Ischemic preconditioning was described by Murry et al1 in 1986. From the pioneer studies carried out by Downey,2,3 much has been found concerning its cellular mechanism. We are certain that surface receptors, mitochondrial K+ATP, free radicals, and protein kinase C all play pivotal roles in the signaling pathways. However, to be protective, preconditioning must be applied before an ischemic event occurs, thus limiting its clinical utility. In 2003 Zhao et al4 reported that several brief coronary occlusions after 60 minutes of ischemia significantly reduced infarct size and attenuated endothelial dysfunction in dogs, naming ischemic postconditioning to this new physiopathological entity.
The underlying mechanisms suggested by ischemic postconditioning include an attenuated generation of reactive oxygen species4 and attenuation of mitochondrial calcium overload.5 Furthermore, Yang et al6 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 al7 demonstrated that ischemic postconditioning activates the prosurvival kinases PI3K-Akt, eNOS, and p70S6K, in accordance with the RISK pathway. Last, Yang et al8 demonstrated that the adenosine receptors are involved in the ischemic postconditioning mechanism. Thus, there are some parallels between ischemic preconditioning and postconditioning.
Additionally, it is important to consider that all the mechanisms mentioned in the studies were described for healthy animals. However, this is far from clinical reality because hypercholesterolemia is an important risk factor related to the evolution of ischemic heart disease in patients. Studies that have assessed the effect of hypercholesterolemia on the injury from ischemia/reperfusion show contradictory results.9,10 Only 1 study11 assessed the effect of ischemic postconditioning under these experimental conditions, but these authors analyzed an advanced stage of atherosclerosis with negative results.
For this reason, the main objective of this study was to determine if ischemic preconditioning and postconditioning were present in hypercholesterolemic animals. A second objective was to determine if the administration of an A1 adenosine receptor blocker causes inhibition of the protective effect of postconditioning in hypercholesterolemic animals.
It was shown6 that the administration of glybenclamide, a K+ATP channel blocker, causes the abolition of the postconditioning protective effect; however, this was not shown in hypercholesterolemic animals. Therefore, and as a final objective, we administered glybenclamide to observe the effect of potassium channel block on postconditioning in the hypercholesterolemic animals.
New Zealand rabbits (1.8 to 2.0 kg) were randomly assigned to 2 different dietary groups: 36 of them were fed standard rabbit food and 25 others received a cholesterol-supplemented diet (1% cholesterol) for 4 weeks. The experimental procedures were conducted in compliance with the American Physiological Society guiding principles.
After 4 weeks of receiving the normal or cholesterol-enriched diet, the rabbits were anesthetized with ketamine (75 mg/Kg) and xylazine (0.75 mg/kg) and then sacrificed with a lethal dose of thiopental (35 mg/kg). The thorax was rapidly opened and the heart was excised. Once each heart was removed, it was placed in a perfusion system according to the modified Langendorff technique.
The heart was perfused with a Krebs-Henseleit buffer containing the following: NaCl 118.5 mM, KCl 4.7 mM, NaHCO3 24.8 mM, KH2PO4 1.2 mM, Mg SO4 1.2 mM, CaCl2 2.5 mM, and glucose 10 mM, pH 7.4 ± 1.4 and gassed with 95% O2-5% CO2 at 37°C. Two electrodes were sutured and connected to a pacemaker with a constant heart rate of 200 beats/minute.
A latex balloon connected to a pressure transducer (Deltram II, Utah Medical System) via polyethylene cannula was inserted into the left ventricle (LV) for measurement of LV pressure. The latex balloon was filled with water to achieve an LV end diastolic pressure of 8-10 mm Hg. We also recorded the coronary perfusion pressure (CPP) through a pressure transducer connected to the perfusion line. All hearts were perfused with constant flow. Coronary flow was adjusted to obtain a CPP of 70.5 ± 4.2 mm Hg during the initial stabilization period. Thereafter, this flow level remained constant throughout the experiment.
