Extracellular Ubiquitin Inhibits β-AR-Stimulated Apoptosis in Cardiac Myocytes: Role of GSK-3β and Mitochondrial Pathways

Aims: β-Adrenergic receptor (β-AR) stimulation induces apoptosis in adult rat ventricular myocytes (ARVMs) via the activation of glycogen synthase kinase-3β (GSK-3β) and mitochondrial pathways. However, β-AR stimulation induces apoptosis only in a fraction (∼15-20%) of ARVMs. We hypothesized that ARVMs may secrete/release a survival factor(s) which protects 80-85% of cells from apoptosis. Methods and results: Using two-dimensional gel electrophoresis followed by MALDI TOF and MS/MS, we identified ubiquitin (Ub) in the conditioned media of ARVMs treated with β-AR agonist (isoproterenol). Western blot analysis confirmed increased Ub levels in the conditioned media 3 and 6 h after β-AR stimulation. Inhibition of β1-AR and β2-AR subtypes inhibited β-AR-stimulated increases in extracellular levels of Ub, whereas activation of adenylyl cyclase using forskolin mimicked the effects of β-AR stimulation. Incubation of cells with exogenous biotinylated Ub followed by western blot analysis of the cell lysates showed uptake of extracellular Ub into cells, which was found to be higher after β-AR stimulation (1.9 ± 0.4-fold; P < 0.05 vs. control, n = 6). Pre-treatment with Ub inhibited β-AR-stimulated increases in apoptosis. Inhibition of phosphoinositide 3-kinase using wortmannin and LY-294002 prevented anti-apoptotic effects of extracellular Ub. Ub pre-treatment inhibited β-AR-stimulated activation of GSK-3β and c-Jun N-terminal kinase (JNK) and increases in the levels of cytosolic cytochrome c. The use of methylated Ub suggested that the anti-apoptotic effects of extracellular Ub are mediated via monoubiquitination. Conclusion: β-AR stimulation increases levels of Ub in the conditioned media. Extracellular Ub plays a protective role in β-AR-stimulated apoptosis, possibly via the inactivation of GSK-3β/JNK and mitochondrial pathways.