Journal of Cardiovascular Pharmacology:
Post-Ischemic Cardioprotection by A2A Adenosine Receptors: Dependent of Phosphatidylinositol 3-Kinase Pathway
Boucher, Matthieu MSc; Pesant, Stéphanie BSc; Falcao, Stéphanie BSc; de Montigny, Chantal TSA; Schampaert, Érick MD; Cardinal, René PhD; Rousseau, Guy PhD
From the Centre de Biomédecine, Hôpital du Sacré-Cœur de Montréal, Département de pharmacologie and Groupe de Recherche sur le Système Nerveux Autonome, Faculté de médecine, Université de Montréal, Montréal, Québec, Canada.
Received for publication June 9, 2003; accepted December 3. 2003.
Supported by the Fondation des Maladies du Coeur du Québec.
Reprints: Guy Rousseau, PhD, Centre de Biomédecine, Hôpital du Sacré-Cœur de Montréal, 5400, boul. Gouin Ouest, Montréal (Québec), Canada, H4J 1C5 (e-mail: firstname.lastname@example.org).
Activation of myocardial A2A adenosine receptors during reperfusion has been shown to be cardioprotective. The intracellular mechanisms underlying this protection remain unknown. To understand the beneficial effects of activated A2A adenosine receptors in such a state, we investigated whether the enzymes phosphatidylinositol 3-kinase (PI3K) and caspase-3 can account for this post-ischemic cardioprotective effect in an anesthetized rabbit model of myocardial infarction (30 minutes ischemia; 5 hours reperfusion). Administration of the A2A agonist CGS21680 (0.2 μg/kg/min) 5 minutes before reperfusion began (Early) reduced infarct size expressed as a percentage of the area at risk (25.7 ± 5.3% versus 46.5 ± 5.3% for the control group; * P < 0.05). Treatment with the A2A agonist 5 minutes after the onset of reperfusion (Late) had no effect on infarct size (38.2 ± 6.2%). In the presence of a selective inhibitor of PI3K (LY294002), the beneficial effects of CGS21680 on infarct size was no longer observed (43.9 ± 7.9%). After 5 hours of reperfusion, higher PI3K activity in the ischemic region was observed in the Early group compared with the other experimental groups. Caspase-3 activity was not observed in these different groups. In another set of experiments, PI3K activity was significantly higher during the first 15 minutes of reperfusion in the Early group as compared with the Control group. Caspase-3 activity increased rapidly during the first 15 minutes of reperfusion in the Control group and remained stable in the Early group. These results indicated that post-ischemic cardioprotection afforded by A2A adenosine receptor activation is PI3K-dependent and modulate rapidly other signaling pathways such as caspase-3.
Cardioprotection can be afforded before or after ischemia by adenosine receptors activation. Activation of A1 or A3 adenosine receptor subtypes before the onset of sustained ischemia reduces infarct size significantly. 1,2 Intracellular signaling pathways involved in these beneficial effects have been identified to involve kinases followed by phosphorylation and activation of membrane end-effectors possibly via KATP channels. 3 Activation of A2A adenosine receptors is effective at doing so during reperfusion. 4 A2A adenosine receptor activation post-ischemia may exert beneficial effects by interfering with the deleterious events associated with neutrophil accumulation, 5,6 oxygen-free radical production, 6,7 or apoptosis. 4 The molecular signaling pathways involved in any post-ischemic cardioprotection afforded by A2A adenosine receptor activation remain unknown.
Experimental evidence suggests that cell death occurring during reperfusion is due to apoptosis. 8,9 Early after the onset of reperfusion, 10 apoptotic stimuli activate the caspase cascade that involves caspase-3, which cleaves substrates important to the maintenance of cellular integrity. 11 Post-ischemic pharmacological interventions that reduce infarct size may thus interfere with apoptosis. 4,12 To determine the intracellular signaling pathways involved in the cardioprotection afforded by A2A adenosine receptor activation, we studied two enzymes that can be modulated by stimulation of G protein-coupled receptors and involved in cell survival.
PI3K is a key signaling enzyme that modifies downstream effectors such as kinases that are involved in cell growth and protein synthesis, thus cell survival. 13 Among the different downstream effectors, PKB/Akt can inhibit caspase-9, a protease crucial in the initiation of apoptosis. PKB/Akt can also phosphorylate Bad, which can no longer, as a consequence, interact with Bcl-2 or Bcl-xl, allowing them to inhibit apoptosis.
