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Protection Mechanisms of Adenosine
in Postischemic Dysfunction and
Myocardial Infarction

Ricardo J. Gelpi, MD

Laboratorio de Fisiopatología Cardiovascular, Departamento de Patología,
Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

   We could define the "stunned myocardium" as a mechanical, systolic and diastolic postischemic dysfunction with absence of irreversible injury and with preserved contractile reserve (1).

   This phenomenon was described by Heyndrickx et al in 1975, while working in the laboratory of Dr. Vatner at the University of Harvard (2). These authors observed, in conscious and chronically instrumented dogs, that when provoking a 15 minutes occlusion of the anterior descending coronary artery with subsequent reperfusion, a partial recovery of the contractile state took place that was only completed 24 hours later (Fig. 1). This discovery was denominated "systolic postischemic dysfunction", thus assigning importance to the mechanical alteration of the contractile state.

Fig. 1: modifications in the systolic parietal thickening in conscious dogs subjected to 15 minutes of occlusion of the anterior descending artery, and later reperfusión. (Modified from: Heyndrickx et al. Am J Physiol 234(6): H653-H659; 1978)

   The term "stunned myocardium" was coined in 1982 by Braunwald and Kloner (3). Later works, showed (4) that there is a simultaneous alteration in the diastolic function that is manifested as an early dysfunction with quick recovery of isovolumic relaxation and a late (but persistent) dysfunction in myocardial stiffness.

   Since the ' 80s diverse hypotheses have been proposed to attempt an explanation of the mechanisms involved in the pathogenesis of this entity; however, many aspects are still controversial.

   Currently, the accepted hypotheses are: 1) the generation of free radicals derived from oxygen (1); and 2) alteration of the homeostasis of cellular Ca++ that leads to a cytoplasmic overload of this ion (1). Both theories do not exclude each other, and they probably represent different aspects of the same physiopathologic process.

   Since the early ' 80s some researchers (5) have postulated that the generation of free radicals could be involved, at least partly, in the genesis of the postischemic ventricular dysfunction.

   However, these first reports have not given direct evidence of the participation of free radicals in the postischemic dysfunction. Therefore, to validate this hypothesis it was necessary to demonstrate and quantify the production of free radicals in the "stunned myocardium". Thus, Bolli et al (6) using more specific and more sensitive techniques proved the generation of these agents, particularly during reperfusion. They also showed a direct relationship between the production of free radicals and the degree of decrease in blood flow during ischemia, indicating that the generation of these compounds during reperfusion is directly proportional to the severity of the ischemic precedent.

   Free radicals can attack diverse cellular components, particularly they could denaturalize proteins and inactivate enzymes, as likewise peroxidize polyunsaturated fatty acids of the cellular membranes, altering their permeability (7). Also, it has been demonstrated that free radicals can inhibit the Na+ K+ ATPase pump (8). The inactivity of this pump causes an increase in the intracellular concentration of Na+, with consequent activation of the Na+ Ca++ exchanger and the development of Ca++ overload. On the other hand, the response of myofilaments to Ca++ could be attenuated (9), as product of a selective damage of the free radicals in the thiole groups of these contractile proteins. Finally, in another study (10) the possibility that these compounds alter the function of the sarcoplasmic reticulum is raised. This would be an extremely important mechanism, since it would involve the homeostasis of the Ca++ ion and it could be the connecting point to another studied hypothesis that will be described in the following point.

   The production source of free radicals in the "stunned myocardium" has not been totally elucidated. In dogs, the xanthine-oxidase enzyme seems to be responsible for the generation of free radicals, while the role played by neutrophiles in this species has not been convincingly established. In human beings and in rabbits the xanthine-oxidase concentration is scarce, and thus its relevance in the physiopathogenesis of the "stunned myocardium" is virtually non-existent. However, other sources of generation of free radicals that could be involved are the cascade of arachidonic acid, the autoxidation of catecholamines and other components, and alteration of the transport chain of mitochondrial electrons.

   Summarizing, strong experimental evidence exists (11) regarding the participation of free radicals derived from O2 in the etiopathogenesis of postischemic ventricular dysfunctions, in models that use both regional as well as global ischemia, and also in vivo and in vitro situations.

   Some researchers (12) have postulated that the physiopathologic mechanism of the postischemic dysfunction would be an alteration in the cellular homeostasis of Ca++.

   This hypothesis includes three possible mechanisms that involve the mentioned ion: 1) a decrease in the response of the contractile apparatus to Ca++ (13), 2) an overload of Ca++ (14), and 3) an alteration at level of the sarcoplasmic reticulum with disconnection of the excitation-contraction process (15).

