ns and there is an influx of Ca2+ ions into the mitochondria. The accumulation ruptures the outer mitochondrial membrane and Rhodamine-123 leaks out from the mitochondria into the cytosol. Rhodamine-123 and DCF fluorescence were observed to co- 6 Erythropoietin Protects Cardiomyocytes from Hypoxia/Reperfusion Injury localize exclusively in the perinuclear regions of the control and EPO treated cells. While in H/R induced cellsRhodamine-123 colocalized PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19672638 in the perinuclear and the cytoplasmic regions. The scatter plot illustrating the degree of co-localization between the two dyes showed a significant increase of about 94.7% in H/R induced cells. Whereas, the control and EPO pretreated cells showed 52% and 58%co-localization, and it is significantly lower when compared to H/R induced cellsas seen in the scatter plot. These results clearly indicate that EPO stabilizes DYm in H/R-induced cells. EPO treatment maintains mitochondrial membrane integrity and intracellular Ca2+ homeostasis Normal cells maintain DYm by accumulating Ca2+ both in the cytosol and the nucleus, as visualized by Fluo-4 AM in live cell imaging. In cells with 8 hrs of hypoxia exposure, Ca2+ accumulates more in the mitochondria and the nucleus compared to the cytosol. At the start PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19673983 of reperfusion MPTP open and the Erythropoietin Protects Cardiomyocytes from Hypoxia/Reperfusion Injury 8 Erythropoietin Protects Cardiomyocytes from Hypoxia/Reperfusion Injury mitochondrial membrane ruptures due to a Ca2+influx intothe mitochondria and the nucleus. In contrast, cells pretreated with EPO maintained mitochondrial membrane integrity and intracellular Ca2+ homeostasis EPO induces phosphorylation of Akt and reduces caspase-3 activity in H/R-induced cells Akt is involved in cellular CJ-023423 chemical information survival pathway by inhibiting apoptotic processes. On the other hand, 
caspase-3 is playing an important role in activating the final step of the proapoptoticsignaling pathway in many cell lines. To examine the role of Akt and caspase-3 in EPO treatment mediated protection in H/Rinduced cell injury; we performed Western and immunofluorescence analysis for p-Akt and spectrophotometric analysis for caspase-3. The immunofluorescence analysis showed an increase in levels of p-AKT as compared to the H/R-induced cells and normoxic controls. The increase in p-Akt was further confirmed by Western analysis from H9C2 cells pretreated with 20 U/ml EPO. Caspases are cysteine proteases, which play an essential role in apoptosis, necrosis and inflammation. To examine whether EPO exerts anti-apoptotic actions on H/R-induced H9C2 cells, we used caspase-3 specific peptide reporter molecule, p-nitro aniline, to cell’s lysate and quantified by spectrophotometer at 405 nm. The color intensity is directly proportional to the level of caspase-3 activity in the cell lysate. Caspase-3 activity increased two fold in H9C2 cells after exposure to H/R, whereas, there was a significant decrease in caspase-3 activity in cells that were pretreated with 20 U/ml of EPO. To further validate the significance of p-Akt and caspase-3 in EPO-treatment mediated protection, we next inhibited Akt using specific inhibitor Wortmannin. We observed that EPO-treatment induce phosphorylation of Akt was inhibited by the treatment Wortmannin. Furthermore, reduced caspase-3 activity by H/R-induced EPO treatment effect was blunted on treatment with Wortmannin. Together, these data indicate that protective effect of EPO-treatment in H/ R-induced cell injury