Abstract 633: Suppression Of Mitochondrial GSK3β Activity By Its Ser9-phosphorylation Contributes To Cardiomyocyte Protection Afforded By Erythropoietin Against Oxidative Stress-induced Apoptosis
Objective Activation of the erythropoietin (EPO) receptor induces phosphorylation of numerous protein kinases, including glycogen synthase kinase 3β (GSK3β), and affords myocardial protection against ischemia/reperfusion injury. However, contribution of GSK3β to EPO-induced protection against apoptosis has not been directly demonstrated. Here, we examined whether alterations in activity and/or localization of GSK3β are critically involved in EPO-induced protection against oxidant stress-induced apoptosis.
Methods and Results Apoptosis was induced in H9c2 cardiomyocyte by H2O2 (50 μM) or angiotensin II (150 μM). Pretreatment with EPO (10 U/ml) suppressed H2O2-induced apoptosis (26.0± 2.7% vs. 38.3±2.9%, p<0.05) and also angiotensin II-induced apoptosis (21.5±0.9% vs. 31.9±1.4%, p<0.05) compared with that in untreated controls. Next, to examine the role of Ser9-phosphorylation of GSK3β, which suppresses GSK3β activity, H9c2 cells were transfected with EGFP-tagged wild-type (WT), constitutively active (S9A) or dominant negative (K85R) GSK3β. Apoptosis by H2O2 was increased in S9A-transfected cells compared with that in WT-transfected cells (44.4±5.7% vs. 30.6±2.1%, p<0.05) and decreased in K85R-transfected cells (26.1±4.3%). EPO suppressed apoptosis in WT-transfected cells (21.9±2.5% vs. 30.6±2.1%, p<0.05) but not in S9A- or K85R-transfected cells. The effect of EPO was mimicked by suppression of GSK3β expression by its siRNA. Finally, to examine the effects of EPO on intracellular localization of GSK3β, H9c2 cells were transfected with WT and mitochondria were stained with MitoTracker. EPO did not change % of total GSK3β cololcalized with mitochondria but increased phospho-GSK3β colocalized with mitochondria by 55%.
Conclusion: Together with reported inhibition of mitochondrial permeability transition by phospho-GSK3β, the present results suggest that suppression of GSK3β activity by its Ser9-phosphorylation in mitochondria plays a crucial role in cardiomyocyte protection by EPO against oxidant stress-induced apoptosis.