Adenosine and Cardioprotection in the Diseased Heart

SPECIAL ARTICLE Jpn Circ J 1999; 63: 231 243 Adenosine and Cardioprotection in the Diseased Heart Masafumi Kitakaze, MD; Tetsuo Minamino, MD; Koichi Node, MD; Seiji Takashima, MD; Hiroharu Funaya, MD; Tsunehiko Kuzuya, MD; Masatsugu Hori, MD Biological and mechanical stressors such as ischemia, hypoxia, cellular ATP depletion, Ca 2+ overload, free radicals, pressure and volume overload, catecholamines, cytokines, and renin-angiotensin may independently cause reversible and/or irreversible cardiac dysfunction. As a defense against these forms of stress, several endogenous self-protective mechanisms are exerted to avoid cellular injury. Adenosine, a degradative substance of ATP, may act as an endogenous cardioprotective substance in pathophysiological conditions of the heart, such as myocardial ischemia and chronic heart failure. For example, when brief periods of myocardial ischemia precede sustained ischemia, infarct size is markedly limited, a phenomenon known as ischemic preconditioning. We found that ischemic preconditioning activates the enzyme responsible for adenosine release, ie, ecto-5'-nucleotidase. Furthermore, the inhibitor of ecto-5'-nucleotidase reduced the infarct size-limiting effect of ischemic preconditioning, which establishes the cause-effect relationship between activation of ecto-5'-nucleotidase and the infarct size-limiting effect. We also found that protein kinase C is responsible for the activation of ecto-5'- nucleotidase. Protein kinase C phosphorylated the serine and threonine residues of ecto-5'-nucleotidase. Therefore, we suggest that adenosine produced via ecto-5'-nucleotidase gives cardioprotection against ischemia and reperfusion injury. Also, we found that plasma adenosine levels are increased in patients with chronic heart failure. Ecto-5'-nucleotidase activity increased in the blood and the myocardium in patients with chronic heart failure, which may explain the increases in adenosine levels in the plasma and the myocardium. In addition, we found that further elevation of plasma adenosine levels due to either dipyridamole or dilazep reduces the severity of chronic heart failure. Thus, we suggest that endogenous adenosine is also beneficial in chronic heart failure. We propose potential mechanisms for cardioprotection attributable to adenosine in pathophysiological states in heart diseases. The establishment of adenosine therapy may be useful for the treatment of either ischemic heart diseases or chronic heart failure. (Jpn Circ J 1999; 63: 231 243) Key Words: Adenosine; Clinical trial; Ecto-5'-nucleotidase; Heart failure; Infarct size; Proten kinase C Adenosine, produced not only in cardiomyocytes but also in endothelial cells, is known to be cardioprotective via activation of adenosine receptors, 1,2 which results in: (1) attenuation of release of catecholamines, -adrenoceptor-mediated myocardial hypercontraction, and myocardial Ca 2+ overload via adenosine A1 receptors; and (2) increases in coronary blood flow and inhibition of platelet and of leukocyte activation via adenosine A2 receptors. Furthermore, adenosine inhibits both renin and tumor necrosis factor (TNF- ) production in experimental models. 3,4 The various effects of adenosine synergistically inhibit the deleterious results of ischemic heart diseases. In addition, the most powerful cardioprotection has recently been found to be afforded by ischemic preconditioning. When brief periods of ischemia precede sustained ischemia, infarct size is markedly limited. 5 Liu et al 6 experimentally demonstrated that an exposure to 8- sulfophenyltheophylline reduces the infarct size-limiting effect of ischemic preconditioning, and Thornton et al 7 showed that adenosine A1 receptor activation is responsible for the infarct size-limiting effect of ischemic preconditioning. Indeed, it has been reported that adenosine contributes to the attenuation of either infarct size or the severity of myocardial stunning. 8,9 In addition to ischemic disorders, (Received December 28, 1998; accepted December 28, 1998) The First Department of Medicine, Osaka University School of Medicine, Suita, Japan Mailing address: Masafumi Kitakaze, MD, The First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita 565-0871, Japan. E-mail: kitakaze@medone.med.osaka-u.ac.jp another very serious heart disease is heart failure. Chronic heart failure is characterized by a reduction in cardiac performance, and several neurohormonal factors are reported to be activated during and to worsen chronic heart failure; 10 catecholamines, renin-angiotensin, and cytokines are thought to be involved in the pathophysiology of chronic heart failure. 11 13 Indeed, chronic heart failure is effectively treated by -adrenoceptor antagonists and angiotensinconverting enzyme (ACE) inhibitors, 11 13 and these drugs have been proved to be effective in the treatment of chronic heart failure in mass studies. Because adenosine antagonizes the harmful factors that may increase the severity of chronic heart failure, it is interesting and important to examine the role of endogenous and exogenous adenosine in chronic heart failure as well. Therefore, we discuss here the role of adenosine in the pathophysiology of acute myocardial infarction 14 17 and chronic heart failure. 18,19 Adenosine in the Ischemic Heart Although adenosine can directly enter the cardiomyocytes and modulate cellular function as the substrate for the ATP resynthesis, the physiological actions of adenosine are mainly attributable to the activation of adenosine receptors, which are classified into 3 subtypes. 20 Adenosine A1 receptors are responsible for the inhibition of adenylate cyclase activity via activation of Gi proteins, and A2 receptors are responsible for stimulation of this enzyme activity via activation of Gs proteins. 21 A3 receptor activation is

