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Mechanisms of erythropoietin-mediated cardioprotection during ischemia-reperfusion injury: role of protein kinase C and phosphatidylinositol 3-kinase signaling

Paul R. Hanlon^*, Ping Fu, Gary L. Wright^*,1, Charles Steenbergen, Murat O. Arcasoy^,2 and Elizabeth Murphy^*

^* Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA; and Departments of
Medicine and
Pathology, Duke University Medical Center, Durham, North Carolina, USA

²Correspondence: Department of Medicine, Divisions of Hematology and Medical Oncology, Duke University School of Medicine, DUMC Box 3912, Durham, NC 27710, USA. E-mail: arcas001{at}mc.duke.edu

SPECIFIC AIMS

The aims of the present study were to determine 1) which erythropoietin (EPO) -stimulated signaling pathways in the heart mediate the cardioprotective effect of EPO administration prior to ischemia-reperfusion injury in the isolated, Langendorff-perfused rat heart; and 2) whether postischemia EPO treatment is also cardioprotective and, if so, whether this effect is through the same signaling mechanism that mediates the cardioprotection afforded by EPO treatment prior to ischemia.

PRINCIPAL FINDINGS

1. Activation of the protein kinase C (PKC) pathway is required for the cardioprotective effect of EPO administered prior to ischemia-reperfusion injury
Erythropoietin (EPO) is a hematopoietic cytokine required for the regulation of mammalian erythropoiesis. In a series of preclinical studies investigating effects of EPO in nonhematopoietic tissues, recombinant EPO administration has been shown to exert a major tissue-protective effect in experimental models of ischemic injury involving neurons, retina, and the kidney. Studies in our laboratory, as well as others, have shown that recombinant EPO also protects cardiac myocytes against ischemic injury in a number of different model systems. In previous work, we showed that recombinant EPO exerts a cardioprotective effect when administered directly to the isolated, Langendorff-perfused rat heart immediately prior to ischemia-reperfusion injury. The short period of EPO treatment suggested that EPO-mediated protection occurred through a rapid mechanism, such as posttranslational modification of second messengers, rather than through mechanisms that required initiation of gene transcription and new protein synthesis. In the present study we investigated mechanisms of the acute cardioprotective effect of EPO in the isolated Langendorff-perfused heart model focusing on specific signaling pathways including the phosphatidylinositol-3-kinase (PI3K)/Akt, mitogen/extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK), and PKC pathways.

Treatment of isolated, perfused adult rat hearts with recombinant human EPO (Procrit) resulted in a significant increase in the percentage of the PKC isoform located in the membrane fraction, whereas EPO treatment had no effect on PKC translocation (Fig. 1 A, B). When chelerythrine, an inhibitor of the PKC catalytic site, was administered to hearts along with EPO treatment prior to ischemia, EPO-mediated improvement in postischemic recovery of function, measured by left ventricular developed pressure (LVDP), was significantly decreased (71%±3 for EPO-treated vs. 53%±4 in chelerythrine+EPO-treated hearts, P<0.0005, Fig. 1C ). Although treatment of the hearts with chelerythrine alone produced a small but significant increase in the postischemic recovery of LVDP (36%±3 in control vs. 50%±5 in chelerythrine-treated hearts, P<0.01), in the presence of chelerythrine EPO treatment did not improve recovery of LVDP (53%±4 for EPO+chelerythrine-treated vs. 50%±5 for chelerythrine-treated hearts). In separate experiments using myocardial 2,3,5-triphenyltetrazolium chloride (TTC) staining after ischemia-reperfusion injury, we determined that the ability of EPO pretreatment to improve functional recovery was associated with a significant reduction in infarct size (62%±6 in control vs. 42%±5 in EPO-treated, P<0.05), through a mechanism that was also blocked by the addition of PKC inhibitor chelerythrine (56%±2 in EPO+chelerythrine vs. 42%±5 in EPO-treated, P<0.05). Thus, we conclude that the rapid cardioprotective effect of EPO is mediated at least in part by the activation of the PKC-signaling pathway prior to or during ischemia.

