Ser9 phosphorylation of mitochondrial GSK-3β is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis

Ohori K, Miura T, Tanno M, Miki T, Sato T, Ishikawa S, Horio Y, Shimamoto K. Ser9 phosphorylation of mitochondrial GSK-3 beta is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis. Am J Physiol Heart Circ Physiol 295: H2079-H2086, 2008. First published September 19, 2008; doi: 10.1152/ajpheart.00092.2008.-The aim of this study was to determine the role of GSK-3 beta in cardiomyocyte protection afforded by erythropoietin (EPO) against oxidant stress-induced apoptosis. Treatment with EPO (10 units/ml) induced Ser473 phosphorylation of Akt and Ser9 phosphorylation of GSK-3 beta and significantly reduced the proportion of apoptotic H9c2 cardiomyocytes after exposure to H2O2 from 38.3 +/- 2.7% to 26.0 +/- 2.9%. This protection was not detected in cells transfected with constitutively active GSK-3 beta (S9A), which lacks Ser9 for inhibitory phosphorylation. The antiapoptotic effect of EPO was mimicked completely by GSK-3 beta knockdown using small interfering RNA and partly by the transfection with kinase-deficient GSK-3 beta (K85R). The level of colocalization of intracellular GSK-3 beta with mitochondria assessed by enhanced green fluorescent proteintagged GSK-3 beta or immunocytochemistry was not altered by EPO treatment. However, EPO increased the level of Ser9-phosphoGSK-3 beta colocalized with mitochondria by 50% in a phosphatidylinositol 3-kinase-dependent manner. Mitochondrial translocation of Bcl2-associated X protein (BAX) after exposure to H2O2 was inhibited by EPO pretreatment and by GSK-3 beta knockdown. These results suggest that the suppression of GSK-3 beta activity by Akt-mediated Ser9 phosphorylation in the mitochondria affords cardiomyocytes tolerance against oxidant-induced apoptosis, possibly by inhibiting the access of BAX to the mitochondria.