232 KITAKAZE M et al. thought to activate Go or Gq proteins, which may increase phospholipase C activity. Because A1 and A3 receptors are distributed mainly on myocardial cells and A2 receptors are on coronary vascular smooth muscle cells in the heart, 22 adenosine may substantially modulate cardiac function as a whole. The Role of Adenosine in the Regulation of Coronary Blood Flow It has been thought that stimulation of adenosine A2 receptors activates adenylate cyclase in the coronary arteries to produce cyclic adenosine monophosphate (camp) and that it relaxes coronary vascular smooth muscle. 23 However, it is not yet established whether camp mediates adenosine-induced vasodilation. In helical strips of canine coronary arteries, cellular camp levels increase only when the vessels are exposed to high concentrations of adenosine (1 10 3 mol/l), 24 which may not necessarily support the role of camp in the adenosine-induced relaxation of coronary smooth muscle cells. Several studies suggest that low concentrations of adenosine relax vascular smooth muscles primarily by decreasing intracellular Ca 2+ levels, through either reduction of sarcolemmal permeability to Ca 2+ 25 or enhancement of Ca 2+ sequestration. 26 Indeed, adenosineinduced coronary vasodilation is attenuated by glibenclamide, an inhibitor of ATP-sensitive K + (KATP) channels. 27,28 Furthermore, the opening of KATP channels plays a crucial role in coronary vasodilation during hypoxia in isolated guinea pig hearts; glibenclamide inhibits the vasodilation produced by hypoxia. 29 Adenosine produced during decreased intracellular ATP concentration can augment the outward current through KATP channels and cause hyperpolarization of smooth muscles, thereby causing coronary vasodilation. Endothelium is also involved in the vasodilator action of adenosine. 30 The vasodilatory effect of adenosine is attenuated by removal of the endothelium in the isolated canine coronary artery, and this effect is greater when adenosine is applied to the luminal side than when applied to the adventitial side. 31 Furthermore, NO is released by the stimulation of adenosine A1 receptors. 32,33 Indeed, adenosine activates guanylate cyclase and increases the intracellular cyclic guanosine monophosphate (cgmp). 32 These observations are compatible with the concept that endogenous adenosine released from the cardiomyocytes may act on the coronary vascular smooth muscle (A2 receptor mediated) in a different way from the exogenous adenosine acting on the endothelial cell receptors (A1 receptor mediated). Furthermore, we have shown that adenosine is required for maintenance of NO synthase (NOS) activity in endothelial cells; in cultured endothelial cells, adenosine A2 receptor blockade decreased NOS activity. In summary, adenosine-induced coronary vasodilation is attributable to either increases in camp or NO or the opening of KATP channels. The importance of each factor may depend on the pathophysiological condition of the heart. Adenosine Production in the Heart Adenosine is mainly released from the heart when the oxygen supply is insufficient for the oxygen requirement, ie, during ischemia, hypoxia, or enhanced oxygen consumption. 34,35 Conversely, adenosine release is decreased when excess oxygen is supplied by overperfusion. 36 These observations suggest that adenosine plays a crucial role in the local regulation of blood flow in the ischemic heart. Major pathways of adenosine formation are the dephosphorylation of 5'-AMP by 5'-nucleotidase (EC 3.1.3.5) and the hydrolysis of S-adenosylhomocysteine (SAH) by SAH hydrolase (EC 3.3.1.1). 37 During normoxia, a major source of adenosine is SAH, formed from S-adenosylmethionine (SAM) through the transfer of the methyl group to a variety of methyl acceptors. 38 SAH is hydrolyzed by SAH hydrolase to adenosine and homocysteine. Adenosine is phosphorylated by adenosine kinase or deaminated by adenosine deaminase. The rate of adenosine production is reported to be approximately 800 pmol min 1 g 1 in the isolated perfused guinea pig heart, which is very close to the hydrolysis rate of SAH (750 pmol min 1 g 1 ). 39 This result suggests that most of the synthesized adenosine is derived from SAH during normoxia. The basal level of adenosine does not play a major role in the regulation of coronary blood flow (CBF) because the basal CBF does not change during infusion of an adenosine receptor antagonist or of adenosine deaminase. During ischemia or hypoxia, however, the major pathway of adenosine production is shifted to the 5'- AMP pathway 40 because adenosine production is markedly reduced by the inhibitor of ecto-5'-nucleotidase. In perfused heart experiments, a close relationship has been found between tissue levels of adenosine, the rate of release of adenosine into the perfusate and coronary blood flow during hypoxia. 41 The adenosine receptor antagonist theophylline decreases coronary blood flow during hypoperfusion. 42 44 Also, a significantly reduced increase in coronary flow during systemic hypoxia has been observed after intracoronary administration of adenosine deaminase. 45 47 These results indicate that adenosine plays a major role in the regulation of coronary blood flow in the ischemic heart. In liver and polymorphonuclear leukocytes, a decrease in the adenylate energy charge (ATP + 1/2ADP) / (ATP + ADP + AMP) may trigger the activation of cytosolic 5'- nucleotidase and enhance adenosine production. 48 It has been reported that 5'-nucleotidase is present as ecto-5'-nucleotidase bound to the membrane and as cytosolic 5'-nucleotidase in the cytoplasm. 49 Because cytosolic 5'-nucleotidase has a higher Km for AMP than ecto-5'-nucleotidase, ecto-5'-nucleotidase may be a primary source of adenosine derived from 5'-AMP. 50 Also, because extracellular adenine nucleotides derived from endothelium, adrenergic nerves, and erythrocytes can be a substrate of ecto-5'-nucleotidase, 51,52 both cytosolic and ecto-5'-nucleotidase may play an important role in adenosine production. We have previously reported that the 1-adrenoceptor antagonist prazosin markedly reduces the release of adenosine from ischemic myocardium either during hypoperfusion 53 or after coronary microembolization 54 (Fig1); administration of a low dose of prazosin that did not affect basal coronary blood flow reduced coronary blood flow and further exaggerated the ischemic damage, ie, caused an increase in lactate production and a decrease in regional myocardial segment shortening. Because the contribution of -adrenergic activity to the release of adenine nucleotides is also reported in endothelial cells, 55 it is likely that activation of protein kinase C (PKC) by 1-adrenergic stimulation is involved in the production of adenosine in hypoxic hearts. 56,57 We found that ecto-5'-nucleotidase is activated by PKC in rat cardiomyocytes (Fig 2), and ecto-5'-nucleotidase then increases the adenosine levels in these cells. Furthermore, PKC may affect the enzymes responsible for adenosine degradation or for production of AMP. Herman and Feigl 58 also observed that adrenergic receptor blockade reduces