   The intrinsic mechanism by which the response of myofilaments to Ca++ would be altered has not yet been fully described. Studies carried out on isolated ventricular trabeculae (16) have shown a decrease of the force developed in response to Ca++, and lesser sensitivity of the contractile apparatus to this ion. These findings are extremely important since they could be the connecting point to the hypothesis that involve to the free radicals derived from the O2. Free radicals, as has been mentioned, would alter the myofilaments response to Ca++ by oxidizing the thiole groups of myofibrillar proteins; thus, the structural modification of these proteins could influence the sensitivity to Ca++. However, it has not been possible to demonstrate an alteration of myofilaments in all the models of "myocardial stunning"; also, this hypothesis does not explain the positive response to the inotropics that act through Ca++.

   On the other hand, it is well-known that during the early phase of reperfusion there exists an overload of Ca++ that could contribute to the pathogenesis of the stunned myocardium. The mechanisms by which Ca++ would produce a prolonged ventricular dysfunction have not been completely clarified, but it is recognized that Ca++ can activate enzymes, among them the phospholipases that affect intracellular organelles (17). Also, the calcic overload can stimulate the formation of free radicals through xanthine oxidase (18).

   A third postulated mechanism linked to the homeostasis of Ca++ is the inadequate liberation of this cation from the sarcoplasmic reticulum toward the cytoplasm (10), and would allow to explain why the exogenous reinstatement of Ca++ and inotropics increase the contraction force of the stunned myocardium, but this hypothesis is not compatible with a study (19) carried out in vitro where instead of decreasing, the transit of Ca++ toward the cytosol was normal or had increased.

   Finally, and as previously mentioned, the increase of the concentration of Ca++ in the cytosol causes the activation of diverse proteins (phospholipases, proteases, etc.). During the last few years it has been determined that some of the mentioned proteins belong to the group of the calpaines. These enzymes have the capacity to cleave other proteins when the intracellular concentration of Ca++ is high (17). The calpain II causes lesions of the myofilaments, but the specific substrate of this enzyme is still unknown. On the other hand, experimental evidence exists (18, 19) that diverse contractile proteins, among which are included troponin T, troponin C, tropomyosin and myosin, are all sensitive to the action of calpaines. Thus, calpaines could be those responsible for the damage of myofilaments during myocardial stunning; however, the activation of these enzymes during the postischemic dysfunction has still not been demonstrated and also their specific substrates should be identified.

   Summarizing, the stunned myocardium is a multifactorial process that involves a complex sequence of cellular alterations which pathogenesis has still not been totally elucidated, since the theories until here developed do not totally explain the cascade of events that culminate in the postischemic ventricular dysfunction. Also, it is necessary to emphasize that, as mentioned previously the hypotheses that involve both free radicals and Ca++ ions do not mutually exclude each other, and they could represent different facets of the same physiopathologic process. In connection with this, some authors (1) mention the possibility that the damage produced by free radicals at level of the sarcoplasmic reticulum is related to an alteration in the homeostasis of Ca++, and that these actions could cause a disconnection of the excitation-contraction response and a cytosolic overload of Ca++. Similarly, free radicals could damage the contractile proteins thus reducing the response to Ca++.

   Some experimental works have shown that the postischemic ventricular dysfunction can be attenuated by carrying out pharmacological interventions prior to the period of ischemia. However, it is of supreme relevance, for a potential therapeutic application, to find this protection and to know the involved mechanisms when procedures are applied during early reperfusion.

   Thus, Olafsson et al. (20) and Pitarys et al. (21) demonstrated that intracoronary and intravenous administration of adenosine, respectively, significantly reduces the infarct size after 90 minutes of regional ischemia in dogs. Although the canine model has been frequently used to study the effects of diverse pharmacological interventions on the ischemic heart, it presents great variability in the infarct size, due fundamentally to the presence of an important collateral circulation. Therefore, the effectiveness of any therapeutic intervention, in this model, is difficult to prove and to interpret.

   Later works (22, 23) were carried out in experimental models using a different species that presents great similarity with the human heart, such as rabbits. This similarity is evidenced in what concerns to low collateral circulation and deficit of the xanthine-oxidase enzyme. These studies demonstrated that the administration of adenosine, an agonist of receiving A1, and A1 receptors, reduces the infarct size after 30 minutes of coronary artery occlusion. However, these investigations were carried out in models of regional ischemia and did not evaluate the ventricular function.

   On the contrary, Goto et al. (24), working with a model of instrumented rabbit, and Vander Heide et al., using conscious dogs, (25) were unable to prove a decrease in the infarct size.

