Abstract 361: ER Stress Contributes to Dysregulation Of GSK-3β-mediated Suppression of Mitochondrial Permeability Transition in Diabetic Hearts
We recently found that myocardial response to protective effects of erythropoietin (EPO) is lost in diabetic rat hearts by increased endoplasmic reticulum (ER) stress. In this study, we tested the hypothesis that ER stress-mediated activation of GSK-3β underlies the failure of EPO to elevate the threshold for opening of the mitochondrial permeability transition pore (mPTP) in diabetic hearts. Sodium tauroursodeoxycholic acid (TUDCA, 100 mg/kg/day i.p. for 7 days) was employed to reduce ER stress in type 2 diabetic rats (OLETF). LETO rats were used as normal controls. Tissues were sampled before and after EPO treatment for immunoblotting. Mitochondria were isolated from the myocardium after 20-min ischemia/5-min reperfusion, and extra-mitochondrial Ca2+ was monitored by a Ca2+-sensitive microelectrode. mPTP opening was induced by repetitive Ca2+ loading, and the amount of Ca2+ (μM Ca2+/mg of mitochondrial protein) required for induction of massive Ca2+ release from the mitochondria was used as an index of mitochondrial susceptibility to mPTP opening. Levels of myocardial GRP78 and GRP94, ER stress markers, and non-phosphorylated GSK-3β in the mitochondria were significantly higher by 30%, 29% and 45%, respectively, in OLETF than in LETO. EPO significantly increased the level of phospho-GSK-3β in LETO but not in OLETF. TUDCA normalized levels of ER chaperones and mitochondrial GSK-3β and restored EPO-induced phosphorylation of GSK-3β in OLETF. The amounts of Ca2+ required for mPTP opening after EPO treatment were 483.3±3.3 μM/mg in LETO, 411.1±19.8 μM/mg in OLETF (p<0.05 vs. LETO) and 510.0±26.5 μM/mg in TUDCA-treated OLETF (p=ns vs. LETO). The levels of Ca2+ required for mPTP opening were positively correlated with phosphorylation levels of mitochondrial GSK-3β at reperfusion (p<0.05, r=0.74). Increased ER stress in diabetic hearts results in both disruption of signaling for GSK-3β phosphorylation and increase in constitutively active non-phosphorylated GSK-3β in the mitochondria. These changes in GSK-3β regulation are responsible for the inability of EPO to suppress mPTP opening and infarction of the myocardium in diabetic hearts.