). Another line of research which led to the recognition of the relationship between Ca2§ and cell injury was the discovery of the "calcium paradox" phenomenon (Zimmermann and Htilsmann, 1966). It was shown that Ca2§ have deleterious effects on reperfusion of the isolated rat heart after a Ca2§ perfusion period. As soon as Ca 2§ is readmitted to the heart, there is an influx of Ca 2§ into the cardiac myocyte and exhaustion of high-energy phosphates. In addition, a rapid myocardial contracture occurs with massive release of cell constituents and excessive ultrastructural damage. As to the mechanism, it has been shown that Ca2§ perfusion of the rat heart produced a distinct separation of the membrane external lamina from the surface coat of the sarcolemma. This alteration was irreversible and correlated with increased cellular 45Ca2§ content and contracture upon reperfusion with normal Ca 2§ (Crevy et al., 1978). There was also considerable disruption of the myofilaments, swelling of the mitochondria with formation of electron-dense particles and disruption of the sarcolemma and intercalated disks. These results clearly emphasize the point that the sarcolemma is the critical structure involved in the initiation of cell injury. This may also be true for the pathophysiology of ischemia and reperfusion. There was no significant uptake of 45Ca2§ after 60 minutes of ischemia which was induced by permanent occlusion of a coronary artery in a dog. However, 40 minutes of ischemia followed by 10 minutes of arterial reflow resulted in an 18-fold increase in Ca 2§ uptake. Reversible myocardial injury induced by 10 minutes of ischemia was not followed by Ca 2§accumulation during a period of 20 minutes of reperfusion (Shen and Jennings, 1972). Addition of free Ca 2§ ions to the perfusate during low flow ischemia in the isolated rat heart accelerated the onset of ventricular failure during the postischemic recovery period (Neely and Grotyohann, 1984). Thus, in ischemia and reperfusion, Ca 2§ ions are also involved in the deterioration of heart function. Oxygen Free Radicals The superoxide free radical has been implicated in several pathophysiological conditions, such as oxygen toxicity, inflammation, and ischemia-related tissue injury (McCord and Roy, 1982). In the myocardium, superoxide free radicals may be involved in reperfusion damage. It is believed they are produced in the mitochondria, in the endothelium, and by invading leucocytes. A small amount of radicals is physiologically produced in the mitochondria, where they are quickly scavenged by the glutathione-glutathione peroxidase system. Another possible source seems to be the xanthine oxidase reaction (Figure 3). During ischemia, xanthine dehydrogenase has been shown to be converted to xanthine oxidase (McCord and Roy,

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Cardiac Adenine Nucleotide Metabolism ATP

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Figure 3. Schematic diagram illustrating the possible sources of oxygen free radicals from the mitochondria, from invading leukocytes, and from the degradation of hypoxanthine resulting from the breakdown of ATP. 1982; Chambers et al., 1985). When oxygen is supplied during reperfusion, there appears to be a burst of superoxide radical production. Because polymorphonuclear neutrophils move into the reperfused tissue, these cells may also be a source of superoxide free radicals. There have been conflicting results as to the effects of allopurinol, an inhibitor of xanthine oxidase, on infarct size. Although the infarct size in dog hearts was reduced subsequent to one hour of total occlusion of the left anterior descending coronary artery followed by four hours ofreperfusion (Chambers et al., 1985), there was no effect when the circumflex coronary artery was occluded for 40 minutes followed by reperfusion for four days (Reimer and Jennings, 1985). Thus, the role of oxygen free radicals in determining infarct size is controversial at least, if not at all doubtful.

NONUNIFORMITY OF THE HEART A particular feature of some organs such as the brain and kidney, is cellular diversity. The heart, is also inhomogeneous, and is not a symmetrical organ. Quite to the contrary, it is heterogeneous and asymmetrical. The latter is the result of ontoge-

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netic development and then, of course, a manifestation of the different resistances characteristic of both the peripheral and pulmonary circulation. After birth, pulmonary vascular resistance is much lower than total peripheral resistance. Therefore, the right and left heart are completely different hemodynamically and morphologically. There is also nonuniformity with regard to atria versus ventricles. In each ventricle there are regional differences between the apex and base and transmural differences between the subendocardium and subepicardium. Furthermore, there are two functionally different systems in the heart, the conduction system and the working myocardium which constitutes the major mass of the heart. In addition, nonuniformity extends to the cellular aspect. Apart from cardiac myocytes there are endothelial cells, smooth muscle cells of the vessel wall, interstitial cells, and possibly white blood cells that originate from the circulation. Thus, the heart is asymmetrical and heterogenous both at the macroscopic and cellular level. Atrium-Ventricle

A characteristic feature of the atrium is that it contains specific endocrine cardiac cells that are rich in secretory granules first described by Kisch in 1956. They contain the atrial natriuretic factor (ANF), a family of peptides (Thibault et al., 1983) present in the saline extract of cardiac atria (deBold et al., 1981). It has been shown that ANF is synthesized in and secreted from the atria myocytes, circulates in the bloodstream, and acts on receptor sites in the kidney and the blood vessels. ANF is thus an endocrine system localized in the atria that is important for body fluid and blood pressure regulation. It is involved in the increase in sodium excretion and in the reduction of blood pressure (Sonnenberg, 1987). Although the regulatory roles of this hormonal system still remain speculative, it is interesting to note that elevated filling cardiac pressure is associated with increased concentrations of ANF and that congestive heart failure is characterized by its elevation (Burnett et al., 1986). In certain pathophysiological situations, such as the development of cardiac hypertrophy, ANF also appears in the ventricle. It has been shown in mice that banding of the thoracic aorta for seven days led to a marked increase in the expression of the ANF gene in the hypertrophied ventricle. The ANF mRNA levels were significantly increased more than 20-fold compared with the control level. This finding is consistent with the activation of an embryonic program of gene expression in the ventricle (Rockman et al., 1991). Differences between atria and ventricles have also been detected in metabolic terms. In rat and rabbit, the atria contain a higher concentration of glycogen than the ventricles (Davies et al., 1947; Weisberg and Rodbard, 1958). However, the total glycogen content of the ventricles was greater than that of the atria in frogs and pigeons (Davies et al., 1947). In all animals tested, the percentage of total glycogen in the free form was higher in the atria than in the ventricles. In contrast, the ventricles were found to contain more nucleotides than the atria per g wet weight. Also the to-

Cardiac Adenine Nucleotide Metabolism

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tal creatine content of the ventricles was greater than that of the atria (Davies et al., 1947).

Apex-Base Differences have also been found within the ventricles with respect to the apex and base. Glycogen concentration was shown to be always higher in the apex region compared with the base of the heart. This was true for the left ventricle, for the right ventricle, and for the septum. Moreover, the same tendency was found within the endocardial and epicardial layer of the respective parts of the heart (Jedeikin, 1964). In the upper part of the septum (base) of the beef heart, a higher content of iron and copper and a greater succinate-cytochrome c reductase activity has been found compared with the lower part of the septum (apex). Because most of the iron is present in the mitochondrial fraction and incorporated into the myoglobin, its presence is indicative of the oxidative function of a myocardial region. Thus, there seems to be a decreasing oxidative metabolic gradient from the base to the apex of the septum (Tota, 1973). This finding agrees quite well with the presence of the skeletal muscle type isoenzyme of lactate dehydrogenase, a specific indicator of anaerobic metabolism in the apex of the heart (Basile and Tota, 1971). Furthermore, as already indicated, the glycogen content was found to be higher in the apex (Jedeikin, 1964).

