Erythropoietin Protects Cardiomyocytes Not Only during Hypoxic Injury but Also Against Doxorubicin-Induced Apoptosis: A Novel Effect Mediated by Phosphatidylinositol 3-Kinase Pathway.
Authors: Fu Ping , Arcasoy Murat O. 
Published: April 2020

Erythropoietin (EPO) is a tissue-protective cytokine that exerts cytoprotective effects against various types of injury in non-hematopoietic tissues. Our previous work using Langendorff-perfused adult rat hearts demonstrated that EPO improves left ventricular function and reduces infarct size during ischemia-reperfusion injury. 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 this study, we characterized the activation of EPO-mediated intracellular signal transduction in primary cardiac myocytes and determined the specific pathways required for protection of cardiomyocytes against hypoxic injury. We also investigated the ability of EPO to protect against cell death induced by doxorubicin (DOX), a chemotherapeutic agent associated with well-established cardiotoxic effects. We hypothesized that protective effects of EPO during hypoxic or DOX-mediated cardiomyocyte injury may be inhibited by specific kinase inhibitors targeting pathways activated by EPO. Primary cardiac myocytes were isolated from neonatal rat ventricles (NRVM) establishing cultures containing &gt;97% cardiac myocytes as determined by immunostaining of cells with anti-cardiac muscle sarcomeric actinin. We found that EPO induces the rapid phosphorylation of PI3K substrate Akt and p44/42 MAP kinases Erk 1/2 in a time-dependent fashion. Constitutive tyrosine phosphorylation of Jak2 was observed that did not increase following EPO treatment of cardiomyocytes. Pre-incubation of cells with PI3K inhibitor LY294002 or MEK inhibitor U0126 prior to EPO treatment inhibited the increased phosphorylation of Akt and Erk1/2, respectively. EPO treatment of NRVMs significantly reduced hypoxia-induced apoptosis by 44% assessed by immunoflurescence microscopy of cardiomyocytes double-labeled with TUNEL and DAPI to quantify apoptotic nuclei (P&lt;0.001 by ANOVA; hypoxia-treated versus hypoxia+EPO, n=12 fields of ~4,000 cells in each group). The ability of EPO to protect against hypoxia-mediated apoptosis was also confirmed by a cell-death ELISA that quantifies mono- and oligo-nucleosomes associated with apoptosis. Treatment of cells with LY294002 or protein kinase C inhibitor chelerythrine abolished the anti-apoptotic effect of EPO during hypoxia, whereas treatment with U0126 or Jak2 inhibitor AG490 did not. We also found that EPO exerts a significant protective effect during DOX-induced cell death in a dose-dependent manner, reducing apoptosis by 36% in TUNEL assays (P&lt;0.001 by ANOVA; DOX-treated versus DOX+Epo, n= 6 fields of ~2,000 cells in each group) and by 29% in ELISA assays (P&lt;0.001 by ANOVA, DOX-treated versus DOX+Epo, n=14). Treatment of cells with LY294002 abrogated the anti-apoptotic effect of EPO in NVRMs treated with DOX. These findings demonstrate that 1)-EPO induces rapid phosphorylation of Akt and Erk1/2 but not Jak2 in primary cardiac myocytes in a time-dependent fashion; 2)-EPO protects cardiomyocytes against apoptosis induced by hypoxia or DOX; 3)-The protective effect of EPO during hypoxia-mediated injury of cardiomyocytes requires the PI3K and PKC pathways, but not MAP kinase or Jak2 pathways; 4) The protective effect of EPO during DOX treatment of cardiomyocytes requires the PI3K pathway.