The second enzyme, caspase-3, is rapidly activated during the early phase of apoptosis, thereby contributing to cell death. Inhibition of caspase-3 during reperfusion induces a significant reduction of infarct size. This reduction suggests an important role for this enzyme in reperfusion injury. 12,14 Interestingly, it has been reported that activation of PI3K reduces caspase-3 activity. 15–17 According to these findings, PI3K seems to be upstream of caspase-3 and thus may act as an important enzyme in post-ischemic cardioprotection.
The present study was conducted to determine the role of A2A adenosine receptors in modulating PI3K and caspase-3 activities and thus the role of the former in cardioprotection afforded by A2A adenosine receptor activation.
All chemicals were obtained from Sigma (St. Louis, MO) unless specified. 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) was obtained from Calbiochem (La Jolla, CA). Leupeptin and Microcystin LR were obtained from Alexis Biochemicals (San Diego, CA). Anti-phospho-tyrosine (PY99) and secondary antibodies were obtained from Santa-Cruz Biotechnology (Santa-Cruz, CA). Tween 20 and [γ32P]-ATP were purchased from Amersham Pharmacia Biotech (Baie-d'Urfé, Canada). Dimethylsulfoxide (DMSO), Tris(Hydroxymethyl)aminomethane (Tris), sodium chloride, and sodium dodecyl sulfate (SDS) were obtained from Laboratoire MAT (Beauport, Canada). Ac-DEVD-AMC was obtained from BD Biosciences (Toronto, Canada). Lowry protein assay reagents were obtained from Bio-Rad Laboratories (Hercules, CA). Enhanced Chemiluminescence Reagents was obtained from Perkin Elmer (Markham, Ontario).
New-Zealand white rabbits (Charles River Canada, Saint-Constant, Québec) weighing between 2.3 and 3.1 kg were randomly assigned to 1 of the 4 following groups (8–10 animals/group): (1) Control group receiving vehicle (DMSO); (2) Early-treated group (Early) where 2-p-(2-Carboxyethyl) phenethylamino-5`-N-ethylcarboxamidoadenosine hydrochloride (CGS21680; A2A adenosine receptor agonist) was administered 5 minutes before the beginning of reperfusion (0.2 μg/kg/min) into the marginal ear vein; (3) Late-treated group (Late) where the same agonist was administered 5 minutes after the beginning of reperfusion (0.2 μg/kg/min); and (4) Early-treated group with the A2A agonist and selective PI3K inhibitor (Early + LY) where CGS21680 (0.2 μg/kg/min) and LY294002 (selective PI3K inhibitor; 1.66 μg/kg/min) were administered simultaneously 5 minutes before the beginning of reperfusion. CGS21680 and LY294002 were solubilized in DMSO. Treatments were administered for 120 minutes in all groups. Dosage of the LY294002 used in the present study was determined from results obtained in a previous pilot study. Dosage of CGS21680 was chosen in accordance with the protocol used by Zhao et al. 4
Three groups were constituted: (1) Control group receiving vehicle (DMSO); (2) Early-treated group (Early) where CGS21680 was administered 5 minutes before the onset of reperfusion (0.2 μg/kg/min); and (3) Late-treated group (Late) where CGS21680 was administered 5 minutes after the onset of reperfusion.
Rabbits were handled in compliance with the procedures of the Animal Care Local Committee. Ketamine/xylazine (35–50 mg/kg and 5 mg/kg i.m., respectively) was used to induce anesthesia, which was maintained with isoflurane (1.5%). Rabbits were intubated and placed on an artificial respirator to maintain physiologic levels of blood gases. Catheters were inserted into the carotid and femoral arteries to collect blood and monitor arterial pressure. Electrodes were placed on front paws to record ECG and heart rate. A left thoracotomy was performed at the fifth intercostal space and the left anterior descending coronary artery was occluded with a silk suture. Ischemia was confirmed by ST segment alterations and cyanosis on the myocardial surface. After 30 minutes of occlusion (ischemia), the ligature was loosened and the myocardial tissue was reperfused. Reperfusion was confirmed by the disappearance of cyanosis. The suture used to occlude the artery was left in place to localize the site of occlusion and to assess infarct size later. In protocol A, the reperfusion period lasted 5 hours. In protocol B, the reperfusion period was limited to 0, 5, or 15 minutes.
