| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 1, 2004 as doi:10.1096/fj.03-1289fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA;
Biosciences Division, SRI International, Menlo Park, California, USA; and Departments of
¶ Pathology and
|| Medicine, Duke University Medical Center, Durham, North Carolina, USA
4Correspondence: Department of Medicine, Division of Hematology, 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 1) investigate for erythropoietin receptor (EPOR) expression in cardiac myocytes, 2) characterize direct effects of EPO on the heart to test the hypothesis that recombinant EPO may exert an acute cardioprotective effect during ischemia-reperfusion injury, and 3) determine whether EPO administration may modulate intracellular pH and/or high-energy phosphate levels in ischemic myocardium. Adult male Sprague-Dawley rats weighing 
250–300 g were used.
PRINCIPAL FINDINGS
1. Cardiac myocytes express EPOR and recombinant EPO administration to the isolated, perfused heart protects the myocardium from ischemia-reperfusion injury
EPOR expression has been described in the hearts of the developing mouse and human fetus by immunohistochemistry, but EPOR expression in adult myocardium has not been reported to date. We first characterized expression of EPOR in adult rat cardiac myocytes and found EPOR mRNA transcript and protein expression by RT-PCR and immunohistochemistry. Next we investigated the ability of recombinant EPO to exert a direct cardioprotective effect during ischemia-reperfusion injury using an experimental model of isolated rat hearts perfused in the Langendorff mode with Krebs-Henseleit (KH) buffer. All isolated, perfused hearts were stabilized for 30 min, followed by a 10 min treatment protocol with either vehicle (group I, controls) or recombinant EPO (group II). The treatment was followed by 20 min of normothermic global ischemia and 25 min of reperfusion with KH buffer. EPO was administered directly to the isolated, perfused heart in the perfusion buffer (final concentration 10 units/mL) for 10 min immediately prior to global ischemia. Left ventricular-developed pressure (LVDP) was measured continuously to assess contractile function. Postischemic recovery of contractile function was examined by measuring LVDP at 25 min of reflow, expressed as a percentage of the baseline pretreatment measurement. Figure 1
A illustrates the results of a representative set of experiments. In control hearts (group I), LVDP recovered to 26 ± 5% of the pretreatment LVDP; hearts pretreated with EPO (group II) exhibited significant improvement of contractile function recovery to 57 ± 7% (P<0.001). In the next set of experiments, we performed concentration response studies to search for the presence of a dose-dependent increase in the cardioprotective effect of recombinant EPO. Isolated, perfused hearts were treated for 10 min with increasing concentrations of recombinant EPO at a final concentration of 1, 5, or 10 units/mL. Results of these studies are illustrated in Fig. 1B
. We found a concentration-dependent increase in the cardioprotective effect of EPO. Control hearts were treated with vehicle and recovered to 35 ± 5% of pretreatment LVDP whereas hearts treated with 1 unit/mL of EPO recovered to 53 ± 11% (not significant); hearts that received 5 units/mL or 10 units/mL of EPO exhibited significantly improved recovery of LVDP compared with controls to 69 ± 9% and 79 ± 3%, respectively (P<0.01).
|
2. EPO administration immediately prior to global ischemia preserves high-energy phosphates (ATP) in ischemic myocardium
Using 31P nuclear magnetic resonance (NMR) spectroscopy, we determined whether modulation of intracellular pH (pHi) and/or high-energy phosphate levels during ischemia—mechanisms that have been implicated in myocardial protection associated with ischemic preconditioning—contributed to the rapid cardioprotection mediated by recombinant EPO pretreatment. As illustrated in Fig. 2
A, addition of EPO before ischemia did not alter pHi; pHi measurements were not significantly different between control and EPO-treated groups during ischemia. To determine the effect of EPO on high-energy phosphates, we monitored phosphocreatine (PCr) and ATP levels. PCr levels during ischemia were similar between control and EPO-treated experimental groups. During reflow, although PCr recovery (% of initial) at 10 min in EPO-treated hearts was higher at 93 ± 7% compared with 62 ± 7% in control hearts, overall PCr levels were not significantly different in EPO and control groups (Fig. 2B)
. Measurements of ATP levels during ischemia-reperfusion revealed significantly better preservation of ATP in EPO-treated hearts. At the end of global ischemia, mean ATP level in EPO-treated hearts was 14 ± 2% compared with 0.09 ± 0.06% in untreated control hearts. During reperfusion, ATP levels recovered to higher levels at 15 min of reflow in EPO-treated hearts to 48 ± 8% vs. 28 ± 6% in controls (P<0.05, Fig. 2C
).
|
CONCLUSIONS AND SIGNIFICANCE
The novel contributions of our studies include the following:
1) We have characterized EPOR expression in adult rat cardiac myocytes.
2) Recombinant EPO administration directly on the isolated, perfused heart confers an acute cardioprotective effect during ischemia-reperfusion injury.
3) Our studies show that EPO-mediated cardioprotection involves preservation of myocardial ATP levels during global ischemia of the isolated perfused heart.
