Abstract
Doxorubicin (DOX) is an effective antineoplastic agent whose use has been limited by its cardiotoxic side effects. Recent studies have established that erythropoietin (EPO), a cytokine essential for red blood cell production, protects against ischemic injury in the heart and other organs. The purpose of this study was to assess whether EPO protects the heart against cardiotoxicity induced by DOX. We found that DOX-induced apoptosis and impaired heart function in mice were largely prevented by EPO administration. To investigate the mechanism of protection by EPO, cultured neonatal mouse ventricular myocytes were treated with EPO at therapeutic levels (i.e., 1 U/ml), before application of DOX (0.1–1.0 μM). EPO protected against DOX-induced cardiomyocyte death (by ≈50%) and apoptosis assessed by annexin-V labeling, DNA fragmentation, and caspase-3 activity. DOX-mediated increases in reactive oxygen species, which trigger cardiotoxicity, were also reversed by preconditioning with EPO. These functional effects of EPO correlated with increased Akt/protein kinase B (∼2-fold) and glycogen synthase kinase 3 (GSK-3; ∼1.3-fold) phosphorylations, suggesting protection by EPO was mediated by phosphatidylinositol 3-kinase activation. Indeed, preventing Akt and GSK-3β phosphorylations by phosphatidylinositol 3-kinase (PI3K) inhibition abolished protection by EPO against cardiomyocyte loss, apoptosis, and oxidative stress. Thus, pretreatment with therapeutic levels of EPO can protect the myocardium against DOX-induced impaired heart function and cardiomyocyte apoptosis by activating PI3K-Akt cell survival pathways.
Footnotes
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This study was supported by a grant from the Canadian Institutes for Health Research (to P.H.B). P.H.B is a Career Investigator of the Heart and Stroke Foundation of Ontario. K.H.K is the recipient of a studentship from the Heart and Stroke Richard/Lewar Centre of Excellence at the University of Toronto. Some of the results were presented at the American Society of Clinical Oncology 41st Annual Meeting in May 2005, Orlando, FL. American Society of Clinical Oncology, Alexandria, VA.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.125773.
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ABBREVIATIONS: DOX, doxorubicin; EPO, erythropoietin; EPOR, erythropoietin receptor; PI3K, phosphatidylinositol 3-kinase; NMVM, neonatal mouse ventricular myocyte; SFM, serum-free media; LDH, lactate dehydrogenase; pNA, p-nitroaniline; AMVM, adult mouse ventricular myocyte; PKB, protein kinase B; GSK-3, glycogen synthase kinase-3; ROS, reactive oxygen species; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester; DCF, 2′, 7′-dichlorofluorescein; TUNEL, terminal deoxynucleotidyl transferase nick-end labeling; bpm, beats per minute; CTRL, control; HR, heart rate; P-, phosphorylated-; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; circ/s, circumferences/second.
- Received May 14, 2007.
- Accepted October 9, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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