ropoietin. EPO, known for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19675486 its protective role in hypoxic conditions, has shown protective properties against I/R injury by effectively reducing apoptotic renal cell death in in-vivo and in-vitro models. EPO also showed a protective effect in neural cells by maintaining DYm and intracellular Ca2+ concentration under pathological conditions. It inhibits caspase-3, 8, 1 like activities and has been shown to protect against 1235481-90-9 supplier apoptosis and necrosis in in-vitro and in-vivo models of brain and spinal cord ischemic injury. Studies have shown that in-vivo administration of recombinant human EPO reduces apoptosis and increases functional recovery after coronary artery occlusion/reperfusion. EPO treatment prevented apoptosis of endothelial cells in-vitro through PI3K/Akt phosphorylation during Hypoxia. It also activates the phosphorylation of STAT-5 and MAPK in these cells. Recent work suggests that EPO application in microglia maintained the expression of Wnt1 thereby regulating the mitochondrial membrane potential, phosphorylation of BAD, inhibits caspase-1 and caspase-3 activation. EPO has been shown to play an important role in cardio protection in rats, pigs and rabbits which are subjected to reperfusion injury by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19674121 inhibiting apoptosis via activation of PI3K, Akt and Erk. The cardioprotection against necrotic cells and the rise in intracellular Ca2+ homeostasis, ROS, DYm and the signaling by which this occurs is also not clear in cardiomyocytes. This study serves to investigate and elucidate the above-mentioned mechanisms of Hypoxia/Reperfusion injury protection in H9C2 cells. We for the first time showed the protective effect of EPO in maintaining DYm and intracellular Ca2+ homeostasis in live H9C2 cells, which were subjected to H/R Erythropoietin Protects Cardiomyocytes from Hypoxia/Reperfusion Injury to simulate the conditions of I/R. In summary, the EPO treatment attenuated apoptosis and necrosis through decrease in ROS, maintained DYm and intracellular Ca2+ homeostasis via modulation of Akt pathway. Detection of Apoptosis by Confocal microscopy Cells were grown to about 7080% confluency and subjected to H/R or normoxia with or without pre-treatment with EPO as described earlier. The cells were then washed with PBS and collected by centrifugation and stained with a combination of Acridine Orange ;Ethidium Bromide 1:1 ratio for 10 mins and 10ml of the cell suspension was taken on slide and fluorescent images were 
scanned using a confocal laser scanning. Materials and Methods Characterization of H9C2 by Cardiac Specific Marker H9C2, myoblast cell line derived from embryonic BD1X rat heart tissue was purchased from National Centre for Cell Science, Pune. Dulbecco’s Modified Eagle’s Medium with Fetal Bovine Serum to a final concentration of 10% was used for this cell line. Cells were grown to about 7080%confluency in a chamber slide and were fixed by using 3% paraformaldehyde and permeabilized in 0.1% of Triton-X 100 for 15 mins. Cells were incubated for 45 mins at 37uC with monoclonal a-sarcomeric actininin at a dilution of 1:800 in PBS. Samples were washed and incubated with anti-mouse biotinylated secondary antibodies for 30 mins at 37uC. After washing with PBS, samples were incubated for 15 mins at 37uC with avidin-conjugated 1:500 FITC. Samples were washed again with PBS, and the cover slip was placed over the chamber slide. Images were captured by confocal microscopy using appropriate filters for visualization of Fluorescein isothiocyanat