Abstract 097: Recombinant Human Erythropoietin Prevents Reactive Oxygen Species - Induced Apoptosis Via Akt and p38 MAPK Pathway During Hypoxia/ Reperfusion Injury

Abstract

Erythropoietin (EPO) is a cytokine produced primarily in the kidney that is essential for red blood cell production. Apart from playing a role in hematopoiesis, EPO also has a protective role in heart myocytes, ovarian, glial cells brain and retinal diseases. In this study we observed that recombinant human EPO (rhEPO) reduces Hypoxia/ Reperfusion (H/R) injury by virtue of its effect on EPO receptor prosurvival signaling pathway, which ultimately leads to reduced expression of apoptotic proteins and increased survival of cardiomyocytes. H9C2 cells were exposed to H/R with or without pretreatment using 10, 15 and 20 U/ml of rhEPO. We determined viability using MTT, nuclear fragmentation by Hoechst staining, apoptotic nuclei by Acridine orange and Ethidium bromide, Reactive Oxygen Species (ROS) by Dicholorofluoresin Diacetate and activity of late apoptotic protease, Caspase-3 by colorimetric Caspase-3 assay. The expression of mitochondrial superoxide dismutase (MnSOD) by RT-PCR and Western blot, phospho-Akt and p38 MAPK by Confocal microscopy were analyzed. Cell viability is increased in cells pretreated with rhEPO compared to cell exposed to H/R. Cells subjected to H/R showed early apoptotic and late apoptotic cells but showed normal nuclei with intact cell membrane in cells pretreated with rhEPO. Intracellular production of ROS and Caspase-3 activity was decreased in cells pretreated with rhEPO compared to cells exposed to H/R. The expression of MnSOD RNA and protein was up-regulated in response to rhEPO, but not in H/R. The phosphorylative activation of Akt, p38MAPK progressively diminished during H/R but increased in rhEPO pretreated cells. We show that rhEPO prevents apoptosis in cardiomyocytes, subjected to H/R injury via phosphorylation of Akt and p38MAPK. These results it is hoped would help us distinguish the cell signaling pathways involved in cardioprotection and thus would open new avenues in cardiovascular therapy.