Mechanisms of Cardioprotection:
Ischemic Postconditioning

	Ricardo J. Gelpi Institute of Cardiovascular Pathophysiology, Faculty of Medicine, University of Buenos Aires, Argentine; Member of the National Council of Scientific Research of Argentina, Buenos Aire, Argentina.
	Martín Donato Institute of Cardiovascular Pathophysiology, Faculty of Medicine, University of Buenos Aires, Argentine

Introduction
Ischemic preconditioning was described for the first time by Murry et al [1] in 1986. Since the pioneering studies by Downey´s group [2,3], much has been found concerning its cellular mechanism. We can be certain that surface receptors [3], mitochondrial K + ATP [4], free radicals [5] and protein kinase C [6] all play pivotal roles in the signaling pathways. However, preconditioning must be applied before an ischemic event to be protective, thus limiting its clinical utility. Therefore, an intervention is needed that, when applied during reperfusion, can attenuate reperfusion injury or limit necrosis in the heart.

Thus, Zhao et al. [7] reported that several brief coronary occlusions after 60 minutes of ischemia significantly reduced infarct size and attenuated endothelial dysfunction in dogs. This "novel" mechanism of cardioprotection named ischemic postconditioning is essentially a form of modified reperfusion, which previous studies from 1980s have established to be cardioprotective [8,9]. Whatever the case, the concept of ischemic postconditioning has clearly elicited renewed interest in the reperfusion phase as a potential target for cardioprotection. Thus, ischemic postconditioning was as effective as preconditioning in reducing infarct size in dogs [7] and rabbits [10], but was lesser than that observed in a rat model [11].

Since ischemic postconditioning has been recently described its cardioprotection mechanisms are still unknown. Nevertheless, a few papers have shown, at least partly, its intracellular mechanism and have stated a hypothesis with regards to this. Hence, the underlying mechanisms suggested by ischemic postconditioning include an attenuated generation of reactive oxygen species [7,11] and attenuation of mitochondrial calcium overload [12]. Furthermore, Yang et al [10] showed that ischemic postconditioning triggers intracellular signaling kinases (ERK/Akt), mitochondrial K + ATP channels and release of nitric oxide (NO) in a rabbit model of ischemia/reperfusion. Similarly, Tsang et al [13] demonstrated that ischemic postconditioning activates the prosurvival kinases PI3K-Akt, eNOS, and p70S6K in accordance with the RISK pathway. Lastly, Yang et al [14] and Kin et al have recently demonstrated that the adenosine receptors are involved in the ischemic postconditionig mechanism. Thus there are some parallelism between ischemic preconditioning and postconditioning.

This concept has its fundamentals on the fact that, on one hand it is well known that a relationship exists between the activation of adenosine receptors [3], the activation of intracellular kinases [6] and ischemic preconditioning mechanism and on the other hand, some studies, not without some degree of controversy, have shown that the administration of adenosine [15,17] or A 1 and A 2 receptor agonists [18,19] during reperfusion, protects from ischemic/reperfusion damage, in particular with regards to the infarct size and myocardial stunning [20]. Since A 1 receptors can be activated during reperfusion, we have hypothetized that there could be a relationship between ischemic postconditioning and the activation of the A 1 receptors.

Additionally, it is important to take into account that all the mechanisms mentioned in the studies were described on healthy animals without coronary risk factors. Nevertheless, it is also known that the risk factors, among them hypercholesterolemia, are significant determinants of the evolution of ischemic heart disease. There are very few and contradictory [21,23] works in the literature that have studied the effect of hypercholesterolemia upon the ischemia/reperfusion phenomena; and there are no studies that have studied ischemic postconditioning in these experimental conditions.

For this reason, it would be important determine if ischemic preconditioning and postconditioning are present in hypercholesterolemic animals. And how this compares to animals fed on a normal diet, which would in turn allow a comparison between the two forms of physiological protection in animals with high cholesterol levels.

It is important to mention that most of the experimental studies performed to know the pathophysiology of the different entities related with the ischemia/reperfusion phenomena, such as stunned myocardium, ischemic preconditioning, hibernated myocardium and ischemic postconditioning used healthy animals [1,7,10,14,20,27]. Not withstanding that these studies have permitted a notorious increase in the knowledge that we have about these important phenomenon, we must recognize that the healthy animals are not representative for 30 to 40% of patients with ischemic heart disease having different degrees of hypercholesterolemia.

In this regard, the studies about ischemic preconditioning on hypercholesterolemic animals are particularly scarce, and the data is highly contradictory [21, 23]. On the other hand, and at least to our knowledge, there are no studies that have evaluated ischemic postconditioning on hypercholesterolemic animals. The lack of studies on this subject matter in hypercholesterolemic models is important as this new kind of physiological protection could have direct clinical applications, not only as in ischemic preconditioning, but particularly in patients which have been submitted to reperfusion therapies (PTCA, thrombolytic).

