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Am J Physiol Heart Circ Physiol 291: H748-H755, 2006. First published March 24, 2006; doi:10.1152/ajpheart.00837.2005
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Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3beta

Masahiro Nishihara,1 Tetsuji Miura,1 Takayuki Miki,1 Jun Sakamoto,1 Masaya Tanno,1,2 Hironori Kobayashi,1 Yoshihiro Ikeda,1 Katsuhiko Ohori,1 Akari Takahashi,1 and Kazuaki Shimamoto1

1Second Department of Internal Medicine and 2Department of Pharmacology, Sapporo Medical University School of Medicine, Sapporo, Japan

Submitted 5 August 2005 ; accepted in final form 21 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The aim of this study was to determine whether erythropoietin (EPO) affords additional cardioprotection to the preconditioned myocardium by enhanced phosphorylation of Akt, STAT3, or glycogen synthase kinase-3beta (GSK-3beta). Preconditioning (PC) with 5-min ischemia/5-min reperfusion and EPO (5,000 U/kg iv) reduced infarct size (as % of area at risk, %IS/AR) after 20-min ischemia in rat hearts in situ from 56.5 ± 1.8% to 25.2 ± 2.1% and to 36.2 ± 2.8%, respectively. PC-induced protection was significantly inhibited by a protein kinase C inhibitor, chelerythrine (5 mg/kg), and slightly blunted by a phosphatidylinositol-3-kinase inhibitor, wortmannin (15 µg/kg). The opposite pattern of inhibition was observed for EPO-induced protection. The combination of PC and EPO further reduced %IS/AR to 8.9 ± 1.9%, and this protection was inhibited by chelerythrine and wortmannin. The additive effects of PC and EPO on infarct size were mirrored by their effects on the level of phosphorylated GSK-3beta at 5 min after reperfusion but not their effects on the level of phospho-Akt or phospho-STAT3. To mimic phosphorylation-induced inhibition of GSK-3beta activity, SB-216763 (SB), a GSK-3beta inhibitor, was administered before ischemia or 5 min before reperfusion. Infarct size was significantly reduced by preischemic injection (%IS/AR = 40.4 ± 2.2% by 0.6 mg/kg SB and 34.0 ± 1.8% by 1.2 mg/kg SB) and also by prereperfusion injection (%IS/AR = 32.0 ± 2.0% by 1.2 mg/kg SB). These results suggest that EPO and PC afford additive infarct size-limiting effects by additive phosphorylation of GSK-3beta at the time of reperfusion by Akt-dependent and -independent mechanisms.

infarct size; phosphatidylinositol-3-kinase; protein kinase C; Akt

HUMAN RECOMBINANT ERYTHROPOIETIN (EPO) has been used for decades as a standard therapy for renal anemia. However, recent studies have shown that EPO has potent cell-protective effects in various tissues, including myocardium, in addition to its effect on erythropoiesis (5–7, 12, 16, 25, 26, 33–35, 43). The presence of the EPO receptor in cardiomyocytes has been indicated in RT-PCR and immunoblot experiments (40), and administration of EPO before or at the time of ischemia has been shown to reduce necrosis, apoptosis, and ventricular dysfunction after ischemia-reperfusion (6, 7, 12, 16, 25, 33–35, 43). These features of EPO-induced cardioprotection are similar to those of ischemic preconditioning (PC). Furthermore, it is interesting to note that there are overlaps in signaling pathways provoked by activated EPO receptors and PC: phosphatidylinositol-3-kinase (PI3K)-Akt pathway, ERK pathway, and STAT-mediated pathway (1, 3, 13, 16–18, 21, 30, 34–39, 43). However, it is not clear which signaling molecule primarily determines the level of cardioprotection against infarction. It is also unclear whether signaling pathways uncommon to PC and EPO receptor activation play roles in cardioprotection against infarction.

To get an insight into molecules that determine the level of anti-infarct tolerance, we examined whether effects of PC and EPO receptor activation are additive in terms of protection against infarction and whether there is any correlation between cardioprotection afforded by PC and EPO with activation of Akt, STAT3, or glycogen synthase kinase-3beta (GSK-3beta). We selected GSK-3beta, in addition to Akt and STAT3, as a possible determinant of anti-infarct tolerance because GSK-3beta is under regulation of Akt (9, 29) and is thought to regulate the threshold for opening of the mitochondrial permeability transition pore (23), an intracellular event determining cell viability (15). Because phosphorylation of GSK-3beta suppresses its enzymatic activity, we also assessed the relationship between timing of suppression of myocardial GSK-3beta activity and anti-infarct tolerance by using an inhibitor of GSK-3beta (SB-216763). Results in this study showed that anti-infarct tolerance afforded by PC and EPO is additive and that the level of phospho-GSK-3beta after reperfusion is correlated with anti-infarct tolerance.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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This study was approved by the Committee for Animal Research of Sapporo Medical University.

