Inhibition of glycogen synthase kinase 3β prevents peroxide-induced collapse of mitochondrial membrane potential in rat ventricular myocytes.

1. Preconditioning has been proposed to protect the myocardium by inhibiting glycogen-synthase kinase (GSK) 3β. The aim of the present study was to test whether transfection of ventricular myocytes with inactive GSK3β would mimic preconditioning and whether a constitutively active form of GSK3β would prevent protection by an opioid receptor agonist. 2. Isolated ventricular myocytes from adult rats were infected with live adenovirus containing either a wild-type (wtGSK), constitutively active (caGSK) or dominant-negative (dnGSK) GSK3β plasmid. Cells were loaded with tetramethylrhodamine ethyl ester (TMRE) and exposed to H₂O₂ (100 μmol/L) for 40 min before mitochondrial membrane potential (ΔΨ_m) was assessed using flow cytometric analysis. 3. Fluorescence intensity was reduced in H₂O₂-treated cells compared with untreated cells, presumably because oxidant injury opened mitochondrial permeability transition pores, causing mitochondria to lose TMRE. The selective GSK3β inhibitor SB216763, as well as the δ-opioid receptor agonist [d-Ala²-d-Leu⁵]-enkephalin (DADLE) (1 μmol/L), protected cells against peroxide-induced loss of ΔΨ_m. 4. Cells transfected with dnGSK (1 μmol/L) were equally protected against peroxide stress, when given throughout the TMRE and H₂O₂ treatment, confirming a protective effect of GSK3β with a highly selective inhibition. Cells transfected with wtGSK did not show any difference in responses to H₂O₂, SB216763 or DADLE compared with untransfected cells, suggesting that adenovirus infection itself had no effect. In contrast, caGSK-transfected myocytes could no longer be protected with DADLE, suggesting a role for GSK3β between the surface receptor and the mitochondria. 5. These experiments confirm that inhibition of GSK3β protects the myocytes, but also that the preconditioning mimetic DADLE loses its protective effect when a constitutively active GSK3β is present. [ABSTRACT FROM AUTHOR]