Abstract 1526: Activation of Glycogen Synthase Kinase-3β (GSK-3β) Plays a Pro-Apoptotic Role in β-Adrenergic Receptor (β-AR)-Stimulated Apoptosis in Adult Rat Ventricular Myocytes (ARVM) via the Involvement of Mitochondrial Death Pathway
Stimulation of β-AR induces apoptosis in ARVM via the activation of mitochondrial death pathway. β1 integrins play a protective role in β-AR-stimulated apoptosis. GSK-3β negatively regulates cardiac myocyte hypertrophy. Here, we tested the hypotheses that
β-AR-stimulated activation of GSK-3β plays a pro-apoptotic role via the activation of mitochondrial pathway, and
β1 integrins protect ARVM via the inhibition of GSK-3β. Treatment of ARVM with isoproterenol (ISO; 10 μM; 15 min) increased GSK-3β activity by 4.6±1.2-fold (p<0.01, n=10) as measured by immune-complex kinase assay. Addition of LiCl in the reaction mix or expression of catalytically inactive GSK-3β using adenoviruses inhibited β-AR-stimulated GSK-3β activity. ISO (15 min) increased tyrosine-216 phosphorylation of GSK-3β. To study the role of GSK-3β in β-AR-stimulated apoptosis, cells were pretreated with pharmacological inhibitors of GSK-3β (LiCl, 1 mM; SB216763, 20 μM) or infected with adenoviruses expressing wild-type (WT), constitutively active (S9A) or inactive (KM) forms of GSK-3β. Inhibition of GSK-3β inhibited β-AR-stimulated apoptosis (percent apoptosis; CTL, 7.5±0.8; ISO, 18.6±1.1*; ISO+LiCl, 5.3±1.1#; ISO+SB, 10.3±3.2#; *p<0.01 vs CTL; #p<0.05 vs ISO, n=3–4) as analyzed by TUNEL-staining assay. Expression of WT- or S9A-GSK-3β alone induced apoptosis, while expression of KM-GSK-3β inhibited β-AR-stimulated apoptosis (ISO, 18.6±1.1; KM+ISO, 10.4±0.8#; #p<0.05 vs ISO, n=7). Inhibition of GSK-3β inhibited β-AR-stimulated increases in cytosolic cytochrome C levels. Adenovirus-mediated expression of β1 integrins (Adβ1A) inhibited β-AR-stimulated increases in GSK-3β activity (fold change vs CTL; ISO, 4.6±1.2; ISO+Adβ1A, 1.3±0.2*; *p<0.05 vs ISO, n=4). Wortmannin (Wort), PI3-kinase inhibitor, reversed the effects on GSK-3β activity, and inhibited the anti-apoptotic effect of β1 integrins (percent apoptosis; Adβ1A+ISO, 6.2±2.4; Wort+Adβ1A+ISO, 20.5± 3.2*; *p<0.05 vs Adβ1A+ISO, n=3). Thus, activation of GSK-3β plays a critical role in β-AR-stimulated activation of mitochondrial death pathway and apoptosis. β1 integrin overexpression inhibits β-AR-stimulated activation of GSK-3β and apoptosis, possibly via the activation of PI3K/Akt pathway.