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(Circulation. 2004;109:2050-2053.)
© 2004 American Heart Association, Inc.
Brief Rapid Communications |
From the Institute for Cell Engineering, McKusick-Nathans Institute of Genetic Medicine, and Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Dr Gregg L. Semenza, 733 N Broadway, Suite 671, Baltimore, MD 21205. E-mail gsemenza{at}jhmi.edu
Received November 7, 2003; de novo received January 22, 2004; revision received March 23, 2004; accepted March 23, 2004.
| Abstract |
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Methods and Results rhEPO was perfused into isolated rat hearts over 15 minutes immediately before 30 minutes of no-flow ischemia and 45 minutes of reperfusion. Compared with saline-perfused control hearts, recovery of left ventricular developed pressure was increased in rhEPO-perfused hearts. rhEPO also increased AKT activity and decreased apoptosis. All of these effects were blocked when the phosphatidylinositol-3-kinase inhibitor wortmannin was infused with rhEPO.
Conclusions rhEPO provides immediate protection against ischemia/reperfusion injury in the isolated perfused rat heart that is mediated by the phosphatidylinositol-3-kinase pathway.
Key Words: erythropoietin ischemia reperfusion myocardial infarction
| Introduction |
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| Methods |
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Immunoblot Assays
Hearts were lysed in 50 mmol/L Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, and 1 mmol/L EGTA, and aliquots were subjected to immunoblot assays with the use of antibodies that recognize the following: phosphorylated or total AKT (Biosource International); phosphorylated or total p70S6K (Santa Cruz Biotechnology); cleaved or noncleaved caspase 3 (Cell Signaling Technology); EPO receptor (R&D Systems); phosphatidylinositol-3-kinase (Upstate Biotechnology); and tubulin (Santa Cruz). Signals from scanned immunoblots were quantified with the use of Image J1.30 software (National Institutes of Health). Immunoprecipitation was performed with the use of antiphosphatidylinositol-3-kinase antibody.7
Laddering Assay
DNA was isolated from heart homogenates by phenol/chloroform/isoamyl alcohol extraction followed by ammonium acetate/isopropanol precipitation. DNA aliquots (2 µg) were analyzed by 1.5% agarose gel electrophoresis followed by ethidium bromide staining.
| Results |
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AKT (protein kinase B), a serine-threonine kinase that is activated by phosphatidylinositol-3-kinase, plays a critical role in promoting cell survival by inhibiting the activation of caspases, which function as effectors of the apoptotic program.8 Expression of an activated form of AKT protects against cardiac ischemia in vivo.9 EPO treatment activates AKT and promotes the survival of cultured endothelial cells10 and cardiomyocytes5,6 subjected to O2 deprivation. Phosphatidylinositol-3-kinase signaling, which is stimulated by binding of EPO to its receptor (EPOR),11 leads to the activation of AKT by phosphorylation on threonine-308 and serine-473.8 Analysis of lysates prepared from nonischemic hearts immediately after perfusion with rhEPO revealed association of EPOR with the p85 subunit of phosphatidylinositol-3-kinase, as demonstrated by coimmunoprecipitation (Figure 2A), and a significant increase in the levels of activated (phosphorylated) AKT compared with control hearts (Figure 2B). To demonstrate that phosphatidylinositol-3-kinase activity is required for the activation of AKT in response to rhEPO treatment, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, was added to the perfusate. Wortmannin blocked the phosphorylation of AKT but had no effect on total AKT protein levels or on the association of EPOR and p85.
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Analysis of lysates prepared from hearts that were subjected to ischemia/reperfusion revealed that rhEPO increased activation of AKT, which was completely blocked by addition of wortmannin to the perfusate (Figure 2C). Phosphorylation of p70 S6 kinase (p70S6K), a downstream target of AKT, was induced by rhEPO in a phosphatidylinositol-3-kinasedependent manner (Figure 2D). rhEPO significantly decreased the cleavage of caspase 3 to its activated form, whereas addition of wortmannin increased the levels of activated caspase 3 (Figure 2E). Ischemia/reperfusion induced apoptotic DNA laddering and caspase 3 activation, effects that were blocked by rhEPO in the absence, but not in the presence, of wortmannin (Figure 2F). Finally, the increased recovery of LVDP and CFR and reduction in LVEDP in rhEPO-treated hearts after ischemia/reperfusion were completely blocked by coadministration of wortmannin (Figure 1).
| Discussion |
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Treatment with rhEPO induced an increase in CFR at the onset of reperfusion that may contribute to the reduced LVEDP and increased LVDP observed in rhEPO-treated hearts. This effect of rhEPO on CFR may be due to activation of endothelial NO synthase via AKT-mediated phosphorylation, leading to NO-mediated vascular dilatation.12,13 rhEPO may also promote endothelial cell survival.10
In addition to potential effects of rhEPO on endothelium, our data demonstrate that the rhEPO-mediated reduction in myocardial apoptosis and caspase 3 activation after ischemia/reperfusion is dependent on phosphatidylinositol-3-kinase signaling. The antiapoptotic effect of AKT is well established via its direct phosphorylation and inactivation of multiple proapoptotic proteins, including caspase 9, an upstream activator of caspase 3.8 Phosphatidylinositol-3-kinase/AKT signaling has been implicated in cardiac protection induced by ischemic preconditioning, insulin-like growth factor 1, and insulin.1416 Thus, therapeutic strategies designed to induce activation of the phosphatidylinositol-3-kinase/AKT signal-transduction pathway may protect patients against ischemia/reperfusion injury. In addition to reducing infarct size, EPO may have additional therapeutic effects in vivo, such as recruitment of vascular progenitor cells,17 that may promote tissue repair.
| Acknowledgments |
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| References |
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