Adenosine and Cardioprotection in the Diseased Heart 233 Fig 1. Adenosine release during coronary hypoperfusion during alpha1- (prazosin, 4 g kg 1 min 1, ic), alpha2- (yohimbine, 9 g kg 1 min 1, ic) with propranolol (0.3 mg/kg, ic), non-selective alpha- (phentolamine, 9 g kg 1 min 1, ic) with propranolol (0.3 mg kg 1 min 1, ic), and beta-adrenoceptor (propranolol, 0.3mg/kg, ic) blockade (ic, intracoronary infusion). Because alpha2-adrenoceptor blockade increases norepinephrine release from the presynaptic nerves and increases myocardial contractility through beta-adrenoceptor stimulation, propranolol was concomitantly infused when phentolamine and yohimbine were infused. Propranolol per se did not modify adenosine production in the ischemic myocardium although it abolished the transient enhancements of adenosine production observed 3 7 min after the onset of coronary hypoperfusion. Administrations of prazosin and phentolamine markedly reduced the release of adenosine during coronary hypoperfusion. 53 Fig 2. The dose response relation between phorbol 12-myristate 13- acetate (PMA) and ecto-5'-nucleotidase activity with and without either GF109203X (an inhibitor of protein kinase C) or cycloheximide (an inhibitor of protein synthesis) in rat cardiomyocytes. Ecto- 5'-nucleotidase activity under control conditions was 6.44±0.89, 5.96±0.78 and 5.81±0.44nmol mg protein 1 min 1 in the PMA, PMA with GF109203X and PMA with cycloheximide groups respectively. 56 adenosine concentration and coronary vasodilation during hypoxia in dogs. The underlying mechanism for this phenomenon may be different from that of adenosine release when -adrenoceptors are stimulated. 59 We have also found that 2-adrenergic activity modifies the vasodilatory action of adenosine (Fig 3); a low dose of the 2-adrenoceptor agonist clonidine enhances adenosineinduced coronary vasodilation, 60 62 and low doses of yohimbine and rauwolscine, 2-adrenoceptor antagonists, reduce the coronary flow response to either endogenous or exogenous adenosine. 59 61 This is consistent with the earlier studies of Nayler et al. 63 They observed that phenoxybenzamine, a non-specific -adrenoceptor antagonist, blocks the vasodilatory action of adenosine in isolated rat and guinea pig hearts. 60 62 Furthermore, the reduction in ischemiainduced myocardial damage by administration of clonidine in coronary hypoperfusion and in coronary microembolization strongly suggests that adenosine plays an important role in the dilation of the coronary arterial bed; clonidine significantly increased coronary blood flow in both of the ischemic models without augmentation of adenosine release. 63 In summary, during ischemia, coronary blood flow is regulated by metabolic and neural mechanisms, ie, adenosine-induced coronary vasodilation and -adrenoceptormediated vasoconstriction. Our findings show that - adrenoceptor stimulation increases both adenosine release and coronary vascular sensitivity to adenosine during ischemia. Other Effects of Adenosine on the Ischemic Heart In the ischemic heart, adenosine-induced coronary vasodilation is beneficial for preserving mechanical and metabolic function during myocardial ischemia. 1,64 However, this is not the only effect of adenosine on the ischemic myocardium. Thromboembolism in small coronary arteries, which is believed to be one of the causes of the noreflow phenomenon of the reperfused myocardium, may increase the severity of acute myocardial infarction. Small coronary microemboli are caused by platelet aggregation, and stimulation of the adenosine A2 receptors has been reported to inhibit the platelet aggregation induced by norepinephrine in vitro. 65,66 We have previously investigated whether endogenous adenosine inhibits thromboembolism secondary to platelet aggregation in in vivo ischemic hearts 67 (Fig4). We further examined the cellular mechanisms of platelet aggregation when adenosine receptors were inhibited. The appearance of P-selectin in the platelets increased because of treatment with 8- sulfophenyltheophylline, and the inhibitor of P-selectin inhibited the platelet aggregation with leukocytes and, thus, with endothelial cells. 68 Thus, endogenous adenosine released in the ischemic myocardium inhibited the activation of platelet P-selectin and inhibited the microemboli in the small coronary vessels. Adenosine also inhibits leukocyte chemotaxis 69 and the production of oxygen-derived free radicals 70 through the

234 KITAKAZE M et al. Fig3. Coronary hyperemia in response to the intracoronary infusion of 3 doses of adenosine during moderate (A) or severe (B) alpha2-adrenoceptor blockade, and moderate (C) or severe (D) alpha2-adrenoceptor stimulation. Moderate and severe alpha2-adrenoceptor blockade was accomplished by the intracoronary infusion of yohimbine (9 and 20 gkg 1 min 1 respectively). Moderate and severe alpha2-adrenoceptor stimulation was produced by the combined intracoronary infusion of norepinephrine (0.03 and 0.3 gkg 1 min 1 ic, respectively) and prazosin (6 g kg 1 min 1 ). Because alpha2-adrenoceptor blockade increases norepinephrine release from the presynaptic nerves and increases myocardial contractility through beta-adrenoceptor stimulation, propranolol was concomitantly infused throughout the study. Propranolol per se did not modify adenosine-induced coronary vasodilation. n, number of measurements. 7 stimulation of adenosine A2 receptors. This decrease in the inflammatory response may also be cardioprotective. 71 Interestingly, the activation of leukocytes decreases ecto- 5'-nucleotidase activity, 72 which may decrease adenosine production and further activate leukocytes. These cyclic variations in leukocyte levels may enhance the injury to ischemic hearts by the release of oxygen-derived free radicals and by adhesion to the endothelial cells causing obstruction of the small coronary arteries. Adenosine also reduces the increase in myocardial contractility, which is induced by -adrenoceptor stimulation. 