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Departments of 1Physiology and 2Medicine, University of South Alabama, Mobile, Alabama
Submitted 3 June 2005 ; accepted in final form 26 August 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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adenosine receptors; extracellular signal-regulated kinases 1 and 2; ischemia-reperfusion; ischemic preconditioning; phosphatidylinositol 3-OH kinase
Curiously, several pharmacological or physical interventions that have been reported to be cardioprotective when administered at reperfusion also appear to require ERK1/2 and PI3K activation (3–5, 10, 22, 23, 30, 31, 33). One explanation is that they all might be utilizing the same basic mechanism as IPC. One such agent is the adenosine A1/A2A agonist AMP579 (29). Empirically we noted that AMP579 was not protective if it was present for only the initial 30 min of reperfusion, whereas an infusion of 60 min profoundly limited infarct size (28). Furthermore, delaying the onset of infusion of AMP579 for just 10 min after the beginning of reperfusion also resulted in a loss of protection (28). This indicates that the ischemic heart requires support from this protective agent from the onset of reperfusion and that support must be maintained for >30 min. Because the adenosine agonist AMP579 is so protective when given at reperfusion, we wondered whether adenosine receptor occupancy during the reperfusion period following the lethal ischemic insult might be involved in IPC's mechanism of protection as well. We also wanted to know whether protection from IPC is dependent on prolonged activation of signal transduction pathways during reperfusion as was the case with AMP579. Accordingly, we exposed preconditioned, isolated rabbit hearts to an adenosine receptor blocker, PI3K antagonist, or ERK1/2 blocker with infusions beginning just before or at various times after the onset of reperfusion.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Isolated Rabbit Heart Model
New Zealand White rabbits of either sex were used. Hearts were removed from animals anesthetized with pentobarbital sodium (30 mg/kg), mounted on a Langendorff apparatus, and perfused with modified Krebs-Henseleit bicarbonate buffer (in mM: 118.5 NaCl, 25 NaHCO3, 4.8 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.7 CaCl2, 10 glucose) which was bubbled with 95% O2-5% CO2 to maintain pH 7.4. A latex balloon was inserted in the left ventricle to register developed ventricular pressure, and a snare was placed around the left coronary artery to produce regional ischemia.
As shown in Fig. 1, 21 groups of hearts were studied. All hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion. Control hearts were subjected to regional ischemia-reperfusion only. IPC hearts received one 5-min period of global ischemia and one 10-min period of reperfusion before the lethal ischemic insult. Some IPC hearts were treated with either 8-(p-sulfophenyl)-theophylline (8-SPT, 100 µM), a nonspecific adenosine receptor blocker, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, either 20 or 200 nM), an adenosine A1 receptor antagonist, 8-(3-chlorostyryl)caffeine (CSC, 1 µM), an adenosine A2A antagonist, N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide (MRS1754, 20 nM), an adenosine A2B antagonist, wortmannin (100 nM), a PI3K inhibitor, or U-0126 (0.5 µM), a MEK1/2 and therefore ERK1/2 inhibitor. All drugs were added to the perfusate for 20 min starting either 5 min before (8-SPT, DPCPX, CSC, MRS1754, wortmannin, U-0126) or 10 (8-SPT, wortmannin, U-0126), 30 (8-SPT, wortmannin), or 60 (wortmannin) min after the onset of reperfusion.
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At the end of reperfusion, the coronary artery was reoccluded, and 2- to 9-µm-diameter fluorescent polymer microspheres (Duke Scientific, Palo Alto, CA) were infused into the perfusate to demarcate the ischemic zone (region at risk) as the area of tissue without fluorescence. The heart was weighed, frozen, and then cut into 2-mm-thick slices. The slices were incubated in 1% triphenyltetrazolium chloride (TTC) in sodium phosphate buffer (pH 7.4) at 37°C for 10–12 min. TTC stains noninfarcted myocardium brick red. The slices were then fixed in 10% formalin to preserve the stained (viable) and unstained (necrotic) tissue. The risk zone was identified by illuminating the slices with UV light. The areas of infarct and risk zone were determined by planimetry of each slice, and the volumes were calculated by multiplying each area by the slice thickness and summing them for each heart. Infarct size is expressed as a percentage of the risk zone.