Surgical Preparation

Surgical preparation was essentially the same as in our previous studies using rats (31, 37). In brief, male Sprague-Dawley rats weighing 260–350 g were anesthetized with pentobarbital sodium (40 mg/kg ip), intubated, and mechanically ventilated with a Harvard Respirator (model 683, Harvard Apparatus, South Natick, MA). Oxygen was supplemented for ventilation to maintain arterial blood gases within physiological range. Rectal temperature in each rat was maintained within 37.5–38.5°C by the use of a heating lamp when necessary. The heart was exposed via left thoracotomy, and a coronary snare was placed around the left coronary artery by using 5-0 silk thread. Saline-filled catheters were placed in the jugular vein and the carotid artery for drug infusion and monitoring arterial blood pressure by the use of an SCK-590 pressure transducer (Nihon-Kohden, Tokyo, Japan), respectively. Precordial bipolar electrodes were placed for recording an electrocardiogram.

Infarct Size Experiments

In protocol 1, after a 20-min stabilization period following surgery, rats were divided into 12 study groups as shown in Fig. 1. Rats received no pretreatment, wortmannin (15 µg/kg), chelerythrine (5 mg/kg), EPO with or without wortmannin or chelerythrine, PC with or without wortmannin or chelerythrine, or a combination of EPO and PC with or without wortmannin or chelerythrine. EPO (human recombinant EPO, Chugai-Pharmaceutical, Tokyo, Japan) was administered intravenously at a dose of 5,000 U/kg at 15 min before ischemia, and PC was performed with a single cycle of 5-min ischemia/5-min reperfusion. Wortmannin and chelerythrine were administered 10 min before PC or 5 min before EPO injection. After pretreatment, the coronary artery was occluded for 20 min and then reperfused. In protocol 2, a GSK-3beta inhibitor (SB-216763) was used to mimic the effects of GSK-3beta phosphorylation on infarct size, and the relationship between timing of suppression of GSK-3beta activity and anti-infarct tolerance was analyzed. In this protocol, rats were divided into four study groups, and each rat received no pretreatment, 0.6 mg/kg or 1.2 mg/kg of SB-216763 at 10 min before coronary occlusion, or 1.2 mg/kg of SB-216763 at 5 min before reperfusion (Fig. 1). Occlusion of the coronary artery was confirmed by elevation of the ST segment in an ECG and cyanosis of the ischemic myocardium. After 2 h of reperfusion, 200 U of heparin were administered, and the heart was excised. The excised heart was mounted onto a Langendorff apparatus and perfused with saline to wash out the blood, and then the coronary artery was reoccluded. A saline suspension of fluorescent polymer microspheres (Duke Scientific, Palo Alto, CA) was infused into the aorta to negatively mark the area at risk, and the heart was frozen at –20°C. Frozen hearts were sliced into 2-mm sections and stained with triphenyltetrazolium chloride as previously reported. Areas of infarct and those of region at risk were determined by SigmaScan (SPSS, Chicago, IL).


Figure 1
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  		Fig. 1. Experimental protocols. Left coronary artery was occluded for 20 min and then reperfused for 2 h. Filled arrows indicate the timing of erythropoietin (EPO; 5,000 U/kg iv) injection, and open arrows indicate the timing of injection of protein kinase inhibitors [chelerythrine (Che) or wortmannin (Wort)]. Preconditioning (PC) was performed with a single cycle of 5-min ischemia and 5-min reperfusion. SB (0.6), group that received 0.6 mg/kg of SB-216763 10 min before ischemia; SB (1.2), group that received 1.2 mg/kg of SB-216763 10 min before ischemia; SB-R, group that received SB-216763 (1.2 mg/kg) at 5 min before reperfusion.

 
Immunoblot Experiments

Tissue sampling protocols. In protocol 1, rats were divided into four groups and received no treatment, EPO, PC, or EPO plus PC before 20-min coronary artery occlusion. In protocol 2, rats were assigned into three groups that received EPO plus PC, EPO plus PC with wortmannin, or EPO plus PC with chelerythrine before ischemia. PC and EPO administration with or without wortmannin or chelerythrine were performed as in the infarct size experiments (Fig. 1). At 5 min after reperfusion following 20-min ischemia, hearts were quickly excised, soaked in ice-cold saline, and then perfused with cold saline in a Langendorff mode. The risk region was negatively marked by injection of Evans blue into the aorta, and the tissue in the risk region was sampled and snap-frozen in liquid nitrogen. We selected this time point for analysis of Akt, STAT3, and GSK-3beta phosphorylation on the basis of results of pilot experiments showing that no clear phosphorylation occurred for Akt after 10 min from the onset of ischemia in EPO-pretreated hearts. Tissue samples were stored at –80°C until immunoblotting.