In our Institute we studied the postconditioning phenomenon induced by two brief periods of reperfusion/ischemia after a more extended period of 30 minutes in hypercholesterolemic animals, and secondly we have studied in a comparative manner the herein above, comparing the effect of ischemic preconditioning and postconditioning on infarct size in the same hypercholesterolemic model . Firstly we have detected that the postconditioning phenomenon significantly reduced the infarct size in the hypercholesterolemic animals and that the degree of protection found is of the same magnitude as that of ischemic preconditioning (Figures 1 and 2). In second place, we have observed that the percentage of reduction of the infarct size is greater in the hypercholesterolemic animals than in the normocholesterolemic ones. This last finding is due to that although the infarct size that is achieved with both forms of endogenous protection is the same, the infarct size in the control group animals with high cholesterol was significantly greater.

Figure 1: Normal animals: The effect of a different interventions on the infarct size can be observed. Infarct size is expressed as a percentage of the total left ventricle area. Observed that infarct size diminished with ischemic pre and postconditioning. The administration of DPCPX (A1 receptor blocker) abolished the postconditioning effect. Medium ± standard error. *: p<0.05 vs. control

Lastly, we show experimental evidence that demonstrates the participation of the A 1 receptors as the "triggers" of the ischemic postconditioning mechanism. In this sense Yang et al [14] have recently demonstrated that adenosine receptors participate in the ischemic postconditioning mechanisms. Our study extends this previous concept by demonstrating that the subtype A 1 would actually be the receptor involved (Figures 1 and 2) . To the best of our knowledge these findings have not been previously described in any study.

Figure 2: Hipercholesterolemic animals: The effect of a different interventions on the infarct size can be observed. Infarct size is expressed as a percentage of the total left ventricle area. Noted that the infarct size is larger in the hypercholesterolemic animals than reached by the normocholesterolemic animals. Medium ± standard error.*: p<0.05 vs. control.

Although previous studies have evaluated ischemic preconditioning and postconditioning comparatively [8, 10, 14], it is the first time that this comparison has been done among normo and hypercholesterolemic animals with pre and postconditioning and all in the same study. The fact that the infarct size that was achieved with ischemic preconditioning and postconditioning was similar, suggests that the hypercholesterolemia does not interfere with the protective mechanisms.

Various studies [7,10,14] have agreed in stating that postconditioning is much more applicable to the clinical setting than preconditioning due to the ischemic episode is often unpredictable clinically, therefore, the ability to protect the heart by intervening at the time of reperfusion provides an approach that is more suited to the clinical scenario. Hence, in the current study we have demonstrated for the first time that ischemic postconditioning is even present in hypercholesterolemic animals, which strengthens even more the concept of its potential applicability in the clinical setting, as approximately between 30-40 percent of patients with ischemic heart disease are known to have elevated plasma cholesterol levels [30].

On the other hand, a consistent and convincing body of work has indicated a central role for adenosine, acting via the A 1 receptors, as endogenous mediator of preconditioning in many species. Adenosine has been reported to protect the heart when given both prior to ischemia and at reperfusion. Xu et al. [19] demonstrated that AMP 579, an A 1 and A 2 agonist, administrated during reperfusion, significantly diminish the infarct size. In the same sense, Budde et al [15] have shown that the administration of adenosine, during reperfusion, reduces the infarct size after 60 minutes of ischemia. The effect was only found to be present if the drug was administered for a prolonged period of time. Hence, there could be a relation between ischemic postconditioning and the activation of the adenosine receptors. We showed that the administration of a selective A 1 receptor blocker abolished the protective effect of ischemic postconditioning both in normal and hypercholesterolemic rabbit hearts.

In summary, brief periods of reperfusion/ischemia performed after a prolonged ischemia significantly decreased the infarct size in normo and hypercholesterolemic animals. This cardioprotective effect is comparable, in the same experimental model, with the well-known phenomenon of ischemic preconditioning. Given that the infarct size is larger in the hypercholesterolemic animals, the reduction percentage of the infarct size was greater than that reached by the normocholesterolemic animals. Lastly, it has been suggested [11] that ischemic postconditioning would be nothing more than an effect resulting from a controlled reperfusion, nevertheless, Tsang et al [13] have demonstrated that protection can occur in absence of blood constituents, suggesting that postconditioning may exert a direct effect on the myocyte by activating prosurvival kinases. Interestingly, the activation of the adenosine A 1 receptors has been associated with phosphorylation of the ERK1/2 kinase [31] and Yang et al [14] showed that the mechanism of ischemic postconditioning involved activation of ERK. Since that A 1 receptors are the triggers of ischemic postconditioning perhaps there are other pharmacological agents successful at protecting ischemic myocardium when administered at reperfusion which use the same pathway leading to activation of the final effector. This needs to be verified as well as it is necessary to know if the benefits obtained in the hypercholesterolemic animals.

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Publication: September 2005

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