73 We have shown that this phenomenon actually occurs in the ischemic heart. 74 A reduced increase in myocardial contractility prevents a further increase in the discrepancy between energy supply and demand. This phenomenon appears to be different from adenosine-induced inhibition of norepinephrine release from sympathetic nerve terminals because it was not abolished in hearts denervated with 6-hydroxydopamine. 75 Adenosine-induced inhibition of norepinephrine release may also prevent catecholamineinduced injury caused by excess amounts of norepinephrine. In this sense, norepinephrine release causes 2 opposite effects. The amount of norepinephrine released during ischemia may enhance adenosine production and adenosine-induced coronary vasodilation through 1- and 2- adrenoceptor stimulations. 1,2 However, high norepinephrine concentrations associated with severe prolonged ischemia may mask the adenosine-related cardioprotection. 76 The Role of Adenosine in Reperfusion Injury When ischemic heart muscle is reperfused before irreversible injury occurs, contractility remains impaired for a long period, a phenomenon known as myocardial stunning. We have reported that endogenous and exogenous adenosine reduces myocardial stunning in the canine model via adenosine A1 and A2 receptors. 77,78 Furthermore, the release of norepinephrine during ischemia may modulate the severity of reperfusion injury via adenosine-dependent mechanisms. 78 Administration of prazosin worsened myocardial stunning and methoxamine improved it. The beneficial effects of methoxamine were abolished by administration of 8-phenyltheophylline, and because prazosin decreased adenosine release and methoxamine enhanced it this indicates that enhanced adenosine release due to administration of methoxamine improves myocardial stunning. The high dose of methoxamine that reduces basal coronary blood flow by 10 20% diminished the beneficial effects of the low dose of methoxamine. Thus, we conclude that moderate 1-adrenoceptor activation can reduce the severity of myocardial stunning by increasing adenosine production. However, we should note that a high dose of methoxamine diminishes the beneficial effect because of direct coronary vasoconstriction. It would be of interest to know the mechanisms by which adenosine reduces myocardial stunning. Stimulation of adenosine A1 receptors has been shown to inhibit the adrenoceptor-mediated inotropic response and to inhibit also intracellular Ca 2+ influx. 73,79 However, because propranolol does not affect the severity of myocardial stunning, 78 inhibition of Ca 2+ influx by adenosine 80 appears to modulate myocardial stunning. Indeed, several experiments have shown Ca 2+ overload as featuring prominently in the pathogenesis of myocardial stunning. 81,82 In contrast, stimulation of adenosine A2 receptors augments hyperemia and inhibits the activation of neutrophils and platelets. 65 71 However, when we increased hyperemia with papaverine instead of adenosine, myocardial stunning did not improve, 78 suggesting that the increase in coronary blood flow due to adenosine may not be related to the reduced severity of myocardial stunning. Microcirculatory disturbances in myocardial stunning improve with the administration of adenosine. 83,84 Adenosine-induced inhibition of neutrophil and platelet

Adenosine and Cardioprotection in the Diseased Heart 235 activation may play a role in reducing myocardial stunning, especially as activated neutrophils have been shown to generate oxygen-derived free radicals 71 and the administration of superoxide dismutase diminishes myocardial stunning. Intriguingly, oxygen-derived free radicals have been reported to reduce ecto-5'-nucleotidase activity and adenosine production during ischemia. 85,86 Thus, decreased generation of oxygen-derived free radicals due to adenosine may preserve 5'-nucleotidase activity and the capacity for adenosine production (Fig5). Adenosine may also reduce the irreversible myocardial cell injury after reperfusion in various species of animals; 87 91 intracoronary infusion of adenosine results in a 75% reduction in myocardial infarct size in dogs 87 and diminishes contractile dysfunction in rats. 89 This beneficial effect may be attributed to one or more of the following mechanisms: (1) preservation of ATP; 9,92 (2) inhibition of neutrophil activation; 89 (3) inhibition of platelet aggregation; 67 and/or (4) an increase in coronary blood flow. 83 Most of all, the preservation of myocardial ATP levels seems to be the most important factor in cardioprotection against irreversible myocardial cell injury. When adenosine is administered throughout ischemic and reperfusion periods, a 90- fold increase of ATP synthesis was obtained in the reperfused myocardium. 94 It is known that: (1) adenosine stimulates glycolysis in rat hearts; 95 97 (2) intracoronary infusion of adenosine increases glucose uptake; 98 and (3) dipyridamole enhances glucose uptake accompanied by an increase in myocardial ATP in the newborn lamb. 99 Thus, enhanced glucose metabolism by adenosine may contribute in part to a decrease in the rate of ATP depletion during ischemia. A 90% decrease in ATP co-incidentally develops the irreversible deterioration of the myocardium, 100 leading to the idea that depletion of ATP content in reperfused myocardium may be a critical factor for the process of irreversible injury. However, a decrease in ATP seems to be a concomitant phenomenon in myocardial stunning because the replenishment of ATP does not necessarily restore contractile function. 101,102 Because it is argued that ATP compartmentation may mask the role of ATP depletion as the cause of myocardial dysfunction, 103,104 it is still unclear whether the beneficial effect of adenosine in myocardial stunning is due to the preservation of ATP. Fig4. Photomicrograph of hypoperfused coronary arteries without (upper panel) and with (lower panel) the intracoronary administration of 8-phenyltheophylline during coronary hypoperfusion (38±2 mmhg). 