Immunoblotting. Frozen myocardial tissues were homogenized in ice-cold Tris buffer containing 20 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and protease cocktail (1 tablet/10 ml of buffer; Complete Mini, Roche Applied Science, Penzberg, Germany). The homogenate was centrifuged at 13,000 g for 15 min, and the supernatant was used for electrophoresis. Protein concentration was determined by using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA). Forty micrograms of protein were electrophoresed on a 10% polyacrylamide gel and then blotted onto a polyvinylidene difluoride membrane (Milipore, Bedford, MA). After blocking was performed with a Tris buffer containing 5% nonfat dry milk and 0.1% Tween 20, the blots were incubated with 1,000-fold diluted antibodies against total Akt, GSK-3beta, STAT3, 473Ser-phospho-Akt, 9Ser-phospho-GSK-3beta, or 705Tyr-phospho-STAT3 (Cell Signaling Technology, Beverly, MA). These proteins were visualized by using an ECL Western blotting detection kit (Amersham, Buckinghamshire, UK) and quantified by using SigmaGel, a gel analysis software (SPSS). Tissue samples from protocol 2 of the immunoblot experiments were used for determination of GSK-3beta alone.

Statistical Analysis

Differences in the hemodynamic parameters within and between study groups were analyzed by two-way repeated-measures ANOVA. Intergroup differences in infarct size data and immunoblot data were tested by one-way ANOVA. When overall ANOVA indicated a significant difference, multiple comparisons were conducted by Student-Newman-Keuls post hoc test. A P value <0.05 was considered to be statistically significant. Results are presented as means ± SE.


   RESULTS
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DISCUSSION
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Exclusion of Rats

In this study, 128 rats were used in the infarct size experiments. Because of problems in surgical preparation, five rats were excluded. Seven rats were excluded due to long-lasting ventricular fibrillation during coronary occlusion, the incidence of which was not different between the study groups. Nine rats were excluded because of hypotension (diastolic blood pressure < 50 mmHg) under baseline conditions. An additional 11 rats were excluded because of technical failure to adequately infuse fluorescent particles for risk area delineation. Sixty-one rats were used in the immunoblotting experiments, and two of these rats were excluded: one because the size of risk area was too small and the other because intractable ventricular fibrillation occurred during coronary occlusion.

Infarct Size Experiments

Data on heart rate and blood pressure are summarized in Table 1. There were no significant differences between these parameters before and after ischemia/reperfusion in all study groups in both protocol 1 and protocol 2. Heart weight and the size of area at risk were comparable between the study groups in each protocol (Table 2). As shown in Fig. 2, EPO alone and PC alone significantly limited infarct size as a percentage of risk area size (%IS/AR) from 56.5 ± 1.8% to 36.2 ± 2.8% and to 25.2 ± 2.1%, respectively. The combination of EPO and PC further reduced %IS/AR to 8.9 ± 1.9%. Wortmannin alone or chelerythrine alone did not modify infarct size. Wortmannin inhibited ~60% of EPO-induced infarct size limitation, whereas the effect of chelerythrine (30% inhibition) was not statistically significant. In contrast, chelerythrine suppressed PC-induced protection by 60%, whereas wortmannin tended to reduce the effect of PC by only 10%. Infarct size limitation by the combination of EPO and PC was significantly attenuated by wortmannin and by chelerythrine. In protocol 2 (Fig. 3), SB-216763 limited infarct size in a dose-dependent manner; %IS/AR was reduced from 50.1 ± 1.8% in controls to 40.4 ± 2.2% by 0.6 mg/kg of SB-216763 and to 34.0 ± 1.8% by 1.2 mg/kg of SB-216763. Administration of SB-216763 (1.2 mg/kg) at 5 min before reperfusion also significantly reduced %IS/AR to 32.0 ± 2.0%.


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  		Table 1. Summary of hemodynamic parameters for infarct size experiments

 

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  		Table 2. Summary of infarct size data

 

Figure 2
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  		Fig. 2. Effects of EPO, PC, and EPO-PC combination on infarct size (IS). IS is expressed as percentage of the area at risk (%IS/AR). *P < 0.05 vs. control, {dagger}P < 0.05 vs. EPO, {ddagger}P < 0.05 vs. PC, §P < 0.05 vs. EPO + PC; n = 6–9 rats.