Left ventricular pressure and CPP were recorded in real time by using a personal computer with an analog to digital converter. The left ventricular developed pressure (LVDP) was calculated as the difference between peak systolic pressure and end diastolic pressure. Ventricular function was assessed at baseline and during the first 30 minutes of reperfusion, although the hearts were reperfused after 2 hours to measure infarct size.
Infarct Size Measurement
After 2 hours of reperfusion, the hearts were frozen and cut into 2-mm transverse slices from the apex to the base. Sections were incubated for 20 minutes in 1% triphenyltetrazolium chloride (pH 7.4, 37°C) and then immersed in 10% formalin. With this technique, viable sections were stained red, whereas the nonstained sections corresponded to the infarct area. Sections were traced in acetate sheets and planimetered (Image Pro Plus, version 4.5). The infarct size was expressed as a percentage of the left ventricular area.
Blood samples were drawn from 13 rabbits, out of a total group of 17 animals fed with a 1% cholesterol-enriched diet. These samples were obtained both at the beginning of the protocol and on the day of the sacrifice to determine serum cholesterol. Cholesterol (C) was determined using commercial enzymatic kits (Roche Diagnostics, Mannheim, Germany) in a Hitachi 917 autoanalyzer. C-HDL (high-density lipoprotein) and C-LDL (low-density lipoprotein) were determined by selective precipitation methods.12,13
The administration of cholesterol was not sufficiently prolonged so as to induce important histologic changes in either the coronary arteries or in the intramural vessels. Therefore, we wanted to detect the well-known and early deleterious effect of hypercholesterolemia on the vascular endothelium.14 To achieve this effect, increased doses of acetylcholine were administered to 6 normal animals and 6 animals with cholesterol-enriched supplement. The vascular response was measured through the variations of coronary perfusion pressure. Because we used an experimental model with a constant coronary flow, the decrease or increase in the CPP indicated changes in the vascular coronary resistance.
At the end of the experiment, small segments of the descending aorta and heart were taken for histologic examination. The samples were fixed overnight in 10% buffered formalin and, after routine processing, were embedded in paraffin. The samples were stained with hematoxylin-eosin and oil red (the latter done in fixed frozen material). All the samples were examined under light microscopy to identify the presence of lipid deposition in the descending aorta and epicardial and intramyocardial heart vessels.
Animals were assigned to 10 different experimental groups:
Group 1 (n = 10): Myocardial infarction was induced by 30 minutes of global no-flow ischemia and 2 hours of reperfusion. We induced global no-flow ischemia by abruptly decreasing the total coronary flow provided by the perfusion pump.
Group 2 (n = 5): The same protocol as in Group 1 was performed, but we induced 5 minutes of ischemia followed by 5 minutes of reperfusion before the 30 minutes of global no-flow ischemia (ischemic preconditioning protocol).
Group 3 (n = 9): The same protocol as in Group 1 was performed, but after 30 minutes of global no-flow ischemia, reperfusion was initiated for 30 seconds followed by 30 seconds of ischemia, repeated for 2 cycles (2 minutes total intervention, ischemic postconditioning protocol).
Group 4 (n = 12): The same protocol as in Group 3 was performed, but during the ischemic postconditioning protocol (first 2 minutes of reperfusion) a selective A1 receptor blocker, DPCPX (200 nM), was administered.
Group 5 (n = 5): The same protocol as in Group 3 was performed, but during the postconditioning protocol glibenclamide, a K+ATP (0.3 μM) channel blocker was administered.
In Group 6 (n = 6), Group 7 (n = 6), Group 8 (n = 6), Group 9 (n = 7), and Group 10 (n = 6), all the protocols in Groups 1 to 4 were repeated but on animals that had been fed with a 1% cholesterol-enriched diet during the 4-week period.
In this description of the experimental protocols we have not included the experiments with acetylcholine because these groups were only used to determine the known effect of hypercholesterolemia on coronary vascular response.