8-Phenyltheophylline is a potent antagonist of intracoronary adenosine receptors and induced thrombosis in the small coronary arteries. Tissue was excised after in situ perfusion fixation for 3 min after the onset of ischemia. The bars in the righthand corner of these figures are 50 m (hematoxylin and eosin). 67 Adenosine and Preconditioning Recently, ischemic preconditioning has received much attention from both basic researchers and clinicians; it was first described by the research group of Jennings. 5 Studies to date have shown that ischemic preconditioning limits infarct size to 10 20% of the risk area in the reperfused ischemic myocardium. 5 7,105 Liu et al 6 have implicated endogenous adenosine in ischemic preconditioning by demonstrating that administration of 8-phenyltheophylline abolishes the beneficial effect of ischemic preconditioning. These investigators hypothesized that ischemic preconditioning occurs via adenosine A1 receptor activation. 7 Adenosine A1 receptor activation activates PKC via activation of phospholipase C, and several investigators found that activation of PKC is transiently observed after ischemic preconditioning. Furthermore, the inhibition of PKC diminishes the infarct size-limiting effect of ischemic preconditioning. As PKC activation and adenosine are involved in ischemic preconditioning, we hypothesized that activation of PKC increases ecto-5'-nucleotidase activity and mediates cardioprotection via the enhancement of adenosine production in ischemic preconditioning. 2 This hypothesis is different from the idea of Downey s group. Downey hypothesized that adenosine activates PKC but our hypothesis was that ischemic preconditioning activates PKC, which phosphorylates ecto-5'-nucleotidase and thereby causes cardioprotection. We believe that Downey s hypothesis is true because many investigators suggest the existence of this pathway, but our question is whether PKC can also activate the adenosine-dependent mechanisms. Some investigators confirm our idea, 106 108 and some investigators have questioned our hypothesis. 109 111 The Cellular Mechanisms of the Infarct Size-Limiting Effect of Ischemic Preconditioning Preconditioning and Ecto-5'-Nucleotidase First of all, we tested whether the ischemic preconditioning procedure increases ecto-5'-nucleotidase activity and adenosine release during reperfusion. 14 This is because (1) our hypothesis is that ischemic preconditioning may make the cardiac adenosine levels increase during sustained

236 KITAKAZE M et al. Fig 5. The staining of the activity of myocardial 5'-nucleotidase before myocardial ischemia (A), and after a 1-min coronary occlusion with (C) and without (B) superoxide dismutase. Ischemia increased the 5'-nucleotidase activity, and superoxide dismutase further increased the 5'-nucleotidase activity. Fig 6. The bar graphs showing ecto- and cytosolic 5'-nucleotidase activity in the control and ischemic preconditioned myocardium before the 40 min coronary occlusion. Both ecto-and cytosolic 5'- nucleotidase activity was augmented by ischemic preconditioning. 14 ischemia, and (2) ecto-5'-nucleotidase is known to be responsible for adenosine production in the ischemic heart. In anesthetized open-chested dogs, after the bypass tube to the left anterior descendent (LAD) coronary artery was occluded 4 times for 5 min, both ecto- and cytosolic 5'- nucleotidase activity increased (Fig 6). Ecto- and cytosolic 5'-nucleotidase activity was defined as 5'-nucleotidase activity in the membrane and cytosolic fractions. 14,15 Adenosine concentration in the coronary venous blood was higher in the ischemic preconditioning group than the untreated control group. Therefore, enhanced adenosine release during ischemia and reperfusion from the preconditioned myocardium may decrease infarct size because exogenously administered adenosine has been proved to decrease infarct size. 9 In contrast, there is a report indicating that increases in adenosine concentration in the interstitial space are not augmented during sustained ischemia in the ischemic preconditioning group. 109 We also observed the differences between the interstitial and the coronary venous adenosine levels during sustained ischemia in the ischemic preconditioning group in our experiments. One possible explanation for this difference is that we measured adenosine concentration in coronary venous blood, and the adenosine level in the coronary venous blood is largely affected by endothelial cells. In turn, the interstitial adenosine levels might be mainly affected by myocardial ecto-5'-nucleotidase. Thus, ischemic preconditioning activates differently ecto-5'-nucleotidase located on the endothelial cells and on the cardiomyocytes. Second, it is possible that even if the adenosine concentration in the microenvironment surrounding ecto-5'- nucleotidase on the myocardial cellular membrane is increased by the activated ecto-5'-nucleotidase, the alteration in interstitial volume determined by myocardial cellular swelling and the rate of washout due to the lymphatic stream may change the interstitial adenosine concentration. In any of these possible situations, the temporal and topical increases in the adenosine concentration surrounding ecto- 5'-nucleotidase may be able to directly activate the adenosine receptors located at the same cellular membrane, which may not contradict Van Wylen s observation. 109 This close juxtaposition may explain how ecto-5'-nucleotidase activates the adenosine receptors. Indeed, the regulatory systems, including the ATP receptors, G proteins, ecto-5'- nucleotidase and KATP channels, are closely linked with each other and are present in a single patch of no more than 1mm 2. 111 Role of the Activation of 5'-Nucleotidase in the Salvage of Myocardial Necrosis in Ischemic Preconditioning To test the cause and effect relationship between activation of ecto-5'-nucleotidase and the infarct size-limiting effect in ischemic preconditioning, we examined whether AOPCP (, -methyleneadenosine 5'-diphosphate) diminished the infarct size-limiting effect of ischemic precondi-