 

Figure 3
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  		Fig. 3. Effects of a glycogen synthase kinase-3beta (GSK-3beta) inhibitor, SB-216763, on IS. IS is expressed as percentage of the area at risk (%IS/AR). *P < 0.05 vs. control, §§P < 0.05 vs. SB (0.6); n = 5–6 rats.

 
Immunoblot Experiments

In protocol 1, there was no significant difference in levels of total Akt, GSK-3beta, and STAT3 in the study groups (data not shown). Thus levels of phospho-Akt, phospho-GSK-3beta, and phospho-STAT3 were expressed as their densitometric levels normalized by those levels of each total protein. As shown in Figs. 4–6, EPO alone and PC alone significantly increased levels of phospho-Akt, phospho-GSK-3beta, and phospho-STAT3, respectively. The combination of EPO and PC did not further increase levels of phospho-Akt and phospho-STAT3 (Figs. 4 and 6). However, an additive effect of EPO and PC on phospho-GSK-3beta level was observed (Fig. 5A). In protocol 2, administration of wortmannin and chelerythrine significantly reduced the level of phospho-GSK-3beta in the myocardium treated with EPO and PC by 51% and by 30%, respectively (Fig. 5B). These effects of wortmannin and chelerythrine on phospho-GSK-3beta are in parallel with their effects on cardioprotection afforded by EPO plus PC (Fig. 2). Furthermore, there was a significant correlation between the group-mean level of phospho-GSK-3beta and group-mean infarct size: y = –0.24x + 72.29, r = 0.809, P < 0.05 (Fig. 5C).


Figure 4
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  		Fig. 4. Phosphorylation of Akt at 5 min after reperfusion after 20-min ischemia. Representative immunoblots and summary of phospho-Akt (p-Akt) levels. Level of Akt phosphorylation in each sample was expressed as a ratio of densitometric value of p-Akt to that of total Akt. AU, arbitrary unit. See Fig. 1 for treatment in each study group; n = 8 rats in each group. *P < 0.05 vs. control.

 

Figure 6
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  		Fig. 6. Phosphorylation of STAT3 at 5 min after reperfusion following 20-min ischemia. Representative immunoblots and summary of protein levels of phospho-STAT3 (p-STAT3). Level of STAT3 phosphorylation in each sample was expressed as a ratio of densitometric value of p-STAT3 to that of total STAT3. See Fig. 1 for treatment in each study group; n = 8 rats in each group. *P < 0.05 vs. control.

 

Figure 5
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  		Fig. 5. Phosphorylation of GSK-3beta at 5 min after reperfusion following 20-min ischemia. Representative immunoblots and summary of phospho-GSK-3beta (p-GSK-3beta) level. Level of GSK-3beta phosphorylation in each sample was expressed as a ratio of densitometric value of p-GSK-3beta to that of total GSK-3beta. See Fig. 1 for treatment in each study group. A: protocol 1 (n = 8 rats). B: protocol 2 (n = 9 rats). C: correlation between the group-mean level of p-GSK-3beta and group-mean infarct size (%IS/AR in the EPO + PC group was plotted against both p-GSK-3beta data in protocol 1 and protocol 2). *P < 0.05 vs. control, §P < 0.05 vs. EPO + PC.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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Involvement of PKC and PI3K in PC and cardioprotection afforded by EPO has been suggested by results of earlier studies showing that inhibitors of these kinases significantly suppressed protective effects of PC and EPO (1, 2, 17, 27, 30–32, 37–40). However, the magnitude of the suppression of cardioprotection was variable in those studies by using different experimental models and conditions. In the present study, the effects of a PKC inhibitor and a PI3K inhibitor differed between PC and EPO-induced protection. Chelerythrine eliminated 60% of PC-induced infarct size limitation, but its inhibitory effect on EPO-induced protection (inhibition by 30%) was not statistically significant. In contrast, wortmannin inhibited 60% of EPO-induced protection but had little inhibitory effect on PC. Theoretically, it is possible that failure of chelerythrine and that of wortmannin to abrogate protection by EPO and PC, respectively, were due to insufficient doses of these agents to inhibit a robust activation of PKC by EPO and that of PI3K by PC. If that is the case, cardioprotection afforded by the combination of EPO with PC should be resistant to inhibition by chelerythrine and wortmannin. However, both of these inhibitors substantially eliminated cardioprotection by the EPO-PC combination (Fig. 2). Comparison of calculated peak levels of wortmannin and chelerythrine in the extracellular space (163 nM and 60.7 µM) with reported IC50 data of these inhibitors for PI3K and PKC (5 nM and 0.7 µM) (22, 41, 42) also argues against the possibility that the doses of chelerythrine and wortmannin were insufficient. Although the intermediate effects of the inhibitors on infarct size are not easy to interpret, the present results suggest that the relative importance of PKC and PI3K in the protective mechanism differs between PC and EPO-induced tolerance against infarction.