Subepicardium-Subendocardium Pronounced transmural gradients, that is, differences between the subepicardial and subendocardial layers of the heart have been described. First, there is morphological evidence for this. It has been shown that the sarcomere length in diastole is greater in the subendocardium than in the middle wall of the canine right ventricle (Morady et al., 1973). This is also true when one looks at the sarcomere length's pressure relationships. For a given left ventricular pressure, sarcomeres are greater in the inner than in the outer layer (Spotnitz et al., 1966). There is also a greater degree of subendocardial shortening in systole in dogs. The subendocardial portion of the ventricular wall accounted for 83% of the total systolic change of wall thickness, whereas the subepicardial portion accounted for only 17%. The subendocardial segment showed 18% shortening, whereas the subepicardial segment showed 10% shortening as measured with ultrasonic dimension gauges (Sabbah et al., 1981). This indicates greater work performed in the subendocardial region. It is, therefore, not surprising that there is a significantly lower oxygen tension in the deep myocardium (10 + 1.8 mm Hg) than in the superficial myocardium (18 + 2.3 mm Hg) of the dog heart (Moss, 1968). When oxygen consumption was measured with a micro-Fick method, it was higher in the subendocardial layer of the dog heart and reached statistical significance in the left ventricle (Weiss et al., 1978). In

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addition to that, coronary blood flow was at least 25% lower in the deep compared with the superficial regions of the heart (Kirk and Honig, 1964). This also contributes to the higher risk to which the subendocardial layer is subjected. This is particularly evident during ischemia and the development of myocardial necrosis. When the circumflex coronary artery is ligated, necrosis extends from the subendocardial to the subepicardial region until it has reached the extent of the area at risk. This is the so-called "wavefront phenomenon" (Reimer and Jennings, 1979). The greater susceptibility of the subendocardium to ischemia is also evident from the metabolic changes that occur. When the circumflex coronary artery was ligated in dogs, there was an ATP decline in the posterior papillary muscle that showed transmural differences. In control animals subepicardial ATP was already higher than midmyocardial and subendocardial ATP. The ischemia-induced ATP decline was more pronounced in the subendocardial layer than in the subepicardium. A similar pattern was observed with creatine phosphate. Here, too, the lowest concentration in the normal heart was in the subendocardial layer, and it declined most markedly there during ischemia (Allison et al., 1977). When coronary blood flow was stopped for 30 seconds, a significant lactate gradient, increasing from the outer to the inner region, was present (Dunn and Griggs, 1975). These results suggest that the contracting ventricle uses energy unevenly and that in myocardial ischemia one of the factors that causes greater subendocardial vulnerability is a greater energy need in this region. Transmural gradients have also been observed with adenosine. Adenosine was trapped with homocysteine and then detected as S-adenosylhomocysteine in dog hearts under controled conditions and during 13-adrenergic stimulation with isoproterenol for 30 minutes. At the end of both the 1. and 3. stimulation, there was an increase in Sadenosylhomocysteine that was more pronounced in the subendocardial layer (Deussen et al., 1991). Transmural gradients exist with respect to cardiac adenine nucleotide and also with respect to carbohydrate metabolism. In the isolated perfused Langendorff rat heart, glucose uptake is greater in the subendocardial layer of the left ventricle. Addition of 0.7 mM oleate induced a decline in glucose uptake, but the subendocardial/subepicardial ratio did not change. Perfusion with 5 or 15 mM lactate also reduced glucose uptake but had no influence on the transmural gradient. Glucose uptake was a little lower in the fight ventricle. In the fight ventricle glucose uptake was also reduced by oleate and lactate (Takala et al., 1984). When mechanical work of the isolated rat heart was eliminated by potassium arrest, glucose uptake was much lower and the transmural gradient had dissappeared. Thus, uptake of external glucose is faster in the subendocardial myocardium than in the superficial layer. Because this gradient was abolished during cardiac arrest, it can be explained by the higher rate of mechanical work due to the greater systolic wall stress in the subendocardial layers (Takala and Hassinen, 1981). There is also a transmural gradient for glycogen in the left and fight ventricle and in the septum. The highest content is in the subendocardium, and the lowest is in the subepicardium. When rats were killed by decapitation, glycogen was lower than in

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anesthetized animals. Ether and sodium pentobarbital anesthesia tended to attenuate the glycogen gradient. (Jedeikin, 1964). Total phosphorylase and phosphorylase a activity also exhibited a transmural gradient. The activities were higher in the subendocardial region. Furthermore it has been shown that there is higher activity of several glycolytic enzymes, such as lactate dehydrogenase, in the inner layer of the dog heart. The activity of glucose-6-phosphate dehydrogenase, the first and regulating enzyme of the oxidative pentose phosphate pathway, was also higher in the inner layer than in the outer layer of the dog heart (Lundsgaard-Hansen et al., 1967). On the other hand, it has been shown that there is higher iron and copper content and greater succinate-cytochrome c reductase activity in the subepicardial layers of both ventricles of the beef heart (Tota, 1973). Taken together, these data indicate that the glycolytic capacity is greater in the subendocardium than in the subepicardium. Conversely, there is a decreasing oxidative metabolic gradient from the epicardium to the endocardium in the free ventricular walls. Interestingly, a transmural gradient has also been found for potassium with a higher content in the subendocardium of the cow heart (Tom, 1973). However, under control conditions, no transmural gradient exists for calcium influx in the isolated, perfused rat heart. During the calcium paradox, a steep gradient was observed with the highest values in the subepicardial layer and the lowest in the subendocardium (Leipiilii et al., 1989). This suggests that the calcium paradox induced myocardial injury is unevenly distributed across the left ventricular wall.

Role of Endocardium in Regulating Heart Function Functional nonuniformity of the heart has also been illustrated in investigations of the effect of the endocardial endothelium on cardiac function. The endocardium is the innermost structure of the heart consisting of a monolayer of endothelial cells. Because all blood circulating through the heart has intimate contact with these endothelial cells of the endocardium, an intracavitary autoregulation of cardiac performance has been suggested (Brutsaert and Andries, 1992). During fetal development, endocardial endothelial cells are the first nonmuscle elements entering the inner spongy myocardial layer, thus lining all intertrabecular spaces during trabeculation and possibly functioning as primitive nutrient vessels before the establishment of a true coronary circulation. Interaction between the endocardium and myocardium is also required to form cushion tissue during the development of the atrioventricular and semilunar valves (Brutsaert and Andries, 1992). In functional terms it has been demonstrated in isolated cardiac muscle that selective destruction of endocardial endothelium induced an abbreviation of isometric twitch duration and an earlier decline in isometric force during relaxation. No significant changes in the initial speed of shortening were observed. Conversely, the presence of an intact endocardial endothelium increased the contractile state of subjacent myocardium by prolonging the isometric twitch with a concomitant increase

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in peak twitch. This effect resembled increased isometric twitch performance induced by increasing initial muscle length. In the in vivo dog heart, transient suppression of endocardial endothelium induced premature pressure fall during relaxation (Brutsaert and Andries, 1992). It has been speculated that endocardial endothelium releases two opposing substances, a positive inotropic factor (myocardium contracting factor, MCF) that prolongs contraction possibly by increasing the sensitivity of the myofilaments to intracellular Ca 2§ and an agent that induces premature relaxation of the myocardium (myocardium relaxing factor, MRF). Under normal conditions, the presence of a functional endocardial endotheliuin delays the onset of the isometric tension decline. Removal of the endocardial endothelium irreversibly abolishes this effect. One may therefore expect that the MRF is overridden by the MCE Because the endocardial endothelium is continuously exposed to superfusing blood, one can imagine that it senses, transmits, and participates in regional and global adjustments of the subjacent myocardium to variations in blood homeostasis. Thus, there could well be an intracavitary autoregulation of cardiac function which may be partly dependent and partly independent of endocardial endothelium. It has been shown that endocardial endothelium participates in or modifies the inotropic response to ANF, Ctl-agonists, serotonin, vasopressin, isoproterenol, acetylcholine, endothelin, and angiotensin (Brutsaert and Andries, 1992).