Measurement of Area at Risk and Infarct Size
At the end of the reperfusion period, the heart was removed and the left anterior descending coronary artery was occluded at the same site to determine the area at risk (AR) with infusion of Evans Blue (0.5%) by retrograde perfusion into aorta (protocol A and B). The heart was then placed at −80°C for 5 minutes and sliced in 4 to 5 transverse sections of 2 mm. Each section was incubated 5 minutes at 37°C in a triphenyltetrazolium chloride solution (TTC 1%, pH 7.4) to determine the area of necrosis (protocol A only). Myocardial infarction was expressed as a percentage of necrosis (I) of the AR. Moreover, AR was expressed as a percentage of left ventricle (LV) area. Ischemic central left ventricle sections from slices 2 to 4 were dissected into 2 equal endocardial and epicardial portions regardless of the localization of the myocardial infarction (protocol A and B). The nonischemic portions of the left ventricle were dissected to serve as internal control. Cardiac tissue was then subjected to snap freezing in liquid nitrogen and stored at −80°C until used.
Frozen cardiac tissue samples were homogenized in lysis buffer (50 mM Tris (pH 7.5), 20 mM β-glycerophosphate, 20 mM NaF, 5 mM EDTA, 10 mM EGTA, 1 mM Na3VO4, 10 mM benzamidine, 0.5 mM PMSF, 10 μg/mL Leupeptin, 5 mM DTT, 1 μM microcystin LR, and 1% Triton X-100) with a polytron homogenizer (10 second bursts at maximum speed). The homogenate was centrifuged at 1000 × g for 10 minutes at 4°C. The supernatant was removed, incubated for 30 minutes at 4°C, and centrifuged for 15 minutes at 10,000 × g at 4°C. The protein content was then assessed using the Lowry method (Bio-Rad). Protein extracts were precipitated with anti-phosphotyrosine antibody conjugated to biotin (1:50) overnight at 4°C. The immune complex was pelleted and washed 3 times with lysis buffer and 2 times with phosphate-buffered saline buffer containing 0.1 M Na3VO4. The immune pellet was then suspended in activation buffer (35 mM ATP, 0.2 mM adenosine, 30 mM MgCl2, 10 mg/mL l-α-phosphatidylinositol, and 20 μCi [γ32P]-ATP) and incubated at room temperature for 20 minutes. The reaction was stopped with the addition of 100 μL hydrogen chloride 1M and 200 μL of Chloroform: Methanol (1:1). The aqueous phase was then discarded. Eighty μL of hydrogen chloride:Methanol (1:1) were then added before discarding the aqueous phase. Twenty μL of the organic phase containing 32P-Phosphatidylinositol were resolved by thin layer chromatography on K6 Silica Gel plates (Whatman, Clifton, NJ) in a solvent system containing Chloroform: Methanol:ammonium hydroxide (45:35:10). Plates were exposed to film for 3 to 5 days (−80°C) before revelation.
Frozen cardiac tissue samples were lyophilized and cytosolic proteins were extracted in lysis buffer (1% Triton X-100, 0.32 M Sucrose, 10 mM Tris (pH 8.0), 5 mM EDTA, 2 mM DTT, 1mM PMSF, 10 μg/mL Leupeptin, 10 μg/mL Pepstatin A, 10 g/mL Aprotinin). Enzymatic reactions were performed in reaction buffer (50 mM Tris (pH 7.5), 5 mM MgCl2, 1 mM EGTA, 0.1% CHAPS, 1 mM DTT) with 25 μg of proteins and fluorogenic substrate, Ac-DEVD-AMC (40μM). Reactions were incubated at 37°C for 3 hours and stopped with the addition of 0.4 M NaOH and 0.4 M Glycine buffer. Fluorescence was quantified with a spectrofluorometer (Photon Technology International, Lawrenceville, NJ) at an excitation wavelength of 365 nm and an emission wavelength of 465 nm.
Results are expressed as mean (± standard error of the mean) and were evaluated using analysis of variance and covariance adapted for factorial experimental design. 18 A P value of less than 0.05 was considered statistically significant.
Hematological and Hemodynamic Data
All groups were similar in terms of weight, hematocrit, hemoglobin, leukocyte, and platelet number at occlusion time (Table 1). Heart rate and mean arterial pressure were similar between groups throughout experiments (Fig. 1A, 1B). These results indicated that neither CGS21680 dosage nor LY294002 exerted any significant effect on monitored hemodynamic parameters.
Area at risk (AR), expressed as a percentage of the total left ventricle (LV) area, was similar for all groups, representing more than 45% of the LV area (Fig. 2B). Infarct size, expressed as a percentage of the AR (I/AR), was significantly smaller in the Early group (25.7 ± 5.3%) when compared with the Control (46.5 ± 5.3%) and Late (38.2 ± 6.2%) groups (Fig. 2A; * P < 0.05). In the presence of LY294002, cardioprotection observed with the early administration of CGS21680 (Early + LY) was no longer present (43.9 ± 7.9%). These results suggest that PI3K is involved in infarct size modulation during A2A adenosine receptor activation.