Multiple signaling pathways are activated by PC and also by EPO, and there are overlaps and cross talk in such pathways. Thus it is possible that there is a signaling molecule to which cell-protective signals converge for achievement of cell protection against ischemia-reperfusion injury. However, such a molecule has not been unequivocally identified for PC or EPO. In this study, we selected Akt, STAT3, and GSK-3beta as phosphoproteins that may play a role as a determinant of the level of cardioprotection because these molecules have been shown to play crucial roles in cell survival (1), PC-induced anti-infarct tolerance (36), and elevation of the threshold for opening the mitochondrial permeability transition pore, a key event leading to cell death (15, 20). Elevations in the level of phosphorylation of Akt (Fig. 4) or that of STAT3 (Fig. 6) by EPO and/or PC were not in parallel with reduction of infarct size (Fig. 2). However, elevation of 9Ser-phospho-GSK-3beta level by EPO and/or PC was in parallel with infarct size reduction (Fig. 5, A and B, vs. Fig. 2), and there was a significant correlation between the group-mean level of phospho-GSK-3beta and group-mean infarct size (Fig. 5C). These results support the notion that GSK-3beta is a common target of converging cell-protective signals provoked by different trigger mechanisms. The mechanism by which phospho-GSK-3beta protects reperfused myocardium remains unclear, but one possibility is suppression of permeability transition pores that open at the time of reperfusion in response to oxygen radicals and calcium overload (15, 20).

A difference between Akt and GSK-3beta in phosphorylation response to EPO and PC was unexpected because GSK-3beta is a substrate of phospho-Akt (9, 29). Interestingly, chelerythrine inhibited elevation of phospho-GSK-3beta level by EPO plus PC (Fig. 5B). This finding is consistent with results of earlier studies by Ballou et al. (4) and Fang et al. (11), indicating that PKC phosphorylates 9Ser of GSK-3beta by Akt-independent mechanisms in noncardiac cells. Thus PKC may be responsible for PI3K/Akt-independent phosphorylation of GSK-3beta in hearts treated with the EPO-PC combination, although the possibility of involvement of other kinases cannot be excluded.

Because no selective activator of GSK-3beta is currently available, we could not directly test whether suppression of GSK-3beta activity by increased 9Ser-phosphorylation of this molecule was sufficient for cardioprotection afforded by combination of EPO and PC. Thus we used SB-216763, which inhibits GSK-3beta activity in an ATP competitive manner (8). As reported by Gross et al. (14), SB-216763 administered 5 min before reperfusion was as protective as its injection before ischemia (Fig. 3). Because coronary collateral flow level is very low in the rat heart (28), cardioprotection afforded by SB-216763 administered shortly before reperfusion is attributable to suppression of GSK-3beta activity on reperfusion. Taken together, these results support the notion that enhanced phosphorylation of GSK-3beta, reducing its activity, on reperfusion protects myocytes from necrosis.

Recently, efficacy of EPO for suppressing lethal reperfusion injury was suggested by results of studies by Lipsic et al. (25) and Bullard et al. (5). They reported that EPO injected 5 min after reperfusion or 10 or 5 min before reperfusion limited infarct size in rat hearts in situ and those in vitro by approximately 40–60%. Interestingly, this cardioprotection was not accompanied by activation of Akt after reperfusion but was abolished by coadministration of a PI3K inhibitor (wortmannin or LY-294002) (5). This apparent discrepancy could be due to inappropriate timing of assessment of Akt phosphorylation or due to the presence of a PI3K-dependent but Akt-independent mechanism. Nevertheless, the present study showed that Akt was activated on reperfusion by EPO administered before ischemia and PC. Thus the role of Akt in protection of the myocardium from ischemia-reperfusion-induced necrosis may differ depending on the timing of EPO receptor activation.

There are limitations in the present study. First, we used only a single protocol of PC and a single dose of EPO, both of which afforded modest limitation of infarct size. Thus we cannot exclude the possibility that relative contributions of PKC and PI3K to PC- and EPO-induced protection are not different when a more potent PC protocol (for example, 2 cycles of 5-min ischemia/5-min reperfusion) and a higher dose of EPO are used. Second, we used transmural tissue samples for assessment of changes in levels of phosphorylated protein kinases in the myocardium at risk because separation of viable and necrotic tissues was not technically possible for thin rat ventricles. Accordingly, the increases in the levels of phospho-Akt, -STAT3, or -GSK-3beta by EPO or PC shown in the present study do not necessarily reflect their changes in the myocardium salvaged after ischemia-reperfusion. In fact, a recent study by Darling et al. (10) showed that elevation of phospho-Akt levels by postconditioning was detected in transmural samples but not in viable subepicardial samples from rabbit hearts. However, the findings that inhibitors of GSK-3beta reduced infarct size in this and earlier studies (13, 37, 38) support the notion that elevation of phospho-GSK-3beta level by EPO and/or PC, leading to suppression of its activity, is a mechanism, but not simply the result, of cardioprotection.