Conduction System-Working Myocardium The heart has the intrinsic ability to initiate its own beat (automaticity) and to beat regularly (rhythmicity). The heart continues to beat even when it is totally removed from the body provided the coronary arteries are artifically perfused. The sinoatrial node, the pacemaker of the heart, and the conduction system are responsible for these remarkable properties of the heart. According to its specialized function, this system has characteristic features that differ from those of the ordinary working myocardium. For instance, the sinoatrial node contains three types of myocardial cells: (1) typical nodal cells which are in the center of the node and appear to be empty due to the poor development of the contractile apparatus. (Only 20% of the total cell volume is occupied by myofilaments which are not properly organized.) (2) transitional cells which surround the typical nodal cells, and (3) atrial cells in which more than 50% of the total cell volume consists of well-organized myofibrils. A special feature of these cells is that they contain granules in the perinuclear region (Bouman and Jongsma, 1986). The nucleotide content of the specialized conduction system of the ox heart has been shown to be less than that of the working cardiac muscle (Davies et al., 1947). The glycogen concentration in the conduction system is higher than in the rest of the myocardium. Therefore, the transmural glycogen gradient with its highest content in the subendocardium is probably related to its distribution in the conduction system.

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Furthermore, it has been shown that the activities of enzymes of both glycolysis and oxidative phosphorylation are lower in the conduction system compared with the working myocardium of calf hearts. When one calculates the ratio of oxidative/glycolytic energy supply, it indicates a shift to glycolysis. Because there are fewer mitochondria in the conduction system, the content of cytochrome c and a ,"re also lower. However, enzymes of the pentose phosphate pathway, such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, have higher activity. This has to be taken into consideration when changes in the activity of these enzymes occur as they do subsequent to catecholamine stimulation of the heart. It was thus concluded that the energy production and the energy demand of the conduction system is less than that in the working myocardium (Ktibler et al. 1969).

Left-Right Heart The most marked nonuniformity of the myocardium is the division into two separate units, the left and the right heart. The two ventricular chambers differ markedly in functional and morphological terms so that this creates an extreme asymmetry ("sidedness") of the heart. At the level of the atria, it must be remembered that the pacemaker, the sinoatrial node, is located in the right atrium. The left ventricle has been studied extensively, but there is much less information available about the right ventricle, in particular in small laboratory animals. This was due mainly to the fact that it was not possible until recently to obtain reliable hemodynamic measurements. It was therefore necessary to develop a fast, easy-to-use, and reliable method to measure basic functional parameters in this heart chamber. Due to major progress in miniaturization techniques, new ultraminiature pressure catheter transducers have been developed which can now be applied in the intact, anesthetized rat. Figure 4 shows that the functions of the left and right heart are routinely measured in closed-chest anesthetized rats with Millar ultraminiature catheter pressure transducers (Figure 4, upper panel). The straight catheter is used for left heart catheterization and the bent catheter for fight heart catheterization. These are three French catheters, and hence the catheter tip has an outside diameter of 0.9 mm. The left heart catheter is inserted into the right carotid artery and then advanced upstream in the aorta into the left ventricle. The bent catheter is inserted into the fight jugular vein and is placed in the fight ventricle (Figure 4, middle panel). The catheterizations are done successively. At the bottom of Figure 4 there are original recordings of heart rate, dp/dtma x, and pressure in the two heart chambers. As it should be, dp/dtma x and systolic pressure are much lower in the right heart than in the left heart. These functional differences are paralleled by the morphological characteristics. The right ventricle has a much thinner wall which is wrapped around the left ventricle. This morphological difference, the "sidedness" of the heart, disappears when the pulmonary vascular resistance becomes elevated.

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Figure 4. Millar ultraminiature catheter pressure transducers (top) for catheterization ofthe right (model PR-291, left-hand side)and left heart (model PR-249, right-hand side) in rats. Position ofthe catheters in the heart chambers duringthe measurements (middle) and original records of right and left heart function (bottom).

A convenient place to study this extreme adaptation of the fight ventricle has been Peru, in particular Morococha, located about 100 miles inland from Lima at an altitude of 14,800 feet, about 4500 m. The upper graph in Figure 5 shows the location of Morococha in relation to Lima at sea level, and the bottom graph shows the pO 2 gradient in the tracheal and alveolar air, in arterial, mean capillary, and mixed venous blood in people living in Lima (solid line) and in Morococha (broken line). All values were lower in the Morococha residents. In arterial blood the difference was about 50 mm Hg (Hurtado, 1964).

Cardiac Adenine Nudeotide Metabolism

99

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Figure 5. Location of Lima and Morococha in Peru (upper panel) and pO 2values in the tracheal and alveolar air and in arterial, mean capillary, and mixed venous blood in people living in Lima and Morococha. Modified after Hurtado, A., 1964.

In this context it is appropriate to refer to earlier experimental studies which have shown that hypoxia brought about by having cats respire a gas mixture of 10.5% 02 in nitrogen induces an immediate elevation of pulmonary arterial pressure (von Euler and Liljestrand, 1946). This hypoxic vasoconstriction is a unique feature of the pulmonary vessels. In contrast, the resistance vessels in the peripheral systemic circulation respond to hypoxia by dilating. It is therefore not surprising that a long-term increase in pulmonary arterial pressure in man has been observed at high altitude. Figure 6 shows the respective data in people living at different altitudes. Pulmonary arterial pressure was normal in persons living in Lima at sea level. When people from Lima had lived as temporlary residents (TR) for a year in Morococha, at 4500 m altitude, pulmonary arterial pressure was elevated. It was even higher in people living permanently at this altitude as native residents (NR). If they had developed chronic mountain sickness (CMS),

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Mean pulmonary arterial pressure in people living at sea level at high altitude. TR: temporary residents living at high altitude for one year; NR: Native residents living at high altitude; CMS: native residents at high altitude with chronic mountain sickness. Modified after Rotta et al., 1956.

pulmonary arterial pressure was further increased (Rotta et al., 1956). Such increased pulmonary pressure should have an effect on the right heart. In fact, the "sidedness" of the heart that normally develops after birth has been shown at autopsy to be absent in children living at high altitude (Arias-Stella and Recavarren, 1962). There is pronounced muscularization of the peripheral arterial lung vessels (Arias-Stella and Saldana, 1963). In rats hypoxia also induces an immediate constriction of the pulmonary vessels resulting in an increase in pulmonary arterial pressure and in right ventricular pressure. Hypoxia was induced by exposing female Sprague-Dawley rats to a hypoxic gas mixture ( 13.3% 02, 0.7% CO 2, 86% N2). This mixture was made by mixing carbogen with nitrogen. The functional parameters of the right ventricle were measured at the end of five minutes of exposure to the hypoxic gas mixture (Figure 7). The fight ventricular systolic pressure increased from a control value of 31 + 1.1 to 55 + 2.1 mm Hg. This experimental model was used to test the effects of several pharmacological interventions. Rats were exposed to two successive five-minute

Cardiac Adenine Nudeotide Metabolism

101

hypoxic periods separated by a normoxic interval of 60 minutes during which the animals received an intravenous infusion of 0.9% NaC1 or of several drugs. Hypoxia caused a marked rise in right ventricular systolic pressure. The functional response to the second hypoxic period did not differ from the first when only 0.9% NaC1 was infused. All calcium antagonists tested, for example, verapamil (Figure 8), reduced the hypoxic pressure increase in a dose-dependent manner and ultimately abolished it (Zierhut and Zimmer, 1989). There are several more experimental examples which demonstrate that the right heart is specifically involved. When 3,3',5-triiodo-L-thyronine (T3) was subcutaneously administered in a single daily dose of 0.2 mg/kg for three days, the percent increase in systolic pressure (Figure 9) and in ventricular weight (Figure 10) was greater in the right than in the left ventricle (Zierhut and Zimmer, 1989). When ,.,.-..