To determine A2A adenosine receptor activation effects in the signaling pathways involved during post-ischemic cardioprotection, we measured PI3K activity by in vitro phosphorylation of l-α-phosphatidylinositol. PI3K activity was enhanced significantly within the Early group with CGS21680 (Fig. 3) when compared with the Control and Late groups. Indeed, PI3K was 64.2 ± 8.3% and 64.5 ± 10.1% for endocardial and epicardial ischemic regions respectively (Control group values being 8.4 ± 8.6% and 15.0 ± 5.2% and Late values being 3.3 ± 7.4% and 31.8 ± 8.8% for endocardial and epicardial ischemic regions respectively). In the presence of LY294002 along with CGS21680 (Early + LY), no PI3K activity was detected in ischemic or nonischemic regions.
As a potential key participant in the reperfusion injury, we measured caspase-3 activity in our experimental model. After 5 hours of reperfusion, we were unable to detect any significant activity in tissues derived from the different experimental groups (data not shown).
Kinetics of activation of PI3K and caspase-3 early after onset of reperfusion
To estimate the effect of CGS21680 on the different enzyme activities during the early phase of reperfusion, we killed the animals after 30 minutes of ischemia followed by 0, 5, or 15 minutes of reperfusion. At the end of the ischemia period (0 minutes of reperfusion), PI3K activity measured in the area at risk was similar between the Control and Early groups. However, after 5 minutes of reperfusion, ischemic regions from the Control group demonstrated a significant reduction in PI3K activity (Fig. 4). This activity remained constant in the Early group. This reduction was greater after 15 minutes mainly in the ischemic endocardium. In the Late group, PI3K activity was not significantly different from that observed in the Control group at 15 minutes of reperfusion.
At the end of the period of ischemia, caspase-3 activity was similar among groups (Fig. 5). However, 5 minutes after the onset of reperfusion, caspase-3 activity was significantly increased in ischemic regions of the Control group as compared with the Early group. This effect was maintained during the first 15 minutes of reperfusion in the endocardial ischemic region. In the epicardial ischemic region, no statistical difference was detected. Activity observed in the Late group indicated that treatment was ineffective to reduce caspase-3 activation.
The present study demonstrates that the beneficial effects exerted by A2A adenosine receptor activation on the post-ischemic myocardium are PI3K-dependent. Furthermore, in the presence of a selective PI3K inhibitor (LY294002), A2A adenosine receptor activation imparted no further cardioprotection. We also observed that cardioprotective therapy via A2A adenosine receptor activation inversely modulates the PI3K and caspase-3 activities. CGS21680 therapy, instituted 5 minutes after the onset of reperfusion, did not affect the PI3K and caspase-3 activities as compared with the Control group, and thus was unable to impart any cardioprotection. These results indicate the potential significance of these enzymes in the cardioprotective effects of A2A adenosine receptor activation. The reduction of infarct size by post-ischemic A2A adenosine receptor stimulation that we observed has been demonstrated by other groups in different experimental models, including pigs, 19 rabbits, 20–22 and dogs. 4,6,23 We also observed that the same regimen starting 5 minutes after the onset of reperfusion is ineffective in reducing infarct size. As proposed by Horwitz et al, 24 these data indicate that the optimal timing of therapy administration depends of the pharmacokinetics, diffusibility, and mechanism of action of these selective pharmacological agents.