Because the primary purpose of this study was to get an insight into determinants of anti-infarct tolerance, we selected 5,000 U/kg as a dose of EPO, which has been shown in earlier studies (5, 25, 33) to confer significant cardioprotection against infarction. Whether lower doses of EPO can activate protective signal pathways leading to cardioprotection, similar to those activated by 5,000 U/kg EPO (Figs. 4–6), remains to be determined. The dose of EPO used in this study is much higher than doses of EPO being clinically used for treatment of anemia and will not be directly applicable for cardioprotection in patients because of side effects such as erythrocytemia, hypertension, and thrombosis. However, this problem in clinical application may be overcome by the use of nonerythropoietic derivatives of EPO, such as carbamylated EPO (24).

In summary, the present study showed that additional cardioprotection can be achieved by a combination of PC and EPO before ischemia, and it is likely that the level of phosphorylation of GSK-3beta by Akt-dependent and -independent signaling is a primary determinant of myocardial tolerance against lethal ischemia-reperfusion injury.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 GRANTS
REFERENCES
 
This study was supported by Grant 08670812 (T. Miura) from the Japan Society for the Promotion of Science, Tokyo, Japan; a grant from Sapporo Medical University Academic Foundation, Sapporo, Japan; and a grant from Chugai Pharmaceutical, Tokyo, Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Miura, Second Dept. of Internal Medicine, Sapporo Medical Univ. School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060–8543, Japan (e-mail: miura{at}sapmed.ac.jp)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Armstrong SC. Protein kinase activation and myocardial ischemia/reperfusion injury. Cardiovasc Res 61: 427–436, 2004.[Abstract/Free Full Text]
  2. Baines CP, Wang L, Cohen MV, and Downey JM. Myocardial protection by insulin is dependent on phosphatidylinositol 3-kinase but not protein kinase C or KATP channels in the isolated rabbit heart. Basic Res Cardiol 94: 188–198, 1999.[CrossRef][ISI][Medline]
  3. Baines CP, Zhang J, Wang GW, Zheng YT, Xiu JX, Cardwell EM, Bolli R, and Ping P. Mitochondrial PKC{epsilon} and MAPK form signaling modules in the murine heart: enhanced mitochondrial PKC{epsilon}-MAPK interactions and differential MAPK activation in PKC{epsilon}-induced cardioprotection. Circ Res 90: 390–397, 2002.[Abstract/Free Full Text]
  4. Ballou LM, Tian PY, Lin HY, Jiang YP, and Lin RZ. Dual regulation of glycogen synthase kinase-3beta by the {alpha}1A-adrenergic receptor. J Biol Chem 276: 40910–40916, 2001.[Abstract/Free Full Text]
  5. Bullard AJ, Govewalla P, and Yellon DM. Erythropoietin protects the myocardium against reperfusion injury in vitro and in vivo. Basic Res Cardiol 100: 397–403, 2005.[CrossRef][ISI][Medline]
  6. Cai Z and Semenza GL. Phosphatidylinositol-3-kinase signaling is required for erythropoietin-mediated acute protection against myocardial ischemia/reperfusion injury. Circulation 109: 2050–2053, 2004.[Abstract/Free Full Text]
  7. Calvillo L, Latini R, Kajstura J, Leri A, Anversa P, Ghezzi P, Salio M, Cerami A, and Brines M. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling. Proc Natl Acad Sci USA 100: 4802–4806, 2003.[Abstract/Free Full Text]
  8. Coghlan MP, Culbert AA, Cross DA, Corcoran SL, Yates JW, Pearce NJ, Rausch OL, Murphy GJ, Carter PS, Roxbee Cox L, Mills D, Brown MJ, Haigh D, Ward RW, Smith DG, Murray KJ, Reith AD, and Holder JC. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol 7: 793–803, 2000.[CrossRef][ISI][Medline]
  9. Cohen P and Goedert M. GSK3 inhibitors: development and therapeutic potential. Nat Rev Drug Discov 3: 479–487, 2004.[CrossRef][ISI][Medline]
  10. Darling CE, Jiang R, Maynard M, Whittaker P, Vinten-Johansen J, and Przyklenk K. Postconditioning via stuttering reperfusion limits myocardial infarct size in rabbit hearts: role of ERK1/2. Am J Physiol Heart Circ Physiol 289: H1618–H1626, 2005.[Abstract/Free Full Text]