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Effect of verapamil on the hypoxia-induced increase in right ventricular systr~lic pressure (RVSP). The RVSP increase by hypoxia is taken as the control and expressed as 1OO%. Verapamil reduced the hypoxia-induced elevation in RVSP in a dose-dependent manner. Mean values +SEM; number of experiments in parentheses.

102

HEINZ-GERD ZIMMER

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Figure 10. Effect of T 3 treatment for three days on right (RVW) and left ventricular weight (LVW) in rats. Mean values +SEM; number of experiments in parentheses. T3-treatment was extended over longer periods of time, fight ventricular hypertrophy was also predominant (Van Liere et a1.,1969; Gerdes et al., 1985). This can be explained by the fact that the right ventricle has to handle a pressure overload in addition to volume overload. This also agrees with the finding that in chronic hyperthyroidism the right venticle shows fibrosis and focal necrosis (Gerdes et al., 1985). These typical morphological signs of pressure overload were not observed in the left ventricle.

Cardiac Adenine Nucleotide Metabolism

103

An elevation in pulmonary artery pressure can also be created in male BrownNorway rats by a single bilateral thorax irradiation with X-rays (Zimmer et al., 1988). This leads to vascular inflammation with intimal proliferative lesions and fibromuscular hypertrophy. Ultimately, there is fibrous accumulation around pulmonary blood vessels, obliterative intimal and medial thickening, and narrowing of the vessel lumen. In this experimental model, the right ventricular systolic pressure was elevated stepwise with time subsequent to lung irradiation (Figure 11). The right ventricular weight and the fight ventricular weight/heart weight ratio were also increased in a time-dependent sequence (Figure 12). Thus, selective increases in pulmonary vascular resistance induced pronounced right-heart hypertrophy (Zimmer et al., 1988). Another model in which the fight ventricle plays a dominant role is experimental myocardial infarction in rats induced by ligation of the left anterior descending (LAD) coronary artery. In some of the infarcted hearts the ischemic area is of such a magnitude that it extends over almost the entire left ventricular free wall four weeks subsequent to LAD coronary artery ligation (Zimmer et al., 1990). In this situation the nonischemic heart has to compensate metabolically and functionally for the loss of viable myocardium. The unaffected myocardium therefore develops hypertrophy. There was a severe reduction in the left ventricular systolic pressure developed and a drastic elevation of left ventricular end-diastolic pressure. On the other hand, right ventricular systolic pressure developed had doubled. From the data presented in Figure 13 it is evident that the pressure developed in the fight ventricle is of the same magnitude as the pressure generated by the left ventricle. Thus, the difference that normally exists between pulmonary and peripheral circulation and, thus, the "sidedness" of the heart had been eliminated almost completely. Parallel to the RVSP (mmHg) B0

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Figure 11. Effect of a single bilateral thorax irradiation on right ventricular systolic pressure (RVSP) in Brown-Norway rats after different periods of time. Data are mean values +SEM, number of experiments in parentheses.

104

HEINZ-GERD ZIMMER

pressure changes there was an increase in the fight ventricular weight/body weight ratio so that the right ventricle was even heavier than the left ventricle (Figure 13). Thus, the right ventricular free wall was the part of the infarcted heart that had deRVW x 100

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Figure 12. Changes in right ventricular weight (RVW) and in the ratio of the right ventricular weight to heart weight (RVW/HW* 100) after 40 and 45 days subsequent to a single bilateral thorax irradiation in Brown-Norway rats. Mean values+SEM; number of experiments in parentheses. RIGHT VENTRICLE LEFT VENTRIC'LE ' . uJ

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Figure 13. Changes in pressure developed in the right and left ventricle in sham-operated controls (open bars) and in rats with large myocardial infarctions (hatched bars) four weeks after coronary artery ligation (upper panel). The bottom panel shows the weights of the right and left ventricle related to the respective body weights. Data are mean values+SEM; number of experiments in parentheses.

Cardiac Adenine Nucleotide Metabolism LV

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Figure 14. Effect of norepinephrine (NE) administered intravenously in rats for three days at three concentrations (50,100, and 200 l~g/kg/h) on right (RV) and left ventricular (LV) pressure. Data are means+SEM.

veloped the greatest degree of hypertrophy. This was due to a pronounced increase in pulmonary vascular resistance which was histologically evident as a marked thickening of the tunica media (Zimmer et al., 1990). The right heart is also a specific target for catecholamines. Figure 14 shows right and left ventricular systolic pressures when norepinephrine was applied as a continuous intravenous infusion for three days. Norepinephrine had differential effects on the right and left ventricular pressure. With increasing doses right ventricular systolic pressure became elevated, whereas left ventricular systolic pressure was first moderately increased and then even declined. Thus, norepinephrine obviously has a much greater positive inotropic effect on the right ventricle (Irlbeck et al., 1996).

PATHOPHYSIOLOGICAL ASPECTS OF MYOCARDIAL ISCHEMIA Myocardial ischemia is a very common human pathophysiological condition. Ischemic heart disease is one of the leading causes of death in the Western world. The effects of myocardial ischemia have turned out to be more complex than previ-

106

HEINZ-GERD ZIMMER

ously thought. Only two distinct consequences of myocardial ischemia were recognized initially: the first, following a prolonged period of severe ischemia, was irreversible ischemic injury, clinically presented as myocardial infarction. The second, following a brief ischemic episode, was prompt and total recovery of myocardial function, that is, reversible ischemic injury, clinically presented as angina pectoris. However, several new features have been observed in recent years. Stunning One of these new observations is myocardial stunning. The term "stunned myocardium," derived from experimental studies (Braunwald and Kloner, 1982), has been defined as regional contractile dysfunction (Heyndrickx et al., 1975). Regional dysfunction had already been observed in 1935 by Tennant and Wiggers in that the myocardium in the distribution of the occluded coronary artery changed from a state of active shortening during systole to one of passive lengthening ("bulging"). It was then found that when the ischemic insult was relatively brief, usually up to 15 minutes, regional dysfunction persisted for a long period of time, hours and days, despite complete restoration of coronary blood flow and absence of myocardial necrosis. The decrease in the cardiac ATP and adenine nucleotide pool induced by a brief ischemic period also lasted for days both in the dog (Reimer et al., 1981) and rat heart (Zimmer and Ibel, 1984). Attempts have been made to delineate this mechanism responsible for myocardial stunning, but a single cause has not yet been established. Initially, it had been thought that abnormalities in energy metabolism might be involved (DeBoer et al., 1980). In fact, the restoration of ATP in the previously ischemic myocardium has been shown to be accelerated by ribose, but it was not possible in the rat in vivo to obtain data on regional wall motion to determine whether the metabolic normalization is associated with functional improvement (Zimmer and Ibel, 1984). The view that the impairment of function in the postischemic stunned myocardium is not due to depletion of ATP and purine precursors and a lack of high-energy phosphate reserves has been challenged by the finding that a normal inotropic response could be obtained with postextrasystolic potentiation and with epinephrine. This also suggested that the contractile machinery is intact in the stunned myocardium (Becker et al., 1986). It was further hypothesized that an increase in cellular Ca 2§ loading may cause dysfunction in the form of myocardial stunning (Marban et al., 1989). Sarcoplasmic reticulum isolated from stunned myocardium of the dog showed a 17% reduction in oxalate-supported 45C2§transport compared with the sarcoplasmic reticulum from normal myocardium. There was also a 20% decrease in the maximal activation by Ca 2§of the sarcoplasmic reticulum Ca2§ and downward shift in the Ca 2* activation curve of the Ca2§247 (Krause et al., 1989). These results indicated that myocardial stunning is associated with damage of the calcium transport system of the sarcoplasmic reticulum with resulting slower removal of

