© 2002 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
Does adenosine protect the heart by acting on the sarcoplasmic reticulum?
Centre for Experimental Surgery and Anaesthesiology, University of Leuven, Campus Gasthuisberg, Herestraat 49, Leuven, B-3000 Belgium
kanigula.mubagwa{at}med.kuleuven.ac.be
* Tel.: +32-16-347-132; fax: +32-16-347-139
Received 27 November 2001; accepted 27 November 2001
See article by Zucchi et al. [9] (pages 326–333) in this issue.
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1. Altered ion homeostasis during ischemia: critical role of intracellular Ca2+ overload |
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 Top 1. Altered ion homeostasis... 2. Ischemic preconditioning and...3. Is an altered...4. Does adenosine alter...References |
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Ischemic heart diseases still constitute the leading cause of mortality and morbidity in industrialized countries; hence a great deal of research is devoted at understanding the underlying mechanisms and at finding means to prevent, delay or attenuate ischemic injury. Intracellular ion homeostasis is altered during ischemia, with the concentrations of H+, Na+, Mg2+, Ca2+ and phosphate increasing markedly (see Ref. [1]). This disturbance of cellular homeostasis, especially the intracellular Ca2+ (Cai) overload, is considered to play a key role in the cell damage during ischemia and reperfusion, and procedures that reduce the Cai rise do protect the myocardium by attenuating or delaying ischemic/reperfusion injury. The increased Cai is supposed to cause cell damage by activating excessive ATP utilization as well as lipolytic and proteolytic enzymes. How the Cai overload is brought about is still not fully clear, but it is accepted that both a large cytosolic redistribution of Ca2+ from intracellular stores (especially early during ischemia) and an entry of Ca2+ from the extracellular compartment (especially late during ischemia or during reperfusion) are involved (see Refs. [1,2] and references therein).
Ca2+ is accumulated at high concentration within the sarcoplasmic reticulum (SR), thanks to an active transport by the SR Ca2+ ATPase (SERCA) and to the presence of Ca2+-binding proteins within the SR. Ca2+ release occurs mainly via the ryanodine receptors. IP3 receptors also constitute Ca2+ release channels, but their role in cardiac function remains heretofore unclear. During brief ischemic periods, the ability of the SR to release Ca2+ is largely maintained, with the contractile failure resulting from a desensitization of the myofilaments. However, ischemia causes progressive damage to the SR (see Refs. [3,4]). Maximum rate (Vmax) of Ca2+ uptake and ryanodine binding are both decreased, suggesting changes at the level of both SERCA and the SR Ca2+ releasing channels. Reperfusion following short ischemia results in a recovery of SR function (eventually associated with myocardial stunning), whereas reperfusion following prolonged ischemia is considered to induce further damage to the SR, thus resulting in an exacerbation of the Ca2+ overload and in cell death.
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2. Ischemic preconditioning and the role of adenosine |
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Top1. Altered ion homeostasis...2. Ischemic preconditioning and...3. Is an altered...4. Does adenosine alter...References |
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Preconditioning (PC) the heart by one or many brief periods of ischemia followed by reperfusion [5] has emerged as a powerful method to recruit endogenous mechanisms that attenuate cell death (hence reducing infarct size) and improve the postischemic recovery of mechanical, electrophysiologic and metabolic function. Protection by PC is now recognized to take place in nearly all organs where ischemia can cause substantial cell damage. Since the discovery of PC, intensive research has been carried out to identify the underlying subcellular or molecular mechanisms. A priori, such knowledge should be useful since it could serve as a basis to design cardioprotective measures or pharmacological tools against ischemia. Among the many substances proposed to mediate PC, adenosine (Ado) has emerged as prime candidate. This is not surprising given the well-known increased production of this substance upon metabolic stress as well as its regulatory action on local blood flow and anti-ischemic effects (see Refs. [6,7]). Beside Ado, other mediators that have been proposed include bradykinin, opioids, reactive free radicals, etc. How Ado or these other substances cause protection during ischemia is still unclear (see Refs. [7,8]). Ado possesses many receptor (AdoR) subtypes, nearly all of which are expressed on the surface membrane of cardiac cells. It is now recognized that not only the A1- but also the A3-AdoR subtype can mediate myocardial protection. Overexpression of the A1-AdoR is reported to be associated with increased resistance to ischemia, while having no effect on heart rate and contractility under basal conditions, as also found by the study of Zucchi et al. in the present Journal issue [9]. (However the receptor overexpression is associated with an enhanced bradycardic action of Ado and a lower sensitivity to β-adrenergic stimulation). Hearts from animals overexpressing the A1-AdoR also display a slower ATP depletion and a delay in diastolic contracture during ischemia, as well as an improved recovery of ATP, CrP and pHi upon reperfusion [10]. As far as the A3-AdoR is concerned, conflicting reports on its influence on ischemia have been obtained: whereas pharmacological studies using A3-AdoR-selective agonists and antagonists indicate a protective role mediated by this receptor, a few recent studies using transgenic animals suggest that overexpressing this receptor increases susceptibility to ischemic injury (see Ref. [7]).