Data are expressed as mean ± standard error of the mean (SEM). Intergroup comparisons were performed using analysis of variance and then with the Bonferroni test for multiple comparisons, and P < 0.05 was considered statistically significant.
Table 1 shows the average values for total, HDL, and LDL cholesterol obtained from the plasma of animals fed with a normal diet and of those fed with a cholesterol-enriched diet. An increase in total cholesterol and LDL cholesterol values was observed in the animals fed with the cholesterol-enriched diet versus the control group (P < 0.05).
Table 2 and Figure 1 show the changes induced by high cholesterol on the coronary vessels' functional response to the administration of acetylcholine and the histologic changes in intramyocardial vessels compared with the aorta. It is shown that during acetylcholine infusion there is a significant increase in the CPP of the hypercholesterolemic animals, without histologic changes in the coronary vessels.
Table 3 shows the values of LVDP and left ventricular end diastolic pressure (LVEDP) at baseline and during different times of reperfusion. In all groups, LVDP was significantly lower compared with the respective values but showed no significant group-related differences throughout the procedure. LVEDP (myocardial stiffness) increased during the reperfusion period in all groups, but there were no significant differences between groups.
Figure 2 shows the infarct size after 30 minutes of global no-flow ischemia, expressed as a percentage of the total left ventricular area in normocholesterolemic (Panel A) and hypercholesterolemic rabbits (Panel B).
In control normocholesterolemic hearts, the infarct size was 16.7 ± 1.7%. Ischemic preconditioning and postconditioning significantly decreased the infarct size up to 5.1% ± 1.7% and 5.4% ± 1.2%, respectively (P < 0.05 vs group control). The administration of DPCPX (A1 receptor blocker) and glibenclamide abolished the protection of ischemic postconditioning in healthy animals, increasing the infarct size up to 15.1% ± 1.7% and 15.2% ± 2.1%, respectively.
The infarct size in hypercholesterolemic hearts (Panel B) was 25.7% ± 2.9% (P < 0.05 vs normal control group). Both ischemic preconditioning and postconditioning significantly reduced infarct size by up to 4.1% ± 1.6% and 4.8% ± 0.9%, respectively (P < 0.05 vs normal control and hypercholesterolemic control groups).
Finally, the administration of DPCPX (A1 receptor blocker) and glybenclamide also abolished the protective effect of ischemic postconditioning in hypercholesterolemic animals, increasing the infarct size up to 21.5% ± 1.7% and 18.8% ± 2.5%, respectively (P < 0.05 vs normocholesterolemic and hypercholesterolemic postconditioning).
The results of the present investigation suggest that ischemic postconditioning reduces infarct size in normal and hypercholesterolemic animals through the activation of A1 and K+ATP channels.
The studies about ischemic preconditioning on hypercholesterolemic animals are particularly scarce, and the data are highly contradictory.15-17 However, there is only one study11 that has evaluated ischemic postconditioning on hypercholesterolemic animals, but it did not show protection; also these authors evaluated an advanced stage of atherosclerosis.
An interesting finding was that there was a significant reduction in the infarct size using a protocol of 2 cycles of reperfusion/ischemia. Until this study, experimental protocols of 3-4 or 6 cycles were used; thus we can assume that this is the first study that shows that it is possible to obtain protection with only 2 cycles of reperfusion/ischemia. In the present study we do not use a conventional postconditioning protocol, but in 2005 Vinten-Johansen et al18 showed that the duration of each cycle would be more important to reach protection in postconditioning than the number of episodes. Although we did not repeat the number of episodes used in a previous study, the duration of each cycle was similar to what has been published by other authors.