  11. Fang X, Yu S, Tanyi JL, Lu Y, Woodgett JR, and Mills GB. Convergence of multiple signaling cascades at glycogen synthase kinase 3: Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase C-dependent intracellular pathway. Mol Cell Biol 22: 2099–2110, 2002.[Abstract/Free Full Text]
  12. Fiordaliso F, Chimenti S, Staszewsky L, Bai A, Carlo E, Cuccovillo I, Doni M, Mengozzi M, Tonelli R, Ghezzi P, Coleman T, Brines M, Cerami A, and Latini R. A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia-reperfusion injury. Proc Natl Acad Sci USA 102: 2046–2051, 2005.[Abstract/Free Full Text]
  13. Fryer RM, Pratt PF, Hsu AK, and Gross GJ. Differential activation of extracellular signal regulated kinase isoforms in preconditioning and opioid-induced cardioprotection. J Pharmacol Exp Ther 296: 642–649, 2001.[Abstract/Free Full Text]
  14. Gross ER, Hsu AK, and Gross GJ. Opioid-induced cardioprotection occurs via glycogen synthase kinase-beta inhibition during reperfusion in intact rat hearts. Circ Res 94: 960–966, 2004.[Abstract/Free Full Text]
  15. Halestrap AP, Clarke SJ, and Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion—a target for cardioprotection. Cardiovasc Res 61: 372–385, 2004.[Abstract/Free Full Text]
  16. Hanlon PR, Fu P, Wright GL, Steenbergen C, Arcasoy MO, and Murphy E. Mechanisms of erythropoietin-mediated cardioprotection during ischemia-reperfusion injury: role of protein kinase C and phosphatidylinositol 3-kinase signaling. FASEB J 19: 1323–1325, 2005.[Abstract/Free Full Text]
  17. Hassouna A, Matata BM, and Galinanes M. PKC-{epsilon} is upstream and PKC-{alpha} is downstream of mitoKATP channels in the signal transduction pathway of ischemic preconditioning of human myocardium. Am J Physiol Cell Physiol 287: C1418–C1425, 2004.[Abstract/Free Full Text]
  18. Hattori R, Maulik N, Otani H, Zhu L, Cordis G, Engelman RM, Siddiqui MA, and Das DK. Role of STAT3 in ischemic preconditioning. J Mol Cell Cardiol 33: 1929–1936, 2001.[CrossRef][ISI][Medline]
  19. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M, Matsuura K, Sano M, Nishi J, Iwanaga K, Akazawa H, Kunieda T, Zhu W, Hasegawa H, Kunisada K, Nagai T, Nakaya H, Yamauchi-Takihara K, and Komuro I. G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat Med 11: 305–311, 2005.[CrossRef][ISI][Medline]
  20. Hausenloy DJ, Duchen MR, and Yellon DM. Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia-reperfusion injury. Cardiovasc Res 60: 617–625, 2003.[Abstract/Free Full Text]
  21. Hausenloy DJ, Tsang A, Mocanu MM, and Yellon DM. Ischemic preconditioning protects by activating prosurvival kinases at reperfusion. Am J Physiol Heart Circ Physiol 288: H971–H976, 2005.[Abstract/Free Full Text]
  22. Herbert JM, Augereau JM, Gleye J, and Maffrand JP. Chelerythrine is potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 172: 993–999, 1990.[CrossRef][ISI][Medline]
  23. Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, Ziman BD, Wang S, Ytrehus K, Antos CL, Olson EN, and Sollott SJ. Glycogen synthase kinase-3beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest 113: 1535–1549, 2004.[CrossRef][ISI][Medline]
  24. Leist M, Ghezzi P, Grasso G, Bianchi R, Villa P, Fratelli M, Savino C, Bianchi M, Nielsen J, Gerwien J, Kallunki P, Larsen AK, Helboe L, Christensen S, Pedersen LO, Nielsen M, Torup L, Sager T, Sfacteria A, Erbayraktar S, Erbayraktar Z, Gokmen N, Yilmaz O, Cerami-Hand C, Xie QW, Coleman T, Cerami A, and Brines M. Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science 305: 239–242, 2004.[Abstract/Free Full Text]
  25. Lipsic E, van der Meer P, Henning RH, Suurmeijer AJ, Boddeus KM, van Veldhuisen DJ, van Gilst WH, and Schoemaker RG. Timing of erythropoietin treatment for cardioprotection in ischemia/reperfusion. J Cardiovasc Pharmacol 44: 473–479, 2004.[CrossRef][ISI][Medline]