Cardiac Adenine Nucleotide Metabolism

107

calcium from the myofibrils. In fact, it has been shown that the peak systolic intracellular calcium concentration is higher in the stunned myocardium than in the control, isolated, perfused ferret heart. Furthermore, the slope of the relationship between developed pressure and Ca 2§ transient amplitude was significantly lower in the stunned myocardium, suggesting that contractile failure in the stunned myocardium is due to a decrease in the myofilament sensitivity to Ca 2§ and to the decrease in maximal Ca2§ force. The increase in the amplitude of Ca 2§ transients would require more ATP to be spent in Ca 2§ sequestration by the sarcoplasmic reticulum. This could explain the reported inefficiency of energy utilization in the stunned myocardium (Kusuoka et al., 1990). Oxygen consumption is much higher in the stunned mycocardium than in the control when normalized for the degree of force generation (Stahl et al., 1988). Interestingly, pretreatment with the calcium antagonist verapamil attenuated the severity of stunning in the dog heart both with respect to segment shortening and endocardial ATP stores. Segment shortening which was only 31 _ 8% of the normal baseline value after 3 hours of reperfusion, which followed a period of 15 minutes of coronary artery occlusion, was restored to 115 +_8% of normal with verapamil, and ATP stores were partially preserved. Even at and after reperfusion, verapamil blunted postischemic contractile dysfunction (Przyklenk and Kloner, 1988). All of these results support the view that calcium overload is involved in the stunning process. Apart from disturbances in calcium homeostasis there is another plausible explanation for myocardial stunning, and that is injury induced by free radicals. As will be recalled, invading polymorphonuclear leukocytes are a likely source of oxygen fleeradical production. Leukocytes are large, stiff, viscous cells which easily adhere to vascular endothelium. They are trapped in myocardial capillaries beginning at the time of coronary occlusion. Further delivery and accumulation by collateral flow during myocardial ischemia results in leukocyte capillary plugging which is the major mechanism in the "no-reflow" phenomenon (Kloner et al., 1974). Reperfusion of previously ischemic dog myocardium with whole blood resulted in occlusion of 27% of capillaries in the endocardium, whereas reperfusion in leukocyte-depleted animals was nearly complete with only 1% of capillaries occluded. Furthermore, leukocyte depletion prevented the increase in tissue water content and decreased the incidence of ventricular arrhythmias (Engler et al., 1986). That oxygen free radicals may be partly responsible for some features characteristic of the stunnned myocardium can be deduced from the fact that oxygen freeradical scavengers have beneficial effects in the stunned myocardium. The combination of superoxide dismutase and catalase markedly enhanced the recovery of myocardial segment shortening in the postischemic reperfused subendocardium of the dog heart (Gross et al., 1986). As shown in Figure 3, superoxide dismutase catalyzes the conversion of the superoxide anion to H202, and catalase catalyzes the conversion of H202 to water. A similar beneficial effect of superoxide dismutase in combination with catalase has been confirmed. However, the improvement in con-

108

HEINZ-GERD ZIMMER

tractile function in the treated dog group was not accompanied by increased ATP stores in the previously ischemic zone (Przyklenk and Kloner, 1986). It is probable that multiple mechanisms contribute to the pathogenesis of myocardial stunning (Bolli, 1990). The possible causes outlined above are not mutually exclusive and may represent different steps in the same pathophysiological sequence. It is, however, important to realize that the stunning process is not evenly distributed throughout the heart. After reperfusion following 15 minutes of-LAD coronary artea-y occlusion, functional recovery was delayed in the inner compared to the mid and outer myocardial layers of the dog heart. Under control conditions, the percent thickening fraction was significantly greater in the inner layer than in the mid and outer layers. During LAD occlusion all layers exhibited comparable degrees of paradoxical systolic thinning. After two, three, and four hours of reperfusion, the percent thickening fraction was always lower in the endocardium. Even after 24 hours of reperfusion this parameter was still depressed in the inner layer. So there was a maldistribution of thickening in the stunned myocardium with a slower recovery of function in the inner myocardial layer (Bolli et al., 1989). Myocardial stunning may not be confined to experimental cardiology. In fact, there are numerous clinical conditions in which the myocardium is subjected to transient ischemia, such as unstable or variant angina, acute myocardial infarction with early reperfusion that may Occur either spontaneously or by thrombolytic therapy. Moreover, open-heart surgery with cardioplegic arrest and heart transplantation may also involve myocardial stunning.

Ischemic Preconditioning The concept of ischemic preconditiong was also developed on the basis of experimental studies. It was observed when four occlusions of the circumflex coronary artery of five min duration in the dog were separated by five min of reperfusion (ischemic preconditioning) followed by a sustained 40 min occlusion. The control group underwent only a 40 min occlusion. The animals were then allowed four days ofreperfusion. Ischemic preconditioning limited infarct size to 25% of that seen in the control group. Transmural mean collateral flow was the same in the preconditioned and control hearts. Thus, despite the fact that the preconditioned hearts had undergone 60 min of cumulative ischemia compared with 40 min in the controls, they had much smaller infarcts (Murry et al., 1986). When multiple periods of brief ischemia of 10 minutes duration were applied, the first ischemic period induced a loss of 61% of ATP and 41% loss of adenine nucleotides from the most severely ischemic subendocardial zone. However, two or even four 10 minute periods of ischemia caused no further adenine nucleotide loss. Thus the heart tolerated additional ischemic periods better than the first (Reimer et al., 1986). In the human heart, it has been demonstrated that there is also adaptation to ischemia in patients undergoing percutaneous transluminal coronary angioplasty

Cardiac Adenine Nucleotide Metabolism

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(PTCA). During the second 90 sec coronary arterial occlusion, there was significantly less myocardial lactate production. The fractional lactate extraction during the first inflation was lower than that during the second inflation. In addition, the second inflation was characterized by less subjective anginal discomfort, less ST segment shift, and lower mean pulmonary arterial pressure (Deutsch et al., 1990). That adenosine is involved in the process of ischemic preconditioning is demonstrated in experiments on rabbits. Preconditioning was accomplished by an occlusion of the left coronary artery for five min followed by 10 min of reperfusion prior to 30 min of sustained ischemia. Infarct size was then measured after three hours of reperfusion. Myocardial infarct size, normalized as a percentage of the area at risk, averaged 39% in the control group. Ischemic preconditioning caused infarcts to be much smaller from the same ischemic insult, only 8%. The protection afforded by ischemic preconditioning disappeared when the adenosine receptor blockers SPT and PD 115,199 were given before preconditioning. The blockers themselves had no effect on infarct size in nonpreconditioned animals when compared with controis. Five min of intravenous infusion of adenosine did not affect infarct size. Infarct size in this group was not different from that in the controls (Liu et al., 1991). In isolated rabbit hearts preconditioning also caused infarct size to be much smaller than in controls. A five min infusion of adenosine or phenylisopropyladenosine (PIA) directly into the perfusate to the isolated hearts was as protective as a five min preconditioning ischemic period to limit infarct size. Obviously, the effect of adenosine must be directly on the heart (Liu et al., 1991). In additional in vivo studies, the adenosine A~ receptor agonist PIA was given intravenously for five min instead of the five-min ischemic preconditioning period. All PIA doses reduced infarct size, but the data suggest that only the high dose of the drug resulted in consistent protection (Thornton et al., 1992). There are two points that have to be emphasized. The first is the time at which PIA is given. It has to be well before the sustained ischemic period. The second point is that infarct size in these studies was measured after three hours of reperfusion. It may well be that adenosine or the adenosine A 1 receptor agonist PIA may only cause a delay in the evolution of infarct and not prevention. Additional time studies should therefore be done to solve this problem. Anyhow, adenosine can have cardioprotective effects by stimulating the adenosine A~ receptor. This seems to be significant for the process of ischemic preconditioning.