Biochemical Studies
Three additional isolated rabbit heart groups were used to generate transmural biopsies of the left ventricle. The hearts were mounted on a Langendorff apparatus and perfused with modified Krebs-Henseleit bicarbonate buffer as described above. All hearts were subjected to 30 min of global ischemia by arresting retrograde aortic perfusion. Control hearts were subjected to only 30 min of global ischemia and 10 min of reperfusion. IPC hearts received a 5-min period of global ischemia and a 10-min period of reperfusion before 30 min of global ischemia. In drug-treated hearts, 8-SPT (100 µM) was present in the perfusate during reperfusion following the 30-min index ischemia. Transmural biopsies of the left ventricle, each weighing 
25 mg, were obtained with a motorized biopsy tool after either 5 or 10 min of reperfusion following the index ischemia. The tissue samples were ejected from the tool into liquid nitrogen within 1 s of excision.
Biopsy tissues were homogenized with a Polytron homogenizer in ice-cold lysis buffer containing (in mM) 20 Tris·HCl (pH 7.5), 150 NaCl, 1 Na2EDTA, 2.5 sodium pyrophosphate, 1 
-glycerophosphate, and 1 Na3VO4, with 1% Triton and 1 µg/ml leupeptin. The total cellular lysates were centrifuged for 15 min at 13,000 g. The concentration of total protein in the supernatant was determined by the Lowry method (Bio-Rad Laboratories, Hercules, CA). The protein samples were diluted in Laemmli sample buffer (Bio-Rad Laboratories), heated for 3 min at 100°C, and then stored at –70°C for later analysis. Samples of supernatant equivalent to 20 µg of total protein were added to the lanes of a 10% SDS-polyacrylamide gel and after electrophoresis were transferred to a nitrocellulose membrane. Transfer of proteins was confirmed by Ponceau red staining of the membrane. The membranes were probed with monoclonal antibodies (1:1,000 dilution) for phospho-ERK 1/2 (Thr202/Thr204) and phospho-Akt (Ser473) and visualized with chemiluminescence. The developed films were scanned, and the density of each band was calculated with Sigmagel software. To correct for differences in exposure time, transfer efficiency, and antibody potency between gels, a standard derived from a previously obtained untreated myocardial sample that demonstrated good phosphorylation of ERK and Akt was included in each gel, and all band densities were normalized to this standard. We measured only phosphorylated ERK and Akt. Thus these measurements quantitate the absolute amounts of the phosphorylated protein in the tissue rather than a percentage of the total ERK or Akt pool.
Drugs
Wortmannin, 8-SPT, DPCPX, CSC, and MRS1754 were obtained from Sigma (St. Louis, MO). U-0126 was obtained from LC Laboratories (Woburn, MA). Wortmannin, U-0126, and 8-SPT were dissolved in DMSO before being diluted in Krebs-Henseleit buffer, resulting in a DMSO concentration of <0.01%.
Phospho-Akt antibody to Ser473, cell lysis buffer, and Lumi GLO were purchased from Cell Signaling Technology (Beverly, MA), monoclonal anti-phospho-ERK1/2 from Upstate (Lake Placid, NY), and horseradish peroxidase-linked anti-mouse IgG antibody from Santa Cruz Biotechnology (Santa Cruz, CA).
Statistics
Results are given as means ± SE. One-way ANOVA with Student-Newman-Keuls post hoc test was performed on baseline hemodynamics, infarct measurements, and Western blot band densities. ANOVA for repeated measures was used to test for time-related differences in hemodynamics within groups. Groups treated with different drugs were analyzed as separate cohorts. Groups treated with the same drug were compared with control and with each other. P < 0.05 was considered significant. In rabbits, the amount of infarction of the risk zone is somewhat dependent on the actual size of the risk zone (32). To test for any influence of differing risk zone sizes among the rabbits, all infarct size data were additionally analyzed by plotting the absolute infarct size seen in each heart against its risk zone size. Treatment-related changes in infarct size in a group are revealed by a downward shift from the control group of the infarct vs. risk zone regression line as determined by analysis of covariance.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Table 1 presents hemodynamic data from the isolated hearts. Coronary flow represents total coronary flow from both ischemic and nonischemic zones. No group differences in heart rate, developed pressure, and coronary flow were observed at baseline. As expected, values for developed pressure and coronary flow in all groups were significantly lower than baseline values during coronary occlusion, with partial recovery during reperfusion.
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There were no significant differences in risk zone volume among the groups (Table 2). In the control group the percentage of the risk zone infarcting was 32.1 ± 4.6%, and this was significantly reduced by one cycle of ischemic preconditioning to 7.3 ± 3.6% (P < 0.05; Fig. 2).
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Selective adenosine receptor subtype antagonists were also administered from 5 min before to 15 min after reperfusion. As seen in Fig. 3, both the A1 antagonist DPCPX at high and low concentrations (33.4 ± 4.9% and 31.6 ± 4.1%, respectively, P < 0.05 vs. IPC) and the A2B blocker MRS1754 (30.6 ± 5.3% infarction, P < 0.05 vs. IPC) aborted protection. However, the adenosine A2A antagonist CSC had no effect on IPC protection.