We detected that the infarct size in the control group, with high plasma cholesterol, was significantly greater than in the normocholesterolemic groups. However, ischemic postconditioning significantly reduced the infarct size in the hypercholesterolemic animals down to the same value of preconditioning. Therefore, we conclude that the reduction of the infarct size is greater in the hypercholesterolemic animals than in the normocholesterolemic ones. There appears to be a controversy in the literature as to whether experimental hypercholesterolemia influences the severity of myocardial ischemia/reperfusion injury. Several studies19-22 clearly show that hyperlipidemia leads to a significant worsening of myocardial ischemia/reperfusion injury, whereas others did not show differences.9,23 In our study the infarct size increased in hypercholesterolemic animals without histopathological changes but with endothelial dysfunction. In our rabbits, the latter was measured using the changes in the coronary perfusion pressure. It is known that in an isolated heart, perfused at constant flow, the changes in the coronary perfusion pressure reflect the changes in the coronary resistance. Therefore, an increase in the coronary perfusion pressure would reflect the vasoconstriction that is the consequence of endothelial dysfunction present in the hypercholesterolemic animals. However, the non-decrease in the coronary perfusion pressure by the administration of acetylcholine in normocholesterolemic animals could be reflecting the lack of coronary vasodilator reserve that is observed in the preparation of isolated hearts.24
In the present study, the left ventricular function in the groups with preconditioning and postconditioning had a partial recovery during reperfusion in contrast with the infarct size that was almost the same as the control. This dissociation between the partial recovery of the function and the almost total recovery of the infarct size during preconditioning and postconditioning could be because the noninfarcted myocardium has zones of stunned myocardium25 that prevent total recovery.
We present experimental evidence to demonstrate that A1 receptors “trigger” the ischemic postconditioning mechanism. In related work, Yang et al8 showed that adenosine receptors participate in the ischemic postconditioning mechanisms. Our study expands on this previous concept by demonstrating that the A1 subtype is the receptor involved. To the best of our knowledge these findings have not been previously described in any study. In 2005 Kin et al26 showed, in an in vivo rat model of ischemia/reperfusion, evidence that ischemic postconditioning would activate A2A and A3 adenosine receptors but not A1 receptors. In agreement with these results, Philipp et al showed,27 in an in vivo rabbit model, the involvement of the adenosine A2b receptor in postconditioning signaling pathways. However, the subject matter is controversial because other authors have also found protection during reperfusion mediated by the adenosine A1 receptors activation.28 In accordance with the latter studies, we showed that the administration of DPCPX to our hearts abolished the protective effect, suggesting the participation of adenosine A1 receptors in the phenomenon of postconditioning. In the groups that received DPCPX we observed a dramatic increase of LVEDP during reperfusion. This effect could be explained because the administration of DPCPX also decreases the protective action of the endogenous adenosine released during the ischemia, leading to an increased deterioration of ventricular function that would involve the entire intracellular pathway including the K+ATP channels. This is suggested by the fact that LVEDP deterioration was also observed in experiments where glybenclamide was administered.
As in our study, it is known that protection can occur in the absence of blood constituents, suggesting that postconditioning may exert a direct effect on the myocyte by activating prosurvival kinases.6,29
Finally, Yang et al6 showed that the K+ATP channels participate in the protective mechanism of ischemic postconditioning. However, they used hearts of normal animals. In the present study we used hypercholesterolemic animals. Because hyperlipidemia is associated with impaired activation of vascular K+ATP channels14-which belong to the same family and respond to the same stimuli-as those located in the myocytes, we hypothesized that hypercholesterolemia could abolish the beneficial effects of ischemic postconditioning. However, the results of the present study show that in hypercholesterolemic animals, ischemic postconditioning reduces infarct size through the activation of K+ATP channels as well.
Brief periods of reperfusion/ischemia performed after a prolonged ischemia significantly decreased the infarct size in normocholesterolemic and hypercholesterolemic animals. This cardioprotective effect is comparable, in the same experimental model, to 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 in this group was larger than that reached by the normocholesterolemic animals.
The authors are grateful for the assistance in the experiments afforded by medical students Jimena Medel and Cristina Lorenzo Carrión.
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