Hibernation Another feature of myocardial ischemia that has been described in recent years is hibernation. The term "hibernating myocardium" has been developed on the basis of clinical observations (Braunwald and Rutherford, 1986; Rahimtoola, 1989). It was defined as persistent myocardial dysfunction at rest for months or years due to a reduced coronary blood flow with viable myocardium. The dysfunction can be partially or completely relieved if the myocardial oxygen supply/demand relation-

110

HEINZ-GERD ZIMMER

ship is favorably altered by coronary bypass surgery or by PTCA. Thus, the hibernating response of the heart, that is a reduction of function to cope with reduced coronary blood flow, is an act of self preservation: little blood, little work. The hibernating myocardium has therefore been considered a smart heart. A clinical example illustrates this point. Coronary arteriography in a patient showed that the LAD coronary artery was totally occluded. The distal LAD was filled by collaterals from the circumflex coronary atery and the posterior descending coronary artery. In the preoperative control, the ejection fraction was 37%, and there was a large akinetic area involving the anterioapical wall. After nitroglycerin, there was an improvement in wall motion of the akinetic zone, and the ejection fraction improved to 51%. The patient had no history of myocardial infarction. After eight months following a coronary bypass operation, the graft was patent and there was good filling of the LAD. The patient now showed normal left ventricular wall motion and an ejection fraction of 76%. Thus, the underperfused myocardium was akinetic not because of irreversible ischemia. It had been hibernating but was viable because it resumed normal contraction after coronary blood flow was restored (Rahimtoola, 1989). There are a few experimental studies in intact hearts with prolonged regional ischemia which show the ability of the heart to downregulate its function. In instrumented dogs, chronic ischemia of moderate severity was maintained for five hours by partial circumflex coronary artery stenosis followed by reperfusion. A persistent impairment in wall thickening occurred that normalized over a week with minimal evidence of necrosis (Matsuzaki et al., 1983). In another study using the pig model, reduction in coronary blood flow was maintained for three hours with a decrease in wall thickening and in regional myocardial oxygen consumption. The metabolic indexes of myocardial ischemia improved over time (Fedele et al., 1988). Although hibernation is an interesting phenomenon, there are many questions that remain unanswered at the present time. Chief is among these the mechanism responsible for this remarkable feature which continues to be a challenge for the experimental cardiologist. It is noteworthy that studies on isolated perfused ferret hearts in which perfusion pressure was lowered from 80 to 60 mm Hg suggested that a decrease in Ca 2§ transients underlies the contractile dysfunction of myocardial hibernation (Marban, 1991).

CATECHOLAMINES AND MYOCARDIAL METABOLISM The sympathetic nervous system with norepinephrine as the major transmitter was and is essential for human survival. From an evolutionary point of view it was important to develop mechanisms to enable the fight and flight reactions through stimulation of the cardiovascular system. The positive chronotropic and inotropic effects brought about by sympathetic nervous stimulation were essential in this regard. However, overstimulation of the sympathetic nervous system can also have

Cardiac Adenine Nucleotide Metabolism

111

adverse effects. This is evidenced by the occurrence of focal myocardial cell lesions which have been induced by excessive doses of isoproterenol (Rona et al., 1959; Fleckenstein, 1984). Furthermore, plasma catecholamines are increased in patients with congestive heart failure (Chidsey et al., 1965). Human failing left ventricles had a 50-56% reduction in fl-receptor density, a 45% reduction in maximal isoproterenol-mediated adenylyl cyclase stimulation and a 54-73% reduction in maximal isoproterenol-stimulated muscle contraction (Bristow et al., 1982). 13-Adrenergic blockers were shown to have beneficial effects in congestive cardiomyopathy (Swedberg et al., 1980) thus confirming the cardiotoxic potential of catecholamines. In addition to catecholamines and the down-regulation of myocardial 13-adrenergic receptors, the renin-angiotensin system seems also to play an important role in the pathophysiology of heart failure, in particular, in the elevation of systemic vascular resistance (Curtiss et al., 1978).

Effects at the Metabolic and Molecular Biological Level Norepinephrine affects both ~t- and 13-receptors, resulting in many effects on the heart and circulation (Figure 15). It is well known that 13-receptor stimulation leads to the stimulation of adenylyl cyclase activity and to the elevation of cAMP (Suthefland et al., 1968). Activation of cAMP-dependent protein kinase A (PKA) induces known metabolic effects, such as the increase in lipolysis and glycogenolysis. Furthermore, via phosphorylation of a channel and sarcoplasmic protein (phospholamban), it induces the stimulation of transsarcolemmal and sarcoplasmic Ca 2§transport and thus

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Figure 15. Schematic representation of the cardiac effects of norepinephrine. For details see text.

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HEINZ-GERD ZIMMER

the inotropic and lusitropic effects (Tada and Katz, 1982). Stimulation of oc-adrenergic receptors is associated with elevation of the second messengers inositol trisphosphat (IP3) and diacylglycerol (Berridge and Irvine, 1984; Nishizuka, 1988). IP 3 liberates Ca 2§ ions from the SR and thus induces a positive inotropic effect (Schmitz et al., 1987; Kohl et al., 1989). Diacylglycerol stimulates protein kinase C (PKC). PKA and PKC can both influence, either directly or indirectly, the expression of proto-oncogenes (Coughlin et al., 1985; Tratner et al., 1992; Gauthier-Rouviere et al., 1992). In neonatal rat myocyte cell cultures, it was shown that cz-adrenergic stimulation induced an increase in the mRNA of c-fos, c-jun, c-myc, and the early growth response gene- 1, egr- 1 (Starksen et al., 1986; Iwaki et al., 1990). It has also been demonstrated that stimulation of both oc- and 13-adrenergic receptors increases the steady-state level of c-fos mRNA in the heart of mice, rats, and Syrian hamsters (Barka et al., 1987). In the isolated perfused working rat heart, norepinephrine induced a transient increase in c-fos and c-myc mRNA expression (Zimmer, 1997). Thus, there is an activation of the immediate early gene program. This may be involved in promoting directly or via transcriptional factors the reexpression of the fetal program of genes coding for the main contractile proteins. These are the skeletal isoform of cz-actin (Izumo et al., 1988) and the 13-myosin heavy chain, V 3(Waspe et al., 1990). Also myosin-light chain-2 increases (Lee et al., 1988). These changes are associated with cardiac hypertrophy.

Influence on the Pentose Phosphate Pathway The oxidative pentose phosphate pathway (PPP) is the link between carbohydrate and nucleotide metabolism (Figure 2). Glucose-6-phosphate (G-6-P) originating from glycogenolysis or from glucose taken up by the myocardial cell is metabolized predominantly via glycolysis. A small portion of G-6-P, however, enters the oxidative PPP of which glucose-6-phosphate dehydrogenase (G-6-PD) is the first and rate-limiting enzyme. This pathway serves mainly two functions: (1) It provides reducing equivalents in the form of NADPH which can be used for the synthesis of free fatty acids and for the reduction of oxidized glutathione (GSSG). This is important for detoxification processes such as the conversion of hydrogen peroxide to water. (2) In this pathway, ribose-5-phosphate is generated which can be transformed to 5-phosphoribosyl-1pyrophosphate (PRPP). This is an essential precursor for the de novo purine nucleotide synthesis, for the salvage of adenine and hypoxanthine to build up AMP and IMP, and for the formation of pyrimidine nucleotides such as UTP from orotic acid. There are connections between this pathway and glycolysis at two levels. This pathway is poorly developed in the heart. It is therefore interesting to examine whether it can be enhanced in intact rats so that more PRPP is provided for nucleotide metabolism. One possibility is to bypass the critical step in the pathway, the G-6-PD reaction. This can be done with ribose. Ribose elevates the available pool of PRPP and stimulates the rate of adenine nucleotide biosynthesis in the rat heart. This enhancement is of such a magnitude that the decline of ATP in many patho-