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Biochemical Studies
IPC significantly increased Akt phosphorylation in samples obtained after 10 min of reperfusion, and the increase was blocked by 8-SPT (Fig. 6A). On the other hand, IPC caused no significant changes in phosphorylation of either ERK1 or -2 after 10 min of reperfusion and 8-SPT had no effect (Fig. 6B). Because we found that administration of the MEK/ERK inhibitor after 10 min of reperfusion was without effect on IPC protection, we hypothesized that we failed to see any change in ERK phosphorylation because activation of ERK had occurred earlier in reperfusion. We therefore studied nine hearts (3 in each group) in which biopsies were taken after only 5 min of reperfusion. The pattern was the same as was seen in Fig. 6: Akt phosphorylation was increased in IPC hearts over that in the other two groups, but there were no differences in phosphorylation of either isoform of ERK among the three groups (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Hence signal transduction pathways must be activated at the onset of reperfusion in the ischemically preconditioned heart, and, perhaps even more unexpectedly, that activation must be sustained for an extended period. The need for continued and prolonged activation of the protective kinases during reperfusion is best illustrated by our studies with wortmannin. Blocking PI3K during the first 15 min of reperfusion prevented the protective effect of IPC, which is consistent with the observation of Hausenloy et al. (9) that PI3K must be active during the first minutes of reperfusion in the ischemically preconditioned heart. More importantly, protection was also lost when we transiently blocked PI3K starting either 10 or 30 min after the onset of reperfusion. This indicates that a continuous PI3K signal must be active for at least the first 30–50 min of the reperfusion period for IPC protection to be realized. After 60 min of reperfusion, however, PI3K is no longer needed. We suggest that the postischemic heart needed the support of PI3K only during a critical convalescent period. After 1 h of support the heart could once again function without this protective signaling. Alternatively, PI3K may simply activate a downstream event that then takes over the protective role from PI3K after 60 min. Of course, Akt phosphorylation was used here as a reporter for PI3K. Hausenloy and colleagues (9) first reported that IPC causes Akt activation on reperfusion. Our results indicate that the enhanced PI3K activity was linked to adenosine receptor occupancy because 8-SPT in the first few minutes of reperfusion aborted the increased Akt phosphorylation triggered by IPC.
A similar but less prolonged need for continued activation was seen with the adenosine receptors. 8-SPT administration started 10 min after the onset of reperfusion continued to block protection, indicating that continued adenosine receptor occupancy is required beyond the first 10 min of reperfusion. Interestingly, however, starting the adenosine blocker 30 min after reperfusion no longer affected IPC protection. This indicates that the timing requirements are different for adenosine receptor occupancy and PI3K activation. When we previously (15) gave a 5-min pulse of adenosine to rabbit hearts, PI3K activity persisted long after the adenosine was washed out. Thus the difference in timing may simply be the result of PI3K activation continuing for some minutes after the adenosine receptor signal is withdrawn.
ERK participation displayed the briefest time course of the three signal transduction components studied. Although ERK needed to be active in the first minutes of reperfusion, delay of administration of the ERK antagonist until 10 min after reperfusion rendered it ineffective in blocking IPC protection. We saw ERK phosphorylation when the adenosine agonist 5'-(N-ethylcarboxamido)adenosine (NECA) was given to reperfused rabbit hearts, suggesting that some adenosine receptors do couple to ERK (30). This unanticipated observation suggests that ERK1/2 may somehow act as a transient but necessary trigger event early in reperfusion. Unlike the observations made by Hausenloy et al. (9) in rat hearts, our biochemical studies did not detect increased activation of ERK in preconditioned rabbit hearts. We cannot rule out the possibility that protection was blocked by a nonspecific effect of the drug. The other possibility is that ERK activation may have occurred in a critical compartment that was too small to be seen in the total cytosolic pool.
Why would adenosine receptors be protective at the time of reperfusion only in the preconditioned heart? IPC might have increased the extracellular level of adenosine during the reperfusion period. The effect of IPC on extracellular adenosine levels during ischemia is controversial. Some investigators have indeed presented evidence that IPC increases adenosine generation. Kitakaze et al. (13) hypothesized that the increased adenosine formation is the result of PKC-dependent activation of 5'-nucleotidase. They showed that PKC directly activates ecto-5'-nucleotidase, which is located on the cell membrane, and they also reported that adenosine 5'-(
,-methylene)diphosphate, a blocker of ecto-5'-nucleotidase, can diminish the cardioprotective effect of IPC when given at the time of the preconditioning ischemia-reperfusion cycle as well as during early reperfusion (14). Additionally, Sinclair et al. (26) observed that an increased extracellular adenosine level in metabolically stressed cells can be the result of PKC-dependent inhibition of adenosine kinase. They also showed that adenosine kinase activity is decreased in cells treated with the selective adenosine A1 receptor agonist N6-cyclohexyladenosine.
