Resveratrol And Zinc Protect The Heart From Ischemia/reperfusion Injury By Targeting Mitochondria

Part 1 Resveratrol-induced cardioprotection and the mechanism underlying the protectionAcute myocardial infarction (AMI) is the leading cause of death worldwide. The occlusion of coronary artery followed by revasculation induces myocardial necrosis and the myocardial contractile dysfunction. In spite of substantial investigation, no specific therapy was yet been established to effectively save myocardium from AMI. First isolated from the roots of white hellebore in 1940, resveratrol (3,4', 5-trihydroxystilbene) is a polyphenolic product found abundantly in grapes and red wines. Studies have shown that resveratrol exerts numerous biological effects including antioxidant , anti-ageing , and anti-inflammation. Resveratrol is a key explanation for the "French Paradox" -- the low incidence of heart disease among the French people, who eat a relatively high-fat diet. Resveratrol pretreatment can protect the heart from ischemia/reperfusion injury by inducing pharmacological preconditioning. The mitochondrial permeability transition pore (mPTP) opening has been proposed to play an important role in myocardial ischemia/reperfusion injury. The mPTP remains closed during ischemia but opens at the onset of reperfusion, and modulation of the mPTP opening at early reperfusion can protect the heart from reperfusion injury. Identified as a regulator of glycogen metabolism, glycogen synthase kinase 3β(GSK-3β) is now a well established component contributing to cell signaling, protein synthesis, cell proliferation, cell differentiation, cell adhesion, and apoptosis. Studies have shown that GSK-3βplays a role in ischemic preconditioning and cardioprotection at reperfusion. In addition, a recent study has shown that GSK-3βplays a central role in pharmacological preconditioning induced modulation of the mPTP opening. Therefore, it is intriguing to determine whether GSK-3βis involved in the action of resveratrol, if resveratrol protects the heart at reperfusion by targeting the mPTP. First, we examined the effect of resveratrol on myocardial infarct size when given at reperfusion and investigated the mechanism underlying the effect. Isolated rat hearts were subjected to 30 min ischemia followed by 2h of reperfusion, and myocardial samples were collected from the risk zone for Western blot analysis. Mitochondrial swelling was spectrophotometrically measured as a decrease in absorbance at 520 nm (A520). Resveratrol reduced infarct size, an effect that was reversed by the mPTP opener atractyloside, indicating that resveratrol may protect the heart by modulating the mPTP opening. This finding was further supported by the observation that resveratrol prevented cardiac mitochondrial swelling.Second, Resveratrol enhanced GSK-3βphosphorylation at Ser⁹ upon reperfusion, an effect that was blocked by the guanylyl cyclase inhibitor ODQ, suggesting the cGMP-PKG signaling pathway is involved in the action of resveratrol. Resveratrol also increased phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), implying that resveratrol does activate PKG. Resveratrol translocated GSK-3βfrom cytosol to mitochondria via the cGMP-PKG pathway.Further studies showed that mitochondrial GSK-3βwas co-immunoprecipitated with cyclophilin D but not with VDAC (voltage dependent anion channel) or ANT (adenine nucleotide translocator).These data suggest that resveratrol prevents myocardial reperfusion injury presumably by targeting the mPTP through translocation of GSK-3βfrom cytosol to mitochondria. Translocated GSK-3βmay ultimately interact with cyclophilin D to modulate the mPTP opening. Part 2 Zinc-induced cardioprotection and the mechanism underlying the protectionFound in most tissues, zinc is fundamental to the structure and function of many proteins, enzymes, and transcriptional factors. Zinc has also been demonstrated to play an important role in cellular signaling by modulating signal recognition, second messenger metabolism, protein kinase, and Phosphatase activities. Recent studies have shown that exogenous zinc can regulate activities of several important intracellular signaling elements such as phosphatidylinositol 3-kinase (PI3K), Akt/PKB, p70S6 kinase, mammalian target of rapamycin (mTOR), extracellular signal-regulated kinase (ERK), and glycogen synthase kinase-3β(GSK-3β). Since these signaling kinases have been demonstrated to be involved in the mechanism underlying cardioprotection against reperfusion injury, the treatment of cardiac cells with zinc may lead to cardioprotection against reperfusion injury. 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 3β(GSK.-3β).The treatment of cardiac H₉C₂ cells with ZnCl₂ (10μM) in the presence of zinc ionophore pyrithione for 20 min significantly enhanced GSK-3βphosphorylation at Ser⁹, indicating that exogenous zinc can inactivate GSK-3βin H₉C₂ cells. The effect of zinc on GSK-3βactivity 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-3βphosphorylation. This may indicate an involvement of the mitochondria in the action of zinc. The effect of zinc on mitochondrial GSK-3βphosphorylation was not altered by the mitochondrial ATP-sensitive K_ATP 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-3β(GSK-3β-S9A-HA) mutant, suggesting that zinc prevents reperfusion injury by inactivating GSK-3β. Cells transfected with the catalytically inactive GSK-3β(GSK-3β-KM-HA) also revealed a significant decrease in cell death, strongly supporting the essential role of GSK-3βinactivation in cardioprotection.Confocal imaging study showed that zinc prevented oxidant-induced mPTP opening through the inhibition of GSK-3β.Taken together, these data suggest that zinc prevents reperfusion injury by modulating the mPTP opening through the inactivation of GSK-3β. The PI3K/Akt signaling pathway is responsible for the inactivation of GSK-3βby zinc.