Cardiac Adenine Nudeotide Metabolism

113

physiological conditions is attenuated or even entirely prevented by ribose (Zimmer et al., 1980; Zimmer, 1983; Zimmer et al., 1984). Another approach is to stimulate G-6-PD, the first and regulating enzyme. This can be achieved by all catecholamines tested so far. There was a dose-dependent stimulation of G-6-PD activity in the rat heart measured after 48 hours of continuous intravenous infusion of norepinephrine. In addition, when the highest dose was given, there was also a marked time dependency. Because norepinphrine stimulates both J3- and a-adrenergic receptors it was of interest to examine the relative contribution of these receptors to the enhancement of G-6-PD activity. The norepinephrine-induced stimulation could be partially antagonized by the 13-receptor blocker metoprolol and by the a-receptor blocker prazosin. When both a- and 13-blockers were combined, the norepinephrine-elicited increase in cardiac G-6-PD activity was abolished almost completely. These results show that stimulation of both 13- and a-receptors leads to an increase in cardiac G-6-PD activity. This norepinephrine-induced stimulation occurred within the cardiac myocyte compartment (Zimmer et al., 1992). Norepinephrine also has an effect at the mRNA level. Figure 16 shows that there was a parCONTROL

NOREPINEPHRINE (0,2 mg/kgh) 72h

-6-PD ~RNA 300" % 200"

(6) 1(=0"

)-6-PD

CTIVITY

16)

~

5-

-

119) ~,

Figure 16. Bar graphs showing mRNA levels and activities of glucose-6-phosphate dehydrogenase (G-6-PD) in hearts of control rats and after continuous intravenous infusion of norepinephrine for 24, 48 and 72 hours. Values are mean + SEM; the number of experiments is in parentheses.

114

HEINZoGERD ZIMMER

j

Catecholamines

Heart rate t Contractility t

G - 6 - PD mRNA f G - 6 - PD Activity t

Oxygen consumption t

PRPP-poolt

Figure 17. Simplified diagram showing the homeostatic mechanism by which catecholamines counteract the possible metabolic injury (decrease in ATP) they may inflict on the heart. allel increase in both the mRNA and activity of cardiac G-6-PD under the influence of norepinephrine. The mRNA increase was prevented by carvedilol, a combined ix- and 13-adrenergic blocker. Norepinephrine also had a moderate effect on the mRNA of 6-PGD. But this increase was not affected by carvedilol. It must therefore be regarded as unspecific. The increase in the mRNA and activity of G-6-PD is a new metabolic effect which all catecholamines, tested so far, have (Zimmer et al., 1990). These effects seem to be part of a long-term homeostatic mechanism. A New Homeostatic Mechanism

Catecholamines increase heart rate and contractility and thereby lead to an elevation of oxygen consumption. This tends to decrease the level of ATP in the cardiac myocyte. At the same time, catecholamines stimulate the mRNA and activity of G-6-PD and thereby expand the pool of the available PRPP which is an important precursor for purine and pyrimidine nucleotide synthetic processes (Figure 2). The ultimate effect is an increase in ATP synthesis which contributes to keeping the adenine nucleotide content of the heart in the normal range (Figure 17). Thus catecholamines repair the possible metabolic damage they inflict on the heart by this adaptational process. The ultimate effect is the maintenance of a normal level of cardiac adenine nucleotides.

METABOLIC CONCEPTS FOR TREATING HEART DISEASE The pathways of degradation and synthesis of ATP are the basis for any concept aimed at metabolic intervention to improve energy metabolism and ultimately the function of the heart. During ischemia ATP is catabolized to ADP, and the latter to

Cardiac Adenine Nucleotide Metabolism

115

AMP. Further degradation of AMP may proceed either via IMP or adenosine to inosine, hypoxanthine, xanthine, and uric acid (Figure 2, bottom). As to ATP synthesis, there are two possibilities: de novo purine synthesis from low molecular precursors or utilization of adenine nucleotide degradation products, such as adenosine, inosine, adenine, and hypoxanthine via the "salvage" pathways. Although adenosine is directly converted to AMP by adenosine kinase, adenine and hypoxanthine need PRPP for the formation of AMP and IMP, respectively. Metabolic intervention in these pathways can be made by preventing or slowing down the loss of purine nucleotide degradation products, by providing purine nucleosides or purine bases, or by stimulating the synthetic processes.

Adenosine Many attempts have been made to supply adenosine for preservation or restitution of the cardiac ATP level. In the normal rabbit heart adenosine increased the ATP content considerably. The most pronounced increase in ATP and total adenine nucleotides was observed with a 1% adenosine solution. The increase amounted to about 30%. With the 1% adenosine solution the maximum ATP level was already reached after three hours. After five hours, the 0.5% solution was as effective as the 1% solution (Isselhard et al., 1980). Adenosine also had marked effects on heart function. Isolated rat hearts were perfused at constant flow and subjected to global normothermic ischemia. The time to onset of ischemic contracture was measured as a marker of myocardial ischemic injury. Adenosine (100 ~tM) extended the time to the onset ofischemic contracture by about 100%. The A 1adenosine receptor agonist phenylisopropyladenosine (PIA, 1 laM) also had a similar effect. The A 2adenosine receptor agonist phenylaminoadenosine (PAA, 1 laM) had no effect at all. The addition of the adenosine receptor blocker BW A1433U (10 ktM) attenuated or abolished the effect of adenosine and PIA and, when given alone, shortened the time to the onset of ischemic contracture. Thus, this effect is specific to adenosine A 1 (Lasley et al., 1990). Adenosine had beneficial effects during ischemia and also during postischemic reperfusion. The percent radial shortening was measured in the ischemic zone of dog hearts at the baseline, one hour into occlusion and three hours after reperfusion. Both groups of dogs developed similar degrees of dyskinesis in the ischemic zone at one hour after occlusion. Three hours after reperfusion, animals treated with intracoronary adenosine at 3.75 mg/min had positive wall motion, whereas control animals showed continued dyskinesis. These beneficial changes correlated with improved regional myocardial blood flow and reduced neutrophil infiltration (Babbitt et al., 1989). A serious consequence of ischemia is cardiac cell death. About 15 to 20 min of ischemia are tolerated by the dog myocardium. When coronary artery occlusion is released after this period of time, there is complete recovery. However, when ischemia lasts for more than 20 min, cell death occurs which increases linearly with time.

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After 90 min of proximal LAD occlusion, 20 dogs were randomized for blood reperfusion with or without adenosine into the proximal LAD at 3.75 mg/min for 60 min after reperfusion. At 24 hours the area at risk was similar in both groups. However, infarct size was significantly reduced in adenosine-treated animals when expressed as the percentage of the area at risk and as the percentage of the left ventricle. In addition, there was an improvement in regional ventricular function in the previously ischemic area (Olafsson et al., 1987). Apart from providing adenosine directly to the heart, one can manipulate its transport or catabolism. Dipyridamole influences adenosine transport and EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, 10 mg/kg) inhibits adenosine deaminase. Dogs on cardiopulmonary bypass had the aorta cross-clamped for 20 min at normothermia, and 30 min later cardiopulmonary bypass was discontinued. Ischemia induced an ATP fall. Infusion of adenosine or EHNA alone had no effect under these experimental conditions. However, when EHNA was combined with adenosine, the ATP fall during ischemia was attenuated and recovered much better during the postischemic period (Foker et al., 1980). Adenosine accumulation during ATP catabolism can also be achieved with 5amino-4-imidazole carboxamide (AICA) riboside. At 5 and 55 minutes of ischemia, endocardial blood flow to ischemic canine myocardium was increased by AICA riboside. In coronary venous blood from ischemic tissue, adenosine was elevated in AICA riboside-treated dogs. Ventricular tachycardia and premature ventricular depolarizations were significantly attenuated by AICA riboside. Granulocyte accumulation was less in the ischemic myocardium in treated animals. So, in addition to its vasodilator action, adenosine may also function as an antiinjury autacoid that links ATP catabolism to inhibition of granulocyte adherence, microvascular obstruction, and superoxide anion formation (Gruber et al., 1989).

