The purpose of this study was to determine whether exogenous zinc prevents 
cardiac reperfusion injury by targeting the mitochondrial permeability 
transition pore (mPTP) via glycogen synthase kinase-3beta (GSK-3beta). The 
treatment of cardiac H9c2 cells with ZnCl2 (10 microM) in the presence of zinc 
ionophore pyrithione for 20 min significantly enhanced GSK-3beta phosphorylation 
at Ser9, indicating that exogenous zinc can inactivate GSK-3beta in H9c2 cells. 
The effect of zinc on GSK-3beta activity was blocked by the phosphatidylinositol 
3-kinase (PI3K) inhibitor LY-294002 but not by the mammalian target of rapamycin 
(mTOR) inhibitor rapamycin or the PKC inhibitor chelerythrine, implying that 
PI3K but not mTOR or PKC accounts for the action of zinc. In support of this 
interpretation, zinc induced a significant increase in Akt but not mTOR 
phosphorylation. Further experiments found that zinc also increased 
mitochondrial GSK-3beta phosphorylation. This may indicate an involvement of the 
mitochondria in the action of zinc. The effect of zinc on mitochondrial 
GSK-3beta phosphorylation was not altered by the mitochondrial ATP-sensitive K+ 
channel blocker 5-hydroxydecanoic acid. Zinc applied at reperfusion reduced cell 
death in cells subjected to simulated ischemia/reperfusion, indicating that zinc 
can prevent reperfusion injury. However, zinc was not able to exert protection 
in cells transfected with the constitutively active GSK-3beta (GSK-3beta-S9A-HA) 
mutant, suggesting that zinc prevents reperfusion injury by inactivating 
GSK-3beta. Cells transfected with the catalytically inactive GSK-3beta 
(GSK-3beta-KM-HA) also revealed a significant decrease in cell death, strongly 
supporting the essential role of GSK-3beta inactivation in cardioprotection. 
Moreover, zinc prevented oxidant-induced mPTP opening through the inhibition of 
GSK-3beta. Taken together, these data suggest that zinc prevents reperfusion 
injury by modulating the mPTP opening through the inactivation of GSK-3beta. The 
PI3K/Akt signaling pathway is responsible for the inactivation of GSK-3beta by 
zinc.