The effectors linked to AdoRs are multiple and include enzymes (adenylate cyclase, phospolipase C, NO synthase, etc.) and many transporters and channels (see Ref. [7]). These effectors have been traditionally considered to be located within or near the cell surface membrane. The activation of sarcolemmal ATP-sensitive potassium (KATP) channels is enhanced by Ado, and a role of these channels in mediating PC has been proposed and widely evaluated in many studies [11–13]. More recently, the importance of intracellular effectors as potential mediators of PC and of the Ado cardioprotective action has been emphasized. Thus the activation of mitochondrial KATP channels is now proposed to be more critical than that of sarcolemmal channels in PC [11–13]. However, there is also evidence supporting the contribution of other intracellular effectors, including the SR and cytoskeletal elements.
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3. Is an altered SR function the mechanism underlying protection by ischemic preconditioning? |
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Top1. Altered ion homeostasis...2. Ischemic preconditioning and...3. Is an altered...4. Does adenosine alter...References |
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Given (1) the critical role of Cai overload in the cell death induced by ischemia, (2) the fact that the SR is the major intracellular store for Ca2+, and (3) the fact that an altered SR function has been shown to take place during ischemia and reperfusion, it is surprising that little attention has been paid to a possible role of the SR in PC. Evidence exists that Cai accumulation is attenuated and the SR function better preserved during prolonged ischemia in preconditioned hearts. Rather than being simply a consequence of the better preservation of organelle function in otherwise protected myocardium, the change in SR function is already present after the short preconditioning ischemia (before prolonged ischemia) and is thus a candidate mechanism to account for the decreased Cai accumulation during prolonged ischemia. The few existing studies that have examined an eventual role of the SR in PC have used two major experimental approaches. One approach involved the use of drugs which interact with either SERCA or the ryanodine receptors to test for their potential cardioprotective actions or their influence on ischemic PC. The second approach has involved the direct measurement of SR function (Ca2+ uptake and release) following the brief ischemic periods used for PC. Evidence from the pharmacological approach shows that in isolated hearts, preventing Ca2+ release by ryanodine or ruthenium red, or preliminarily emptying the Ca2+ content of the SR by cyclopiazonic acid or thapsigargin are associated with less myocardial damage during subsequent ischemia (see Refs. [4,14]). Although one study proposed that ischemic PC could still be induced in the absence of a functional SR, its conclusion was based on the questionable assumption that ryanodine or cyclopiazonic acid were able to completely suppress the SR function at the concentrations used [15]. In any case the interpretation of the data obtained using this pharmacological approach remains difficult because of the negative inotropic action exerted by the drugs, which by itself may largely contribute to protection. The direct measurement of SR function shows that SR Ca2+ uptake and release as well as [3H]ryanodine binding are decreased in homogenates and/or microsomal fractions derived from preconditioned hearts before exposure to prolonged ischemia [2,14,16,17]. Zucchi et al. [17] found that the recovery of SR Ca2+ release followed a time course similar to that of the protection by PC. It is therefore plausible that changes in SR function can at least contribute to mechanisms underlying PC.