View larger version (19K): [in this window] [in a new window]   Figure 1. EPO mediates translocation of PKC and PKC inhibitor chelerythrine inhibits the cardioprotective effect of EPO. A, B) Langendorff-perfused hearts were either left untreated (control) or treated with EPO (10 units/mL) for 15 min. A) Representative Western blots for PKC and PKC are shown. Membrane (memb) and cytosol (cyto) fractions from untreated and EPO-treated hearts are indicated. B) Quantitative representation of the amount of PKC protein in the membrane fraction for PKC and PKC isoforms in untreated (control) and EPO-treated hearts (n=3 in each group). The data represent the mean (±SE) protein amount in the membrane fraction expressed as a percentage of the total PKC amount. *P < 0.05 compared with control by t test. C) Recovery of cardiac function in response to EPO in the presence of chelerythrine. Beginning 5 min prior to EPO treatment and continuing during EPO treatment, hearts were perfused with 5 µM chelerythrine. Data represent the mean percentage of LVDP recovery ±SE. a) P < 0.01 compared with control group. b) P < 0.0005 compared with EPO + chelerythrine group. c) P < 0.0001 compared with chelerythrine alone group.

EPO treatment of perfused rat hearts also activated the MEK/ERK and PI3K-signaling pathways, both of which have been implicated in cardioprotection. Administration of U0126, a pharmacologic inhibitor of MEK, 5 min prior to and concomitant with EPO treatment inhibited constitutive and EPO-mediated phosphorylation of ERK 1 and 2 (substrates of MEK), but had no effect on EPO-mediated cardioprotection (71%±3 for EPO-treated vs. 76%±6 for EPO+U0126-treated hearts). Similarly, treatment of hearts with the PI3K inhibitor LY294002 beginning 5 min prior to and concomitant with EPO treatment blocked the EPO-induced increase in phosphorylation of Akt (a substrate of PI3K), but did not inhibit the cardioprotective effect of EPO (71%±3 in EPO-treated vs. 75%±6 in LY294002+EPO-treated hearts). Similar results were observed using a different PI3K inhibitor, wortmannin, administered prior to ischemia along with EPO. When wortmannin was administered both prior to ischemia and during reperfusion, EPO-mediated improvement in postischemic recovery of LVDP was significantly inhibited (71%±3 in EPO-treated vs. 51%±5 in EPO+wortmannin-treated hearts, P<0.001), suggesting that PI3K activity during reperfusion, but not prior to ischemia, mediates the cardioprotective effect of EPO.

2. Postischemia EPO treatment mediates significant cardioprotection through a mechanism that requires PI3K activity during reperfusion
EPO treatment upon reperfusion resulted in significant improvement of LVDP recovery (45%±4 in control hearts vs. 61%±1 in EPO-treated hearts, P<0.01; Fig. 2 ). Concomitant treatment with chelerythrine and EPO during reperfusion did not affect EPO-mediated recovery of function (69%±4 in EPO+chelerythrine-treated vs. 61%±1 in EPO-treated hearts). However, concomitant treatment with LY294002 and EPO at the time of reperfusion significantly inhibited the EPO-induced improvement in postischemic function (48%±3 in EPO+LY294002-treated vs. 61%±1 in EPO-treated hearts, P<0.05). We also found that postischemia EPO treatment significantly reduced infarct size (62%±6 in control vs. 37%±3 postischemia EPO-treated, P<0.05) through a mechanism that was also blocked by the addition of LY294002 (60%±4 in postischemia EPO+LY294002-treated vs. 37%±3 in EPO-treated, P<0.05). These data indicate that postischemia EPO treatment at the onset of reperfusion is cardioprotective, but to a lesser extent than pre-ischemia EPO treatment, and that PI3K activity during reperfusion is critical for the postischemia cardioprotective effect of EPO.

View larger version (24K): [in this window] [in a new window]   Figure 2. EPO treatment upon reperfusion is cardioprotective through a mechanism dependent on PI3K activity. EPO and inhibitor, 5 µM chelerythrine (chel) or 10 µM LY294002 (LY), treatment was administered immediately after global ischemia at the onset of reperfusion. Recovery of LVDP (% initial) was measured in control, inhibitor (no EPO) -treated, postischemia EPO-treated, and postischemia EPO + inhibitor-treated hearts. a: P < 0.01 compared with control group. b: P < 0.0005 compared with EPO + chel group. c: P < 0.05 compared with EPO-treated group.