Several previous studies have suggested that EPO may exert direct cellular effects in embryonic or neonatal cardiac myocytes where EPO has been reported to stimulate myocyte proliferation in culture. Our studies demonstrate that EPOR expression in adult rat myocardium mediates a direct and acute cardioprotective effect for recombinant EPO on the isolated, perfused heart subjected to ischemia-reperfusion injury.
Recent studies have described a cardioprotective effect for systemic EPO using different experimental myocardial infarction (MI) models in rats and rabbits. In these studies, administration of high systemic doses of recombinant human EPO beginning either 24 h before or at the time of coronary artery ligation was associated with significantly improved hemodynamic function on days 3 and 7 post-MI. In another recent study using isolated, perfused rat hearts, pretreatment with systemic, high-dose recombinant human EPO 24 h before ischemia-reperfusion injury resulted in improved recovery of function. Where EPO was added systemically, it is unclear which cell type was responsible for mediating the protection. For example, systemic EPO administration to the animal before ischemia could mediate protection via cytokine release by noncardiac cells or by an altered inflammatory response or other noncardiac mechanisms. To better understand whether EPO has acute, direct effects on myocardium, we added EPO directly to an isolated perfused heart.
Our studies demonstrate for the first time that brief exposure of isolated, perfused hearts to EPO immediately before global ischemia is sufficient to confer a cardioprotective effect during ischemia-reperfusion injury as assessed by the enhancement of postischemic contractile recovery. Our data indicate that preservation of high-energy phosphates (ATP) in ischemic myocardium in EPO-treated hearts may represent one mechanism by which EPO contributes to the improvement of contractile function (Fig. 3
). Our findings indicate there is a very rapid cardioprotective response to EPO; further investigation is needed to establish the early EPOR signaling events in cardiac myocytes required for the beneficial effects of EPO on cardiac function.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1289fje; 
2 G.L.W. and P.H. contributed equally to this work.
3 Present address: Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA.
This article has been cited by other articles:
|
|
 |
|
  H. Kobayashi, S. Minatoguchi, S. Yasuda, N. Bao, I. Kawamura, M. Iwasa, T. Yamaki, S. Sumi, Y. Misao, H. Ushikoshi, et al. Post-infarct treatment with an erythropoietin-gelatin hydrogel drug delivery system for cardiac repair Cardiovasc Res, June 25, 2008; (2008) cvn154v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lundby, Y. Hellsten, M. B. F. Jensen, A. S. Munch, and H. Pilegaard Erythropoietin receptor in human skeletal muscle and the effects of acute and long-term injections with recombinant human erythropoietin on the skeletal muscle J Appl Physiol, April 1, 2008; 104(4): 1154 - 1160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. C. Levy Anemia in heart failure: marker or mediator of adverse prognosis? J. Am. Coll. Cardiol., February 5, 2008; 51(5): 577 - 578. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ramond, E. Sartorius, M. Mousseau, C. Ribuot, and M. Joyeux-Faure Erythropoietin Pretreatment Protects Against Acute Chemotherapy Toxicity in Isolated Rat Hearts Experimental Biology and Medicine, January 1, 2008; 233(1): 76 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Gao, M. Boucher, J. K. Chuprun, R.-H. Zhou, A. D. Eckhart, and W. J. Koch Darbepoetin alfa, a long-acting erythropoietin analog, offers novel and delayed cardioprotection for the ischemic heart Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H60 - H68. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Contaldo, C. Meier, A. Elsherbiny, Y. Harder, O. Trentz, M. D. Menger, and G. A. Wanner Human recombinant erythropoietin protects the striated muscle microcirculation of the dorsal skinfold from postischemic injury in mice Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H274 - H283. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Schneider, K. Jaquet, R. Malisius, S. Geidel, E. Bahlmann, S. Boczor, T. Rau, M. Antz, K.-H. Kuck, and K. Krause Attenuation of cardiac remodelling by endocardial injection of erythropoietin: ultrasonic strain-rate imaging in a model of hibernating myocardium Eur. Heart J., February 2, 2007; 28(4): 499 - 509. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Prunier, O. Pfister, L. Hadri, L. Liang, F. del Monte, R. Liao, and R. J. Hajjar Delayed erythropoietin therapy reduces post-MI cardiac remodeling only at a dose that mobilizes endothelial progenitor cells Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H522 - H529. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Linde, D. Mosier, F. Blecha, and T. Melgarejo Innate immunity and inflammation - New frontiers in comparative cardiovascular pathology Cardiovasc Res, January 1, 2007; 73(1): 26 - 36. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Lipsic, R. G. Schoemaker, P. van der Meer, A. A. Voors, D. J. van Veldhuisen, and W. H. van Gilst Protective Effects of Erythropoietin in Cardiac Ischemia: From Bench to Bedside J. Am. Coll. Cardiol., December 5, 2006; 48(11): 2161 - 2167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Jie, M. C. Verhaar, M.-J. M. Cramer, K. van der Putten, C. A. J. M. Gaillard, P. A. Doevendans, H. A. Koomans, J. A. Joles, and B. Braam Erythropoietin and the cardiorenal syndrome: cellular mechanisms on the cardiorenal connectors Am J Physiol Renal Physiol, November 1, 2006; 291(5): F932 - F944. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Feng Beyond erythropoiesis: The anti-inflammatory effects of erythropoietin Cardiovasc Res, September 1, 2006; 71(4): 615 - 617. [Full Text] [PDF]