In contrast, there is also evidence of decreased adenosine release during ischemia and reperfusion in IPC hearts. Harrison et al. (8) documented inhibition of adenosine release into interstitial and vascular compartments of preconditioned hearts. They concluded that the protective effect of IPC is unrelated to tissue adenosine content during lethal ischemia and after reperfusion. This is consistent with our data (7) and those of others (24) showing that IPC actually diminishes purine release during a subsequent ischemia.
We have demonstrated here that not only must the protective signal transduction pathways be activated at reperfusion but also those signals must be maintained while the heart recovers from its ischemic insult. Thus IPC may act to prolong the duration of adenosine release rather than increase its concentration. To our knowledge, no one has ever tested whether preconditioned hearts have a prolonged adenosine release in the reperfusion period after an ischemic insult.
A third possibility would be that IPC selectively induces adenosine receptor activation during reperfusion because of changes of density and/or affinity of the receptors rather than because of increased extracellular adenosine. Indeed, Li and He (16) demonstrated that in hearts subjected to 60 min of ischemia myocardial membranes from preconditioned hearts had a higher density of adenosine receptors than those from nonpreconditioned hearts. Even more interesting, Altiok et al. (1) found that PKC activation enhanced the cAMP response from an adenosine A2B agonist. This appeared to be at either the receptor or the G protein level.
The present study did not completely determine the adenosine receptor subtype that is responsible for IPC protection at reperfusion. Unfortunately, the infarct size model is too cumbersome and expensive to resolve the receptor subtype with rank order potencies. Thus we were forced to test the inhibitors at a single concentration. We were further hampered by the fact that few of the antagonists have been characterized in rabbit systems, so the precise Ki for the antagonists against rabbit receptors is unknown. Nevertheless, our preliminary observations would suggest that an A1 and/or an A2B receptor is involved.
Clearly the A1 (19) and/or the A3 (18) receptor triggers the protection of IPC during the brief preconditioning ischemia. We could find no report in which an A1-selective agonist infused just before reperfusion yielded cardioprotection. Therefore, it was quite surprising to find that a highly selective adenosine A1 antagonist administered at reperfusion blocked IPC protection. The initial observations in this series of experiments were made with a DPCPX concentration of 200 nM. This dose was selected on the basis of binding studies with rat membranes that indicated that the Ki of DPCPX was 0.3 nM for A1 receptors and 340 nM for A2A receptors (20). However, human receptor binding studies demonstrated a slightly different profile. The Ki for A1, A2A, A3, and A2B receptors has been reported to be 2.5, 156, 509, and 56 nM, respectively (17). Hence we considered the possibility that at the dose used blockade of A2B or even A2A rather than A1 receptors might have caused the loss of IPC protection. Consequently, as shown in Fig. 3, we also examined the effect of 20 nM DPCPX, a dose that should have still blocked A1 receptors but would be less likely to have an effect on A2A or A2B receptors. Because DPCPX continued to block protection at the lower dose, we cannot easily reject the conclusion that A1 receptors are involved.
A surprising finding was that the very selective A2B antagonist MRS1754 also blocked IPC protection, whereas the selective A2A antagonist CSC did not. The Ki of MRS1754 for human A1, A2A, and A2B receptors is 403, 503, and 1.97 nM, respectively (11). On the basis of the human data, the concentration of MRS1754 used would have been expected to block only A2B receptors. Interestingly the A1/A2 adenosine agonists AMP579 (29) and NECA (30) limit infarction when delivered at reperfusion, but their protection could be blocked with the A2A antagonist CSC (27, 30), which had no effect in the present study. Thus although signaling pathways for IPC and reperfusion therapy with NECA or AMP579 may both involve adenosine receptors, their respective subtypes and mechanisms may be very different. Kin et al. (12) recently studied postconditioning, another intervention that protects the heart at reperfusion. Although they found that postconditioning protection in rat hearts was also dependent on adenosine receptors, it seemed to be A3 and A2A mediated. They actually saw less adenosine release during reperfusion in postconditioned murine hearts and suggested that this might have reflected better retention of adenosine by the heart. These investigators used ZM241385 to block A2A receptors. However, at the dose used nearly all A2B receptors would also have been blocked (17), thus making it impossible to exclude the A2B receptor as the biologically important one. Thus, although all of these interventions seem to involve adenosine-dependent protection at reperfusion, it is not yet possible to unequivocally establish involvement of a particular adenosine subtype. Clearly further experimentation is warranted.