CONCLUSIONS AND SIGNIFICANCE

The important contributions of the present studies are: 1) treatment of hearts with EPO prior to ischemia is cardioprotective through a mechanism that is mediated by activation of the PKC pathway associated with EPO-induced translocation of the PKC isoform; 2) postischemia EPO treatment of the perfused heart at the time of reperfusion is cardioprotective with significant improvement of LVDP recovery and reduction in infarct size; 3) activation of MEK/ERK- and PI3K/Akt-signaling pathways prior to ischemia-reperfusion injury is not required for the cardioprotective effect of EPO pretreatment; and 4) PI3K activity during reperfusion is required for the cardioprotective effects of EPO regardless of whether hearts are treated with EPO prior to ischemia or at reperfusion.

The role of PKC in EPO-induced cardioprotection is consistent with a growing body of recent experimental evidence suggesting that PKC protects the heart against ischemic injury. Our studies revealed that EPO treatment of the heart led to translocation of PKC, but not PKC, consistent with the possibility that PKC is the isoform that mediates the cardioprotective effect of EPO. Activation of PKC by ischemic preconditioning has been shown to elicit a cardioprotective effect by opening mitochondrial K-ATP channels that have been shown to be important in other models of cardioprotection. A recent study using perfused infant rabbit hearts has shown that inhibition of the mitochondrial K-ATP channel can prevent EPO-mediated cardioprotection.

Our data indicate that PI3K inhibitor treatment given prior to ischemia and continued during reperfusion eliminated EPO-mediated protection, consistent with the results of a previous study that implicated the PI3K pathway in EPO-mediated cardioprotection in the isolated, Langendorff-perfused rat heart model. Our studies also show that PI3K inhibition only prior to ischemia did not block the protective effect of EPO, consistent with a requirement for PI3K activity during reperfusion for EPO-mediated cardioprotective effect.

Another important finding of our studies is the ability of EPO to protect the isolated, perfused heart when the drug is administered after ischemic injury at the onset of reperfusion. The cardioprotective effect of EPO given after ischemia was dependent on PI3K pathway activity during reperfusion. A similar requirement for PI3K activity during reperfusion has been reported for the cardioprotective effects of ischemic preconditioning that can be blocked by addition of the PI3K inhibitor LY294002 only on reperfusion. Inhibition of the PKC pathway during reperfusion had no effect on EPO-mediated cardioprotection when EPO was given postischemia during reperfusion.

Taken together, these data demonstrate EPO-induced activation of several signaling pathways in the isolated, adult Langendorff-perfused rat heart including MEK/ERK, PI3K/Akt, and PKC pathways (Fig. 3 ). EPO-mediated cardioprotection requires PKC activation prior to ischemia and the activity of PI3K/AKT pathway during reperfusion. Future studies will investigate EPO receptor structure-function relationships in the heart and the identification of receptor domains required for EPO-mediated activation of intracellular signaling and cardioprotection.

View larger version (16K): [in this window] [in a new window]   Figure 3. EPO treatment of isolated, perfused, adult rat hearts activates the PKC-, PI3K/Akt-, and MEK/ERK-signaling pathways. When hearts were treated with EPO prior to ischemia, the cardioprotective effect of EPO was blocked by addition of a PKC inhibitor (chel) but not by inhibitors of PI3K (LY) or MEK (U0) activity. EPO treatment prior to ischemia led to translocation of PKC, but not PKC, suggesting that the cardioprotection afforded by EPO pretreatment may be mediated by EPO-induced activation of PKC. In contrast, when EPO was administered postischemia at the time of reperfusion, EPO-mediated cardioprotection was blocked by inhibition of PI3K (LY) but not PKC (chel), demonstrating that the cardioprotection afforded by EPO treatment at reperfusion is mediated by activation of PI3K. When EPO was administered prior to ischemia, inhibition of PI3K both concomitant with EPO treatment and during reperfusion resulted in elimination of EPO-mediated cardioprotection, demonstrating that cardioprotection afforded by EPO treatment prior to ischemia also requires PI3K activity during reperfusion.

FOOTNOTES

¹ Present address: Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3545fje;

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