Erythropoietin Protects Cardiac Myocytes Against Anthracycline-Induced Apoptosis by PI3K-Akt-Mediated Phosphorylation of Glycogen Synthase Kinase-3β.

Anthracycline antibiotics are an important component of chemotherapy regimens for hematologic malignancies and solid tumors despite their well-documented potential for adverse cardiac effects. Identification of mechanisms of anthracycline-induced cardiotoxicity has been important to develop strategies to prevent the cardiotoxic effects of anthracyclines without interfering with their anti-tumor effects. The hematopoietic cytokine erythropoietin (EPO), frequently used in anemic cancer patients, exhibits significant tissue-protective effects in various non-hematopoietic organs. Recent studies of EPO variants that retain tissue-protective properties of EPO in non-hematopoietic tissues without stimulating erythropoiesis suggested fundamental differences between EPO-mediated cellular signaling in hematopoietic versus non-hematopoietic cells as well as novel clinical applications for recombinant EPO and its derivatives in disorders other than anemia. In the isolated, Langendorff-perfused heart, we previously showed that EPO improves left ventricular function and reduces infarct size during ischemia-reperfusion injury. These protective effects were associated with preservation of myocardial ATP and EPO-mediated activation of specific signal transduction pathways in the isolated, perfused heart. In this study, we investigated the mechanisms of EPO-mediated protection of cardiac myocytes against apoptotic cell death induced by doxorubicin (DOX). Cultures of primary cardiac myocytes isolated from neonatal rat ventricles (NRVM) were established and cells were treated with DOX in the presence or absence of EPO. Using immunofluorescence microscopy of cardiomyocytes double-labeled with TUNEL and DAPI to quantify apoptotic nuclei, we found that EPO treatment significantly reduced by 35% DOX-induced apoptosis in a dose-dependent manner (p<0.001 by ANOVA). The ability of EPO to protect against DOX-mediated apoptosis was also confirmed by a cell-death ELISA that quantifies mono- and oligo-nucleosomes associated with apoptosis demonstrating a significant 33% reduction in cell death. In addition, EPO treatment significantly reduced caspase-3 activation during DOX-induced apoptosis. EPO treatment of cardiomyocytes induced the rapid phosphorylation of PI3K substrate Akt (serine 473 and threonine 308) and the p44/42 MAP kinases Erk 1/2 in a time-dependent fashion. The PI3K inhibitor LY294002 and Akt inhibitor Akti 1/2 or the MEK inhibitor U0126 inhibited the EPO-induced phosphorylation of Akt and Erk1/2, respectively. Treatment of cells with LY294002 or Akti 1/2 abolished the anti-apoptotic effect of EPO during during DOX treatment, whereas treatment with U0126 did not. EPO induced the phosphorylation of GSK-3β, a downstream target of PI3K-Akt. The ability of EPO to phosphorylate GSK-3β was blocked by LY294002 or Akti 1/2. Because phosphorylation is known to inactivate GSK-3β, we investigated whether GSK-3β inhibition is protective. We used two different inhibitors of GSK-3β including lithium chloride and SB216763. Using TUNEL and DNA fragmentation assays, we found that treatment with lithium chloride or SB216763 exerts a significant protective effect in cardiomyocytes during DOX-induced apoptosis (p<0.001), in a manner similar to EPO. These data suggest that erythropoietin may serve as a novel cardioprotective agent against anthracycline-induced cardiotoxicity by inhibiting cardiomyocyte apoptosis through the PI3K-Akt pathway and inactivation of GSK-3β by phosphorylation.