Adenosine and Cardioprotection in the Diseased Heart 237 Fig7. Infarct size in the control group, the ischemic preconditioning group, the AOPCP treatment group, the AOPCP treatment with IP group, the AOPCP pretreatment with IP group, and the AOPCP during reperfusion with IP group. Infarct size markedly decreased in ischemic preconditioning. The infarct size-limiting effect of ischemic preconditioning was completely abolished by administration of AOPCP. Administration of AOPCP during the ischemic preconditioning procedure or during reperfusion after sustained ischemia diminished (p<0.001) infarct size compared with infarct size in the control group. In contrast, infarct size of the AOPCP pretreatment with IP group and the AOPCP during reperfusion with IP group were larger (p<0.01) than that in the ischemic preconditioning group. 15 tioning in dogs. 15 AOPCP is a potent and selective inhibitor of ecto-5'-nucleotidase. We occluded the coronary artery 4 times for 5 min with intracoronary administration of AOPCP. AOPCP was administered into the LAD coronary artery 5 min before the ischemic preconditioning (IP) procedures and was continued for 60min of reperfusion, except for the coronary occlusion period (the AOPCP treatment with IP group). In other dogs, AOPCP was administered into the LAD coronary artery 40 min before ischemia and was continued for 60 min of reperfusion, except for the coronary occlusion period (the AOPCP treatment group). To discriminate between the role of increases in ecto-5'- nucleotidase activity during the ischemic preconditioning procedure (the AOPCP pretreatment with IP group) and the role of its activity during reperfusion (the AOPCP during reperfusion with IP group) on the infarct size-limiting effect, we infused AOPCP only during the ischemic preconditioning procedure or only during reperfusion up to 60 min in the ischemic preconditioned dogs. There were no significant differences in risk area and collateral flow during ischemia between the 6 groups. Fig7 shows the infarct size in the 6 groups. Ischemic preconditioning markedly diminished the infarct size. AOPCP completely abolished the infarct size-limiting effect of ischemic preconditioning. In the AOPCP pretreatment with IP and the AOPCP during reperfusion with IP groups, infarct size was partially diminished compared with the AOPCP treatment with IP group and the AOPCP treatment group. Fig8. Plasma adenosine levels in patients with chronic heart failure. Chronic heart failure was classified by the New York Heart Association functional class. In each class, the plasma adenosine levels were divided by ischemic and non-ischemic heart failure. 18 Infarct size in these 2 groups was smaller than the control and AOPCP groups and larger than that in the ischemic preconditioning group. These results indicate that increased ecto-5'-nucleotidase activity during ischemic preconditioning procedures and during early reperfusion synergistically contributes to the infarct size-limiting effect of ischemic preconditioning. The Role of the Activation of Protein Kinase C via 1-Adrenoceptors in Activation of Ecto-5'-Nucleotidase According to the data of Downey s group, PKC is closely linked to ischemic preconditioning. We have also reported that activation of PKC increases ecto-5'-nucleotidase activity in rat cardiomyocytes. 56,57 Because PKC is activated by ischemic preconditioning, 16 ischemic preconditioning may increase ecto-5'-nucleotidase activity via PKC activation. We observed that activation of ecto-5'-nucleotidase due to ischemic preconditioning is diminished by GF109203X, an inhibitor of PKC, in canine hearts. Furthermore, inhibition of PKC by GF109203X diminished the infarct size-limiting effect of ischemic preconditioning. We have also shown that threonine and serine residues of ecto-5'-nucleotidase are phosphorylated in the preconditioned myocardium. Therefore, we speculate that phosphorylation of ecto-5'-nucleotidase by PKC may change the characteristics of the active site of ecto-5'-nucleotidase or induce a conformational change in the structure of 5'- nucleotidase. The next question was how PKC is activated during ischemic preconditioning. As ischemic preconditioning increases the release of norepinephrine from presynaptic vesicles, we tested the role of 1-adrenoceptor activation in cardioprotection in ischemic preconditioning. 17 In openchested dogs, constant infusion of prazosin (4 g kg 1

238 KITAKAZE M et al. min 1 ) into the LAD coronary artery was performed 5min before ischemic preconditioning and continued throughout the first 60 min of the reperfusion period, except during coronary occlusion (the prazosin with IP group). In the other dogs, prazosin was infused into the LAD coronary artery 40 min before ischemia without ischemic preconditioning and continued for 60 min of reperfusion, except during coronary occlusion (the prazosin group). To test whether 1-adrenoceptor stimulation mimics the infarct size-limiting effect of ischemic preconditioning, we administered methoxamine into the LAD (40 gkg 1 min 1, for 4 cycles of 5 min with 5-min intervals; the methoxamine group). After exposure to methoxamine, 90min of coronary occlusion and 6 h of reperfusion were carried out. In addition, to examine the role of increased ecto-5'-nucleotidase activity as a result of exposure to methoxamine, we concomitantly infused AOPCP 5 min before ischemic preconditioning and continued for 60 min of reperfusion, except during 90 min of coronary occlusion to animals treated with methoxamine (the methoxamine with AOPCP group). In another group, we determined the effect of AOPCP on infarct size (the AOPCP group). In this group, AOPCP was administered 40 min before coronary occlusion and during 1h of reperfusion after 90min of coronary occlusion. Ischemic preconditioning increased both ecto- and cytosolic 5'-nucleotidase activities in the myocardium. Prazosin diminished the increases in ecto- and cytosolic 5'- nucleotidase activities due to ischemic preconditioning, and it completely abolished the infarct size-limiting effect of ischemic preconditioning. Methoxamine increased both ecto- and cytosolic 5'-nucleotidase activities to the levels obtained by ischemic preconditioning, and it reduced infarct size to the level seen with ischemic preconditioning. This observation is consistent with previous studies. It is reported that 1-adrenoceptor activation is closely involved in reducing the severity of ischemia and of reperfusion 2 and in ischemic preconditioning. 