Inosine There are fewer studies available on the effects of inosine, although this nucleoside increases during ischemia to tissue levels that are higher than those of adenosine (Gerlach and Deuticke, 1963). The features of inosine, however, are similar to those of adenosine. Like adenosine, inosine has vasodilator properties, although in much higher doses. This has been shown for the coronary vascular bed (Jones et al., 1981; Schneider and Zimmer, 1991) and for peripheral circulation. In the intact rat, there was a marked decrease in total peripheral resistance and in mean arterial pressure. In addition, inosine has a negative inotropic influence in that left ventricular dp/dtma x and cardiac output were diminished. This effect may be species-specific (Seesko and Zimmer, 1990). Inosine antagonized the norepinephrine-induced positive inotropic effect and the increase in cardiac output. In rats, pretreatment with the 13-receptor blocker metoprolol and the calcium antagonist verapamil aggravated the negative inotropic effect (Seesko and Zimmer, 1990).

Cardiac Adenine Nucleotide Metabolism

117

Inosine also has cardioprotective potential (Goldhaber et al., 1982). It is first degraded to hypoxanthine (Wiedmeier et al., 1972) which uses up the available PRPP to build IMP. This can then be converted to AMP, ADP, and ATP. It has been shown that inosine is incorporated via hypoxanthine into mycoardial adenine nucleotides (Harmsen et al., 1984). Inosine slowed the ischemia-induced decrease in ATP and creatine phosphate (Ingwall, 1982)and accelerated the postischemic ATP recovery of Langendorff-perfused (Harmsen et al. 1984) and working rat hearts (Schneider and Zimmer, 1991). It has also been demonstrated that inosine is taken up by the human heart (de Jong et al., 1989). It has been administered in a limited number of patients with shock (Czarnecki et al. 1989).

Ribose Ribose is a pentose sugar that, when exogenously supplied, affects the synthesis of adenine and uridine nucleotides. Via ribose-5-phosphate it specifically elevates the available pool of PRPP in heart and skeletal muscle (Zimmer and Gerlach, 1978). PRPP can then be utilized for the de novo synthesis of ATP and UTP and for the conversion of adenine to AMP and of hypoxanthine to IMP (Figure 2). It has been demonstrated that ribose stimulates adenine nucleotide de novo synthesis to such an extent that the ATP decline in various pathophysiological conditions is attenuated or even prevented. This had a positive effect on global rat heart function. The metabolic effect of ribose has been observed in a variety of experimental models and conditions from the isolated rat myocyte to the in vivo dog preparation. Ribose has also clinical relevance in patients with myoadenylate deaminase deficiency and in McArdle's disease. Furthermore, it facilitates thallium redistribution and enhances the detection of viable myocardium in the diagnosis of coronary artery disease (Zimmer, 1992). Additionally, it has been shown that ribose improves the tolerance of the heart to ischemia in patients with severe LAD coronary artery stenosis. After three days of oral ribose treatment, treadmill walking time until 1 mm of ST-segment depression occurred was significantly greater in 10 patients than in the placebo group also consisting of 10 patients (Pliml et al. 1992). This first clinical result suggests that ribose can effectively influence cardiac energy metabolism as an adjunct therapy for myocardial ischemia. Interesting effects have been observed with ribose combined with adenine and inosine, respectively. In these studies on intact rats, a depression of cardiac ATP was induced by isoproterenol five hours after subcutaneous injection. Adenine and ribose at the concentrations applied in this study did not affect the isoproterenolelicited ATP decline. Inosine attenuated it to so some extent. Adenine (Figure 18) and inosine (Figure 19) combined with ribose entirely abolished the ISO-induced ATP decline after 5 hours of intravenous infusion. Thus, the reduced cardiac ATP content, which results from the degradation of inosine, can be restored very quickly by exploiting the salvage pathways with adenine and hypoxanthine. A prerequisite for this is that the available pool of PRPP be elevated by ribose (Zimmer and

118

HEINZ-GERD ZIMMER CONTROL

ISO

ISO 9 Ribose

.Adenine

.Adenine

Ril~ose

~5" I]J

"6

(10) P~

4-

E

---=3-

191"

171"

r/A .,-/- ,. ....

N N * p < 0.0005 vs Control

Figure 18. Myocardial contents of ATP in control rats and five hours after subcutaneous injection of isoproterenol (ISO, 25 mg/kg) alone and with intravenous infusion of ribose (200 mglkglh) or adenine (50 mglkglh) or a combination of both for five hours. Mean values + SEM; number of experiments in parentheses.

CONTROL

ISO

I SO ,,Ribose

.Inosine

9 Inosine

Ribose

,,

6 --

~Vl 5 -

(121 ~"

181 181"

$

o

4"

"--"

3"

E 0. I---

(101

19)*

< 2"

0 * p< 0.0005 vs Control

Figure 19. Myocardial content of ATP in control rats and five hours after subcutaneous injection of isoproterenol (ISO, 25 mg/kg) alone and with intravenous infusion of ribose (200 mg/kg/h) or inosine (200 mg/kg/h) or a combination of both for five hours. Mean values + SEM; number of experiments in parentheses. Schneider, 1991). This concept should also be applicable to the synthesis of uridine nucleotides, because orotic acid needs PRPP to build up OMP and ultimately UTP (Figure 20).

Cardiac Adenine Nucleotide Metabolism

RIBOSE - - - -

119

R-S-P

l HY

N,H,N

9- A M P

UN~P

ADP

UDP

ATP

UTP

Schematic diagram illustratingthe effect of ribose, adenine, hypoxanthine, and orotic acid on the synthesis of ATP and UTP, respectively.

Figure 20.

SUMMARY In this chapter various components of cardiac adenine nucleotide metabolism have been analyzed and then integrated into the discussion of two clinically relevant pathophysiological conditions, ischemia and catecholamine actions. The most common form of cell injury in the heart is ischemia. The most likely causes for injury, such as depletion of adenine nucleotides, accumulation of glycolytic products, calcium overload, and oxygen free radicals have been described. These distinct mechanisms have been used to characterize and possibly explain three newly recognized features of myocardial ischemia: stunning, ischemic preconditioning, and hibernation. Apart from the well-known functional and metabolic effects of catecholamines, in particular of norepinephrine, on the heart, several new molecular biological findings have been observed such as the increased expression of proto-oncogenes. Another new aspect is the stimulation of the oxidative pentose phosphate pathway. Both the mRNA and activity of G-6-PD, the rate-limiting enzyme of this pathway, are enhanced by norepinephrine in the entire rat heart and in isolated cardiac myocytes. Stimulation of this pathway leads to more reducing equivalents in the form of NADPH and to more ribose-5-phosphate which is the immediate precursor of PRPP. This is important for both the de novo purine synthesis and the salvage pathways. A new homeostatic mechanism has been formulated in that catecholamines reduce the possible metabolic damage they inflict on the heart by action on the oxidative pentose phosphate pathway. On the basis of adenine nucleotide metabolism, several therapeutic interventions have been developed and partially applied in patients. Among these, adeno-

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sine, inosine, and ribose have been described in m o r e detail. A d e n o s i n e and inosine have p r o n o u n c e d h e m o d y n a m i c effects which m a y not be desired in s o m e patients. In contrast, ribose is entirely neutral in functional terms and can be c o m b i n e d with conventional cardiac therapy without interfering with it. It m a y therefore be an appropriate adjunct in treating cardiac diseases.

ACKNOWLEDGMENTS The results of own studies were obtained in investigations supported by the Deutsche Forschungsgemeinschaft (Zi 199/8-2,3, Zi 199/10-1). I would like to thank Sabine D'Avis for her help in preparing this chapter and Heinz Hofmann for the art work.

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