|
|
|
|
|
|
Y. Li, G. Takemura, H. Okada, S. Miyata, R. Maruyama, L. Li, M. Higuchi, S. Minatoguchi, T. Fujiwara, and H. Fujiwara Reduction of inflammatory cytokine expression and oxidative damage by erythropoietin in chronic heart failure Cardiovasc Res, September 1, 2006; 71(4): 684 - 694. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nishihara, T. Miura, T. Miki, J. Sakamoto, M. Tanno, H. Kobayashi, Y. Ikeda, K. Ohori, A. Takahashi, and K. Shimamoto Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3beta Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H748 - H755. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Tada, Y. Kagaya, M. Takeda, J. Ohta, Y. Asaumi, K. Satoh, K. Ito, A. Karibe, K. Shirato, N. Minegishi, et al. Endogenous erythropoietin system in non-hematopoietic lineage cells plays a protective role in myocardial ischemia/reperfusion Cardiovasc Res, August 1, 2006; 71(3): 466 - 477. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Gross and G. J. Gross Ligand triggers of classical preconditioning and postconditioning Cardiovasc Res, May 1, 2006; 70(2): 212 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Miki, T. Miura, T. Yano, A. Takahashi, J. Sakamoto, M. Tanno, H. Kobayashi, Y. Ikeda, M. Nishihara, K. Naitoh, et al. Alteration in Erythropoietin-Induced Cardioprotective Signaling by Postinfarct Ventricular Remodeling J. Pharmacol. Exp. Ther., April 1, 2006; 317(1): 68 - 75. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Satoh, Y. Kagaya, M. Nakano, Y. Ito, J. Ohta, H. Tada, A. Karibe, N. Minegishi, N. Suzuki, M. Yamamoto, et al. Important Role of Endogenous Erythropoietin System in Recruitment of Endothelial Progenitor Cells in Hypoxia-Induced Pulmonary Hypertension in Mice Circulation, March 21, 2006; 113(11): 1442 - 1450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Moon, M. Krawczyk, D. Paik, T. Coleman, M. Brines, M. Juhaszova, S. J. Sollott, E. G. Lakatta, and M. I. Talan Erythropoietin, Modified to Not Stimulate Red Blood Cell Production, Retains Its Cardioprotective Properties J. Pharmacol. Exp. Ther., March 1, 2006; 316(3): 999 - 1005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Li, G. Takemura, Y. Li, S. Miyata, M. Esaki, H. Okada, H. Kanamori, N. C. Khai, R. Maruyama, A. Ogino, et al. Preventive Effect of Erythropoietin on Cardiac Dysfunction in Doxorubicin-Induced Cardiomyopathy Circulation, January 31, 2006; 113(4): 535 - 543. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P Van der Meer, E Lipsic, R. Henning, K Boddeus, J van der Velden, A. Voors, D. van Veldhuisen, W. van Gilst, R. Schoemaker, J. Leemans, et al. Heart Failure after Myocardial Infarction--Benefit beyond Hemoglobin from Erythropoietin: Erythropoietin Induces Neovascularization and Improves Cardiac Function in Rats with Heart Failure after Myocardial Infarction J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3449 - 3454. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Maloyan, L. Eli-Berchoer, G. L. Semenza, G. Gerstenblith, M. D. Stern, and M. Horowitz HIF-1{alpha}-targeted pathways are activated by heat acclimation and contribute to acclimation-ischemic cross-tolerance in the heart Physiol Genomics, September 21, 2005; 23(1): 79 - 88. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. van der Meer, E. Lipsic, R. H. Henning, K. Boddeus, J. van der Velden, A. A. Voors, D. J. van Veldhuisen, W. H. van Gilst, and R. G. Schoemaker Erythropoietin Induces Neovascularization and Improves Cardiac Function in Rats With Heart Failure After Myocardial Infarction J. Am. Coll. Cardiol., July 5, 2005; 46(1): 125 - 133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Rossert and K.-U. Eckardt Erythropoietin receptors: their role beyond erythropoiesis Nephrol. Dial. Transplant., June 1, 2005; 20(6): 1025 - 1028. [Full Text] [PDF]
|
|
|
|
|
|
T. Rui, Q. Feng, M. Lei, T. Peng, J. Zhang, M. Xu, E. Dale Abel, A. Xenocostas, and P. R. Kvietys Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1 Cardiovasc Res, February 15, 2005; 65(3): 719 - 727. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. O. Arcasoy, K. Amin, S.-C. Chou, Z. A. Haroon, M. Varia, and J. A. Raleigh Erythropoietin and Erythropoietin Receptor Expression in Head and Neck Cancer: Relationship to Tumor Hypoxia Clin. Cancer Res., January 1, 2005; 11(1): 20 - 27. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