17 Therefore, these results may support the hypothesis that 1-adrenoceptor stimulation mediates cardioprotection seen in ischemic preconditioning, which is attributable to activation of ecto-5'-nucleotidase. We also found that the PKC activated by ischemic preconditioning is -PKC in canine hearts. 112 In summary, we hypothesized that the linkage between PKC/ecto-5'-nucleotidase/adenosine production plays a role in mediating the cardioprotection seen in ischemic preconditioning. It is intriguing to know that the backward (PKC adenosine) and forward (adenosine PKC) mechanisms amplify each other and contribute to the cardioprotection afforded by ischemic preconditioning. The next target in the field of ischemic preconditioning was to examine intracellular mechanisms; several investigators have suggested the involvement of MAP kinase and p70 S6 kinase, and sarcolemmal and mitochondrial KATP channels. What is Chronic Heart Failure? Chronic heart failure, the end-state of the diseased heart, is characterized by the reduction of cardiac performance relative to the oxygen demand of the body, however several neurohormonal factors are reported to increase the severity of chronic heart failure. 98 Catecholamine, renin-angiotensin and cytokines are thought to be involved in the pathophysiology of chronic heart failure. 10 13 Indeed, chronic heart failure is effectively treated by -adrenoceptor antagonists and angiotensin-converting enzyme (ACE) inhibitors, 10 12 and these drugs have been proved to be effective in the treatment of chronic heart failure in mass studies. Interestingly, activation of PKC by norepinephrine and by angiotensin II activates ecto-5'-nucleotidase, and cytokines increase transcriptional and protein levels of ecto-5'- nucleotidase; 113 both of which may increase plasma adenosine levels. Adenosine, produced not only in cardiomyocytes but also in endothelial cells, is known to be cardioprotective via adenosine receptors by: 1,2 (1) reduction in the release of catecholamine, -adrenoceptor-mediated myocardial hypercontraction, and Ca 2+ overload via A1 receptors and (2) increases in coronary blood flow and inhibition of platelet and leukocyte activation via A2 receptors. Furthermore, adenosine inhibits renin release and TNF- production in experimental models. 105,106 However, there are no reports on the metabolism of endogenous adenosine in chronic heart failure. The Role of Endogenous Adenosine in the Pathophysiology of Chronic Heart Failure We, therefore, tested the role of adenosine in chronic heart failure. 18 We measured the plasma adenosine levels in 71 patients (mean age 52±2 years, range 25 81). There were 40 patients with chronic heart failure due to ischemic heart diseases, and 31 patients with chronic heart failure due to valvular heart diseases (12 patients with mitral regurgitation and 5 patients with aortic regurgitation) and dilated cardiomyopathy (14 patients). The plasma adenosine levels increased according to NYHA classification (Fig 8). Ejection fractions (EF) assessed by echocardiography or ventriculography were 71±5%, 62±4%, 40±4% and 24±5% in the NYHA classification I IV respectively. However, there was no direct correlation (R=0.193, NS) between EF and the plasma adenosine levels in the patients with chronic heart failure. There were no significant differences in the plasma adenosine levels in the patients with ischemic and non-ischemic (valvular diseases and dilated cardiomyopathy) chronic heart failure (Fig 4). We confirmed in 16 other patients with chronic heart failure (mean age 54±2 years, range 30 68) that the day-to-day differences are not significant (the plasma adenosine levels were 59±6 nmol/l at the first time of the measurement, 53±8 nmol/l at the second time 14 days later, and 63±7 nmol/l at the third time 28 days later). In addition, we measured the plasma adenosine levels at 07.00 h before breakfast (57±6 nmol/l), at 11.00 h (63±9 nmol/l) and 13.00 h (65±8 nmol/l) before and after lunch, 18.00 h (54±9 nmol/l) and 20.00h (67±7nmol/L) before and after dinner, and 22.00h (60±6 nmol/l) just before sleep in 10 other healthy laboratory staff members, and found that the variation of the plasma adenosine levels throughout a day was not significant. There was no correlation between adenosine levels and age in the control subjects. We also measured the plasma norepinephrine levels in 65 patients with chronic heart failure. The plasma norepinephrine levels were also increased according to NYHA classification: NYHA I, 118±15 pg/ml [122±9 pg/ml in patients with ischemic heart failure (n=14), and 115±5 pg/ml in patients with non-ischemic heart failure (n=5)]; NYHA II, 337±26 pg/ml [359±39 pg/ml in patients with ischemic heart failure (n=15), and 316±35 pg/ml in patients with non-ischemic heart failure (n=9)]; NYHA III, 668±44

Adenosine and Cardioprotection in the Diseased Heart 239 Fig9. The changes in plasma adenosine levels and the New York Heart Association functional classification in patients with chronic heart failure treated with dipyridamole for 6 months. 19 pg/ml [672±69 pg/ml in patients with ischemic heart failure (n=8), and 663±56 pg/ml in patients with non-ischemic heart failure (n=11)]; and NYHA IV, 1158±87 pg/ml [1140±174 pg/ml in patients with ischemic heart failure (n=3), and 1173±86 pg/ml in patients with non-ischemic heart failure (n=6); when compared with 84±7 pg/ml in the control subjects, p<0.05]. There was a correlation (R=0.46, p<0.01) between the plasma adenosine and norepinephrine levels in the patients with chronic heart failure. We tested the dependency of the adenosine concentration on drugs for the treatment of chronic heart failure, ie, diuretics, digitalis, -blockers, Ca blockers, isosorbide dinitrate and ACE inhibitors. Plasma adenosine levels were independent of each drug (p=0.98, p=0.24, p=0.65, p=0.15, p=0.59 and p=0.17, respectively, according to multiple regression analysis) used in the present study. These results indicate that plasma adenosine levels increase in patients with chronic heart failure, and that this increase in plasma adenosine level correlates well with the functional classes of the severity of chronic