The present study is, to our knowledge, the first to identify the PI3K signaling pathway as a key target for post-ischemic cardioprotection afforded by A2A adenosine receptors. These receptors are not unique with respect to dependence on PI3K signaling for post-ischemic cardioprotection. Indeed, it has been shown that the beneficial effect of post-infarction insulin can also be blocked via a PI3K inhibitor, 25,26 as can the effects of bradykinin. 27 PI3K is involved in cell survival and limits apoptosis by modulation of downstream target effectors such as PKB/Akt and p70S6 kinase. Both enzymes can phosphorylate Bad, maintaining cytosolic sequestration of this protein in its inactive form. When Bad is activated and translocated to mitochondria, it forms a heterodimer with Bcl-2 or Bcl-xl to promote apoptosis. 28 It has also been suggested that PI3K activation is beneficial to the post-ischemic myocardium by decreasing caspase-3 activity. 16 Data obtained from protocol B demonstrate that caspase-3 activity in the Early group is similar to that observed in nonischemic regions whereas caspases-3 activity in the Control group increases. Stable caspase-3 activation in Early-treated animals during early reperfusion parallels PI3K activity. In contrast, activity of both enzymes diverged in the Control group, presumably acting to promote cell death by apoptosis. Overall, the present data support a previous study indicating that A2A adenosine receptor activation reduces apoptosis as evaluated by TUNEL staining and DNA fragmentation. 4
Interestingly, results obtained in the Late group indicated PI3K and caspase-3 activities are similar to Control group, in contrast to the Early group. These results suggest that coupling between A2A adenosine receptors and these enzymes is not direct and involves other proteins. If indeed direct coupling occurred between receptors and these enzymes, the activity measured in both treated groups (Early and Late) would have been more similar than that which we observed. Although second messenger(s) involved in modulating PI3K and caspase-3 activities remains unknown, recent studies indicate that cAMP may modulate PI3K activity. 29,30 These findings are interesting since A2A adenosine receptors are positively coupled to adenylyl cyclase and thus cAMP production. Further studies are needed to confirm the role of cAMP in this beneficial effect.
Myocardial tissue was separated into two regions (endocardial and epicardial ischemic regions) because of observations demonstrating that endocardium is more sensitive to ischemia than epicardium. 31 Therefore, enzyme activity differences may be more prominent in the former region when comparing treated groups with control groups. Our results confirm that differences occur in both regions, reflecting the relative extent of damage in each area. However, analysis of covariance did not indicate any relation between infarct size and PI3K or caspase-3 activities after 5 hours of reperfusion. In addition, we observed a significant difference in PI3K and caspase-3 activities after 5 minutes of reperfusion. According to Ganz et al, 32 5 minutes of reperfusion are not sufficient to detect any significant progression of infarct size. These results suggest that the extent of damage plays a minor role in the PI3K and caspase-3 activities observed and that other factors, including A2A adenosine receptors activation, are involved.
In the present study isoflurane was used as the anesthetic agent. It has been shown that isoflurane can reduce infarct size with a mechanism similar to preconditioning. 33,34 In our study, however, isoflurane did not reduce infarct size. This discrepancy can be explained by the fact that preconditioning induction can be achieved by stopping administration of isoflurane a few minutes before the ischemic period begins, thereby achieving maximum cardioprotective effect; this was not the case in our study.
A2A adenosine receptors are mediators of vasodilation processes and may induce hypotension. In the present study, mean arterial pressure did not differ throughout the experiment among groups. This suggests that the CGS21680 dosage used had no hypotensive effect. Similar results were also obtained by Zhao et al 4 using the same regimen of CGS21680 treatment. On the other hand, Jordan et al 6 observed a reduction in left ventricular peak systolic pressure with this agent. One possibility may be that isoflurane, which possesses cardiopressant effects, may mask any vasodilator effects of CGS21680. It is also possible that CGS21680 may induce vasodilation when infused directly into the coronary artery 6 versus infusion into the marginal ear vein (our study) or the left atrium. 4 Another possibility is that A2A agonist concentrations are not high enough to induce any vasodilation. Cardioprotection afforded by the A2A agonist might also be due to an indirect mechanism involving circulating cells such as neutrophils as observed by others. 4,6 Further in vivo studies are needed to determine if CGS21680, when so administered, acts directly on cardiomyocytes.
One limitation of our study is that PI3K activities were determined in tissue that has been incubated in TTC (protocol A). Results obtained from pilot studies indicate that these activities were reduced by 25 to 30% when tissues samples were incubated in TTC. Since all ventricular tissue samples (including areas at risk) were in contact with TTC, we expressed our results in percentage terms, comparing data from ischemic to nonischemic tissue. This provides a way to correct enzyme activity levels and thus eliminate inter-animal variability. Moreover, in protocol B different activities were measured in tissues that were not incubated in TTC. The differences observed between these groups were found to be similar to those derived in protocol A, indicating that the methods of evaluating enzyme activity in protocol A was reliable.
Our results indicated that post-ischemic cardioprotection afforded by A2A adenosine receptor activation is PI3K-dependent and may rapidly modulate other signaling pathways such as caspase-3.
The authors thank Dr. Robert Élie for statistical analysis, Dr. Andrew Armour, Dr. Terrence Hebert, Diane Boucher, and Alexandro Zarruk for critical reading of the manuscript as well as Dr. Denis DeBlois and Dr. Eve-Lyne Marchand for caspase-3 assay.
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