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4. Does adenosine alter the SR function? |
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Top1. Altered ion homeostasis...2. Ischemic preconditioning and...3. Is an altered...4. Does adenosine alter...References |
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Given the reported role of Ado in mediating PC, one interesting question is whether Ado influences SR function during ischemia, and whether the action of Ado on the SR can eventually account for the cardioprotection by PC. Under normal conditions, Ado is not known to have any direct action on the SR function, except for the effects mediated by its action on sarcolemmal channels (hence on the action potential duration and on the trigger for the Ca2+ release) or on cAMP. However, given the multiple enzyme effectors coupled to AdoRs, among which many are able to modify (e.g., phosphorylate) SR proteins, it is not unlikely that direct effects of Ado on the SR could become apparent under specific physiological or pathological conditions.
In a previous study carried out in rat, Zucchi et al. [18] demonstrated that treatment of isolated hearts with the non-selective Ado-R agonist R-PIA induced a decrease of the rate of Ca2+ release measured in myocardial crude homogenates, a decrease of the saturation binding of [3H]ryanodine, and a decrease of the cell damage upon a subsequent ischemia. The effects were attributed to A3-AdoRs since they were insensitive to A1- or A2-AdoR-selective antagonists, whereas they could be obtained by the A3-AdoR-selective agonist IB-MECA and suppressed by the A3-AdoR-selective antagonist MRS-1191 [14,18]. In that study, it was surprising that the A1-AdoR was not involved, despite the wide consensus on the role of this receptor subtype in cardioprotection. In their present study (appearing in the current Journal issue), Zucchi et al. [9] now tested the hypothesis that A1-AdoRs induce a similar cardioprotective effect mediated by the SR. For this purpose they used transgenic mice overexpressing the A1-AdoR, and measured oxalate supported 45Ca2+ uptake, which in the presence of ryanodine allows a determination of the activity and Ca2+ affinity of SERCA. Using this technique, the basal activity of SR release channels can also be assessed by measuring the ryanodine-sensitive Ca2+ leak [17,19]. The authors also measured [3H]ryanodine binding by techniques identical to those of their previous study in rats. The data in Zucchi et al.'s present study confirm the higher resistance of transgenic mice to ischemia, since diastolic contracture during ischemia is lower and recovery of systolic function higher in these animals. The study also shows that Ca2+ uptake into the SR is decreased in transgenic mice overexpressing the A1-AdoR compared to wild-type mice. The decreased Ca2+ uptake was associated with a decreased Vmax but unchanged KCa, hence suggesting a downregulation of SERCA, or a change in the catalytic turnover of the enzyme. There was no difference in [3H]ryanodine Bmax between transgenic and wild-type mice. The authors conclude that the decreased Ca2+ uptake in transgenic mice results in a less filled Ca2+ store, which will result into less Cai overload and cell injury during ischemia/reperfusion. Therefore these results suggest that the expression of AdoRs is able to influence SR function. However, the study calls for a few remarks and raises new questions. First, the data suggest species-related differences in the coupling between AdoR subtypes and the SR, since A3-AdoRs are proposed to be involved in rat vs. A1-AdoRs in mouse. The lack of A1-AdoR effect on rat SR indicates that mechanisms not related to this organelle are involved in the A1-AdoR-mediated protection in this species [20]. Secondly, the SR changes linked to AdoRs are also different, since changes in SR release channels are proposed to take place in rat vs. SERCA changes in mouse. Thirdly, given the possibility that the overexpression of a given receptor could be associated with nonspecific changes in other proteins, the finding that an increase in AdoR expression is associated with changes in the SR needs to be substantiated by a demonstration that this effect is due to receptor activation (e.g., by showing that it can be reversed by antagonists). Finally, the study does not clarify the mechanisms by which the AdoRs are linked to the SR: how do the receptors talk to the SR, i.e., what second messengers are implicated? Does Ado cause these effects by antagonizing β-adrenergic stimulation? Do the effects involve protein kinase C or other kinases, etc...? Nevertheless the study opens a new avenue in the search for mechanisms underlying Ado effects and short-term myocardial protection by PC. Given the difficulty inherent to every comparison of two different (but small in size) groups of animals, these results need to be confirmed by other laboratories, and by different techniques. For example, it will be interesting to perform measurements of Ca2+ transients and SR Ca2+ content in myocytes dissociated from mice overexpressing AdoRs or treated with Ado agonist.
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