heart failure in NYHA classification. Myocardial ischemia is a potent stimulator of adenosine release from the heart. Although there were no differences in the plasma adenosine levels in the patients with ischemic and non-ischemic chronic heart failure in the present study, myocardial ischemia may contribute to adenosine production in patients with chronic heart failure. This is because even non-ischemic chronic heart failure may be attributable to the latent myocardial ischemia and hypoxia caused by coronary microvascular spasm, myocardial external compressive forces on the small coronary arteries, increased diffusion distances of oxygen as a result of myocardial distention, or the reduced perfusion gradients from the epicardium and endocardium with increased myocardial stress in dilated cardiomyopathy. Because endogenous norepinephrine levels rise with the increasing severity of chronic heart failure and endogenous norepinephrine increases the activity of ecto-5'-nucleotidase, the enzyme responsible for adenosine production, 17,50 the rise in plasma norepinephrine levels may contribute to the increases in adenosine production. Indeed, we observed high levels of plasma norepinephrine, which correlate with the plasma adenosine levels. Interestingly, norepinephrine is believed Fig 10. The changes in ejection fraction and oxygen consumption during the bicycle exercise test in patients with chronic heart failure treated with dipyridamole for 6 months. 18 to worsen chronic heart failure, and adenosine diminishes the cardiovascular effect of norepinephrine. Therefore, adenosine may contribute to negative feedback mechanisms against the progressive loop between norepinephrine and heart failure. New Therapeutic Strategy of Heart Failure With Adenosine We further tested whether elevation of the adenosine levels by dipyridamole or dilazep for 6 months modulated

240 KITAKAZE M et al. Fig 11. The changes in plasma adenosine levels (A), the New York Heart Association functional classification (B), ejection fraction (C) and oxygen consumption during the bicycle exercise test (D) in patients with chronic heart failure treated with dilazep for 6 months. 19 the pathophysiology of chronic heart failure. 19 Twenty-two patients (mean age 58±4 years, range 42 74) attending a specialized chronic heart failure clinic over 6 months and judged as being New York Heart Association (NYHA) function class II or III were examined. The other drugs used for the treatment of chronic heart failure were not altered during the study. There were 5 patients with chronic heart failure as a result of ischemic heart diseases, and 17 patients with valvular heart diseases and dilated cardiomyopathy. These diseases were diagnosed by catheterization examination and myocardial biopsy. Blood was sampled at least 15 min after bed-rest. We administered 300mg/day dipyridamole (n=17) or 300 mg/day dilazep (n=7) for 6 months, and discontinued the drug for another 6 months. We assessed the NYHA classification, EF and maximal oxygen uptake (V O2) using an ergometer before the onset of administration, 3 and 6 months after commencing the trial, and 6 months after withdrawal of the relevant drug. Dipyridamole increased the plasma adenosine levels for 6 months (Fig 9) and improved the severity of chronic heart failure; both EF and V O2 were also increased (Fig 10). Dilazep also increased the plasma adenosine levels and improved the severity of chronic heart failure after 6 months (Fig 11). Heart rate (range from 70±5 to 67±5 min 1 ) did not change, and mean systemic blood pressure (from 94±3 to 89±3 mmhg, p<0.01) slightly decreased because of administration of either dipyridamole or dilazep. The extent of the increases in adenosine levels was independent of each drug (p=0.91, p=0.32, p=0.42, p=0.18, p=0.26 and p=0.19, respectively, according to multiple regression analysis). These results indicate that plasma adenosine levels increase in patients with chronic heart failure, and that this increase in plasma adenosine level diminishes the severity of chronic heart failure. Because adenosine is reported to reduce the effects of the sympathetic nervous system, the renin-angiotensin system and the cytokine system, 6 8 the elevation of adenosine levels may largely contribute to the beneficial treatment of chronic heart failure. Indeed, the present study revealed that the elevation of adenosine levels caused by the 2 different drugs equally improved chronic heart failure. As myocardial ischemia is one of the causes of chronic heart failure, the elevation of adenosine levels may have diminished the severity of myocardial ischemia, stunning or hibernation. This is because even non-ischemic chronic heart failure may be attributable to the latent myocardial ischemia and hypoxia caused by coronary microvascular spasm, myocardial external compressive forces on the small coronary arteries, increased diffusion distances of oxygen as a result of myocardial distention, and the reduced perfusion gradients from the epicardium and endocardium with increased myocardial stress in dilated cardiomyopathy. However, Ca channel blockers, which may induce coronary vasodilation, may not improve chronic heart failure; it is not likely that elevation of adenosine improves chronic heart failure by an improvement in latent myocardial ischemia. Because we have reported that the plasma adenosine levels increase in patients with chronic heart failure, the present study suggests that this increased adenosine level may be compensatory in chronic heart failure. Therefore, elevation of adenosine levels in patients with chronic heart failure may be a novel and effective strategy for the treatment of chronic heart failure. These drugs need to be tested for decreases in mortality and morbidity in a mass study. Future Direction of Investigation of Adenosine in the Heart Because in the ischemic and in the failing heart many factors that deteriorate the heart are activated, it is important to consider inhibiting the various harmful factors. One strategy is to administer corresponding drugs to diminish these factors, eg, superoxide dismutase for oxygen-derived free radicals. However, this is not realistic in the clinical setting because we need to administer many drugs to