title: Erythropoietin protects from reperfusion-induced myocardial injury by enhancing coronary endothelial nitric oxide production.
abstract: OBJECTIVE
  Cardioprotective properties of recombinant human Erythropoietin (rhEpo) have been shown in in vivo regional or ex vivo global models of ischemia-reperfusion (I/R) injury. The aim of this study was to characterize the cardioprotective potential of rhEPO in an in vivo experimental model of global I/R approximating the clinical cardiac surgical setting and to gain insights into the myocardial binding sites of rhEpo and the mechanism involved in its cardioprotective effect.
  
  
  METHODS
  Hearts of donor Lewis rats were arrested with cold crystalloid cardioplegia and after 45 min of cold global ischemia grafted heterotopically into the abdomen of recipient Lewis rats. Recipients were randomly assigned to control non-treated or Epo-treated group receiving 5000 U/kg of rhEpo intravenously 20 min prior to reperfusion. At 5 time points 5-1440 min after reperfusion, the recipients (n=6-8 at each point) were sacrificed, blood and native and grafted hearts harvested for subsequent analysis.
  
  
  RESULTS
  Treatment with rhEpo resulted in a significant reduction in myocardial I/R injury (plasma troponin T) in correlation with preservation of the myocardial redox state (reduced glutathione). The extent of apoptosis (activity of caspase 3 and caspase 9, TUNEL test) in our model was very modest and not significantly affected by rhEpo. Immunostaining of the heart tissue with anti-Epo antibodies showed an exclusive binding of rhEpo to the coronary endothelium with no binding of rhEpo to cardiomyocytes. Administration of rhEpo resulted in a significant increase in nitric oxide (NO) production assessed by plasma nitrite levels. Immunostaining of heart tissue with anti-phospho-eNOS antibodies showed that after binding to the coronary endothelium, rhEpo increased the phosphorylation and thus activation of endothelial nitric oxide synthase (eNOS) in coronary vessels. There was no activation of eNOS in cardiomyocytes.
  
  
  CONCLUSIONS
  Intravenous administration of rhEpo protects the heart against cold global I/R. Apoptosis does not seem to play a major role in the process of tissue injury in this model. After binding to the coronary endothelium, rhEpo enhances NO production by phosphorylation and thus activation of eNOS in coronary vessels. Our results suggest that cardioprotective properties of rhEpo are at least partially mediated by NO released by the coronary endothelium.
authors.affiliation: Institute for Veterinary Physiology, Vetsuisse Faculty, Switzerland.
authors.forename: Deyan
authors.initials: D
authors.lastname: Mihov
authors.forename: Nikolay
authors.initials: N
authors.lastname: Bogdanov
authors.forename: Beat
authors.initials: B
authors.lastname: Grenacher
authors.forename: Max
authors.initials: M
authors.lastname: Gassmann
authors.forename: Gregor
authors.initials: G
authors.lastname: Zünd
authors.forename: Anna
authors.initials: A
authors.lastname: Bogdanova
authors.forename: Reza
authors.initials: R
authors.lastname: Tavakoli
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D009320:Atrial Natriuretic Factor / Q000097:blood; D001834:Body Water / Q000378:metabolism; D002316:Cardiotonic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D003331:Coronary Vessels / Q000187:drug effects* / Q000378:metabolism; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004921:Erythropoietin / Q000494:pharmacology / Q000627:therapeutic use*; D016027:Heart Transplantation / Q000473:pathology; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D009569:Nitric Oxide / Q000096:biosynthesis*; D018384:Oxidative Stress; D051381:Rats; D011917:Rats, Inbred Lew; D011994:Recombinant Proteins; D018746:Systemic Inflammatory Response Syndrome / Q000097:blood / Q000517:prevention & control; D020107:Troponin T / Q000097:blood
chemical_list: D002316:Cardiotonic Agents; D011994:Recombinant Proteins; D020107:Troponin T; D004921:Erythropoietin; D009569:Nitric Oxide; D009320:Atrial Natriuretic Factor