Inhibition of Phosphodiesterases Leads to Prevention of the Mitochondrial Permeability Transition Pore Opening and Reperfusion Injury in Cardiac H9c2 Cells

We tested if inhibition of phosphodiesterases (PDEs) with IBMX (1-methyl-3-isobutylxanthine) can modulate the mitochondrial permeability transition pore (mPTP) opening by inactivating glycogen synthase kinase 3 beta (GSK-3 beta). H9c2 cells were exposed to 600 mu M H2O2 for 20 min to cause the mPTP opening. Mitochondrial membrane potential (Delta Im) was assessed by imaging cells loaded with tetramethylrhodamine ethyl ester (TMRE). Cell viability was measured with propidium iodide (PI) fluorometry using a fluorescence reader. Ischemia/reperfusion injury was induced by exposing cells to ischemic solution for 90 min followed by 30 min of reperfusion. IBMX reduced loss of Delta Im caused by H2O2, indicating that inhibition of PDEs can prevent the mPTP opening. However, IBMX could not inhibit the pore opening in cells transfected with the constitutively active GSK-3 beta (GSK-3 beta-S9A) mutant, suggesting a critical role of GSK-3 beta in the action of IBMX. IBMX also reduced reperfusion injury in a GSK-3 beta dependent manner. In support, IBMX increased GSK-3 beta phosphorylation at Ser(9), an effect that was reversed by both the PKA inhibitor H89 and the PKG inhibitor KT5823. In support, IBMX activated both PKA and PKG. IBMX failed to prevent the loss of Delta Im in the presence of H89 or PKA siRNA. Similarly, both KT5823 and PKG siRNA reversed the protective effect of IBMX. Inhibition of PDEs prevents the mPTP opening by inactivating GSK-3 beta through PKA and PKG. GSK-3 beta is a common downstream target of PKA and PKG. Inhibition of PDEs may be a useful approach to prevent reperfusion injury.