  26. Maiese K, Li F, and Chong ZZ. New avenues of exploration for erythropoietin. JAMA 293: 90–95, 2005.[Abstract/Free Full Text]
  27. Mayr M, Metzler B, Chung YL, McGregor E, Mayr U, Troy H, Hu Y, Leitges M, Pachinger O, Griffiths JR, Dunn MJ, and Xu Q. Ischemic preconditioning exaggerates cardiac damage in PKC-{delta} null mice. Am J Physiol Heart Circ Physiol 287: H946–H956, 2004.[Abstract/Free Full Text]
  28. Maxwell MP, Hearse DJ, and Yellon DM. Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc Res 21: 737–746, 1987.[ISI][Medline]
  29. Meijer L, Flajolet M, and Greengard P. Pharmacological inhibitors of glycogen synthase kinase 3. Trends Pharmacol Sci 25: 471–480, 2004.[CrossRef][Medline]
  30. Murphy E. Primary and secondary signaling pathways in early preconditioning that converge on the mitochondria to produce cardioprotection. Circ Res 94: 7–16, 2004.[Abstract/Free Full Text]
  31. Nozawa Y, Miura T, Miki T, Ohnuma Y, Yano T, and Shimamoto K. Mitochondrial KATP channel-dependent and -independent phases of ischemic preconditioning against myocardial infarction in the rat. Basic Res Cardiol 98: 50–58, 2003.[CrossRef][ISI][Medline]
  32. Qin Q, Downey JM, and Cohen MV. Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase. Am J Physiol Heart Circ Physiol 284: H727–H734, 2003.[Abstract/Free Full Text]
  33. Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, Walton GB, Thompson RB, Petrofski JA, Annex BH, Stamler JS, and Koch WJ. A novel protective effect of erythropoietin in the infarcted heart. J Clin Invest 112: 999–1007, 2003.[CrossRef][ISI][Medline]
  34. Rafiee P, Shi Y, Su J, Pritchard KA Jr, Tweddell JS, and Baker JE. Erythropoietin protects the infant heart against ischemia-reperfusion injury by triggering multiple signaling pathways. Basic Res Cardiol 100: 187–197, 2005.[CrossRef][ISI][Medline]
  35. Shi Y, Rafiee P, Su J, Pritchard KA Jr, Tweddell JS, and Baker JE. Acute cardioprotective effects of erythropoietin in infant rabbits are mediated by activation of protein kinases and potassium channels. Basic Res Cardiol 99: 173–182, 2004.[CrossRef][ISI][Medline]
  36. Smith RM, Suleman N, Lacerda L, Opie LH, Akira S, Chien KR, and Sack MN. Genetic depletion of cardiac myocyte STAT-3 abolishes classical preconditioning. Cardiovasc Res 63: 611–616, 2004.[Abstract/Free Full Text]
  37. Tanno M, Tsuchida A, Nozawa Y, Matsumoto T, Hasegawa T, Miura T, and Shimamoto K. Roles of tyrosine kinase and protein kinase C in infarct size limitation by repetitive ischemic preconditioning in the rat. J Cardiovasc Pharmacol 35: 345–352, 2000.[CrossRef][ISI][Medline]
  38. Tong H, Chen W, Steenbergen C, and Murphy E. Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. Circ Res 87: 309–315, 2000.[Abstract/Free Full Text]
  39. Tong H, Imahashi K, Steenbergen C, and Murphy E. Phosphorylation of glycogen synthase kinase-3beta during preconditioning through a phosphatidylinositol-3-kinase-dependent pathway is cardioprotective. Circ Res 90: 377–379, 2002.[Abstract/Free Full Text]
  40. Vahlhaus C, Schulz R, Post H, Rose J, and Heusch G. Prevention of ischemic preconditioning only by combined inhibition of protein kinase C and protein tyrosine kinase in pigs. J Mol Cell Cardiol 30: 197–209, 1998.[CrossRef][ISI][Medline]
  41. Von Stebut E, Amon U, Herbert JM, and Wolff HH. Investigations with the selective PKC inhibitor chelerythrine on human basophils. Agents Actions 41: C56–C57, 1994.
  42. Walker EH, Pacold ME, Perisic O, Stephens L, Hawkins PT, Wymann MP, and Williams RL. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin and staurosporine. Mol Cell 6: 909–919, 2000.[CrossRef][ISI][Medline]
  43. Wright GL, Hanlon P, Amin K, Steenbergen C, Murphy E, and Arcasoy MO. Erythropoietin receptor expression in adult rat cardiomyocytes is associated with an acute cardioprotective effect for recombinant erythropoietin during ischemia-reperfusion injury. FASEB J 18: 1031–1033, 2004.[Abstract/Free Full Text]



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