In the present study adenosine receptor blockade with 8-SPT had no effect on infarct size in nonpreconditioned hearts. Zhao et al. (34) reported that administration of 8-SPT at reperfusion to open-chest rabbits or an A1-selective blocker during the ischemic period nearly doubled infarct size in nonpreconditioned hearts. Because an A1-selective blocker could not duplicate this effect at reperfusion, they concluded that endogenous adenosine protects the nonpreconditioned heart through the A1 receptor during ischemia and through another subtype at reperfusion. The discrepancy may be related to use of different experimental models: isolated, buffer-perfused hearts in the present study and blood-perfused, in situ hearts in the report by Zhao et al. (34). In our previous study (19) 8-SPT did tend to increase infarct size in nonpreconditioned in situ rabbit hearts, but this increment was not significant. Furthermore, when nonselective adenosine blockers were administered during ischemia to in situ dog hearts, they had no effect on infarct size (2) nor did abolishing intracardiac adenosine with adenosine deaminase infusion in the in situ pig heart (25).
In conclusion, protection in the ischemically preconditioned heart is dependent on activation of adenosine receptors in the first minutes of reperfusion following termination of the index ischemia. Adenosine exerts its protection through activation of PI3 kinase and possibly ERK as well. Continuous PI3K activity during the first 30 min of reperfusion is required as transient interruption of PI3K at any time during this critical convalescent period resulted in loss of IPC protection. Observations with selective antagonists suggest that both A1 and A2B receptors are involved.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1 during early reoxygenation or reperfusion are mediated by p42/p44 MAPK. J Cardiovasc Pharmacol 38: 930–939, 2001.[CrossRef][ISI][Medline] in activation of ecto-5'-nucleotidase in the preconditioned canine myocardium. Biochem Biophys Res Commun 239: 171–175, 1997.[CrossRef][ISI][Medline]This article has been cited by other articles:
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  A. Kuno, N. V. Solenkova, V. Solodushko, T. Dost, Y. Liu, X.-M. Yang, M. V. Cohen, and J. M. Downey Infarct limitation by a protein kinase G activator at reperfusion in rabbit hearts is dependent on sensitizing the heart to A2b agonists by protein kinase C Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1288 - H1295. [Abstract] [Full Text] [PDF]
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 J. Vinten-Johansen, Z.-Q. Zhao, R. Jiang, A. J. Zatta, and G. P. Dobson Preconditioning and postconditioning: innate cardioprotection from ischemia-reperfusion injury J Appl Physiol, October 1, 2007; 103(4): 1441 - 1448. [Abstract] [Full Text] [PDF]
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R. R. Morrison, X. L. Tan, C. Ledent, S. J. Mustafa, and P. A. Hofmann Targeted deletion of A2A adenosine receptors attenuates the protective effects of myocardial postconditioning Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2523 - H2529. [Abstract] [Full Text] [PDF]
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 P. Wang, C. Zaragoza, and W. Holman Sodium-Hydrogen Exchange Inhibition and {beta}-Blockade Additively Decrease Infarct Size Ann. Thorac. Surg., March 1, 2007; 83(3): 1121 - 1127. [Abstract] [Full Text] [PDF]
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 D. K. Arrell, S. T. Elliott, L. A. Kane, Y. Guo, Y. H. Ko, P. L. Pedersen, J. Robinson, M. Murata, A. M. Murphy, E. Marban, et al. Proteomic Analysis of Pharmacological Preconditioning: Novel Protein Targets Converge to Mitochondrial Metabolism Pathways Circ. Res., September 29, 2006; 99(7): 706 - 714. [Abstract] [Full Text] [PDF]
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 D. J. Hausenloy and D. M. Yellon Survival kinases in ischemic preconditioning and postconditioning Cardiovasc Res, May 1, 2006; 70(2): 240 - 253. [Abstract] [Full Text] [PDF]
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, Individual experiments; 
, group means with SE. 8-SPT blocked the protective effect of IPC when added either 5 min before or 10 min after the onset of reperfusion. It did not block the infarct-limiting effect of IPC when infusion was delayed until 30 min after reflow. 8-SPT itself had no effect on infarction. *P < 0.05 vs. control; #P < 0.05, IPC + 8-SPT vs. IPC.