   Finally, some authors (26) have suggested the possibility that adenosine improves the systolic function by an increase of the coronary flow, by acting on the vascular A1 receptors (Gregg's phenomenon) and that, in a similar way to Frank-Starling's law, conditioning the length of myocardial fiber by increasing the parietal vascular volume and, in consequence, the length of the surrounding myocites ("internal pre-load").

   To our knowledge, only one work (27) showed an improvement of the contractile state and attenuation of the increase of diastolic stiffness during reperfusion, in a model of isolated and isovolumic rabbit heart, administering the adenosine before, during and after the period of ischemia. However, when the intervention was only carried out during reperfusion the protection achieved was not significant. Also, they used a prolonged period of ischemia (60 minutes) because of which it is valid to think that the adenosine acted reducing the infarct size (through the preconditioning phenomenon?) and, in this way, it indirectly improved the ventricular function.

   In works carried out at our laboratory (28, 29), the administration of exogenous adenosine since the beginning of reperfusion attenuated the systolic alterations and the increase of the diastolic stiffness present after 15 minutes of global ischemia (Fig. 2). This protective effect was mediated by the activation of A1 receptors, since it could be abolished by the administration of DPCPX (a selective blocker of A1 receptors) and independent of modifications in the infarct size (Fig. 3).


Fig. 2: The left ventricle developed pressure (LVDP) (UPPER PANEL) and the left ventricle end diastolic pressure (LVEDP) (lower panel), in the control group, in the group treated with adenosine and in the group to which both adenosine and DPCPX (A1 blocker) were administered. It can be observed that adenosine attenuated the systolic alterations of the postischemic dysfunction. This effect was abolished with the administration of DPCPX. Also, adenosine attenuated the increase of diastolic stiffness. *: p <0.05 vs control.

Fig. 3: Infarct size, expressed as a percentage of the area of the left ventricle, in control group, in the group treated with adenosine and in the group to which both adenosine and DPCPX (A1 blocker) were administered. *: p < 0.05 vs control

   With this period of ischemia the infarct size is not very significant and the functional damage is totally reversible. Only two works (30, 31) used short periods of ischemia and evaluated the effects of adenosine in the reperfusion. These works used a different species, the dog, in an experimental model of open thorax, that is to say, hearts that developed external work and that, as has already been mentioned, hinder evaluation of the ventricular function. Furthermore, they were unable to find beneficial actions when administering the drug during reperfusion.

   We have shown experimental evidence demonstrating that adenosine, through the stimulation of A1 receptors, protects the heart from the postischemic systolic dysfunction and from the increase in diastolic stiffness. The stimulation of A1 receptors unleashes diverse metabolic changes that could attenuate the postischemic dysfunction.

   During early reperfusion numerous compounds are released, which could contribute to the presence of injury by way of reperfusion (32); among these substances the endothelines should be highlighted. These compounds possess the most potent vasoconstrictor effect known at present, and they would participate, during reperfusion, by causing a significant deterioration of the vasodilating response and reducing the blood flow in the previously ischemic bed (32). Velasco et al. (33) have described that adenosine can reduce the release of endothelines during early reperfusion, thus improving the ventricular function. The mechanism by which adenosine would reduce the formation of endothelines remains unknown. On the other hand, the powerful vasodilating attributes of adenosine could not only revert the vasoconstriction produced by the endothelines, but rather they would also reduce the contraction of the vascular smooth muscle when reducing the entrance of Ca++ to this muscle through the slow Ca++ channels (33).

   It is well-known that during reperfusion there is an overload of Ca++ (mechanism postulated for myocardial stunning) that would provoke a cellular lesion when activating phospholipases and proteases that would accelerate the degradation of ATP. Also, the increase in the concentration of Ca++ stimulates the production of free radicals through the xanthine-oxidase enzyme, facilitating the development of cellular injury. Adenosine, by activation of A1 receptors, could reduce the entrance of Ca++ through the sarcolemma when inhibiting the adenyl cyclase enzyme. This produces a decrease in the levels of cyclic AMP and in the activity of the protein kinase (PKA) and, as consequence, the phosphorylation of the slow Ca++ channels of the cytoplasm does not take place, thus reducing the influx of Ca++ toward the cytosol (30, 34). Although this possible protection mechanism has not been studied in detail, the present investigation demonstrates that the initial step of this specific mechanism is present, by having shown that protection during reperfusion requires activation of the A1 receptors (34).

   Acknowledgement: we wish to thank Dr. Martin Donato for his important assistance in the preparation of this conference.


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35. Peart J, Headrick J. Intrinsic A1 adenosine receptor activation during ischemia or reperfusion improves recovery in mouse hearts. Am J Physiol 279: H2166-H2175; 2000



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