Jinkun Xi

Postconditioning Prevents Reperfusion Injury by Activating δ-Opioid Receptors

Background:While postconditioning has been proposed to protect the heart by targeting the mitochondrial permeability transition pore (mPTP), the detailed mechanism underlying this action is unknown. The authors hypothesized that postconditioning stimulates opioid receptors, which in turn protect the heart from reperfusion injury by targeting the mPTP. Methods:Rat hearts (both in vivo and in vitro) were subjected to 30 min of ischemia and 2 h of reperfusion. Postconditioning was elicited by six cycles of 10-s reperfusion and 10-s ischemia. To measure nitric oxide concentration, cardiomyocytes loaded with 4-amino-5-methylamino-2′,7′-difluorofluorescein were imaged using confocal microscopy. Mitochondrial membrane potential was determined by loading cardiomyocytes with tetramethylrhodamine ethyl ester. Results:In open chest rats, postconditioning reduced infarct size, an effect that was reversed by both naloxone and naltrindole. The antiinfarct effect of postconditioning was also blocked by the mPTP opener atractyloside. In isolated hearts, postconditioning reduced infarct size. Morphine mimicked postconditioning to reduce infarct size, which was abolished by both naltrindole and atractyloside. N-nitro-l-arginine methyl ester and guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one blocked the action of morphine. Further experiments showed that morphine produces nitric oxide in cardiomyocytes by activating &dgr;-opioid receptors. Moreover, morphine could prevent hydrogen peroxide–induced collapse of mitochondrial membrane potential in cardiomyocytes, which was reversed by naltrindole, N-nitro-l-arginine methyl ester, and the protein kinase G inhibitor KT5823. Conclusions:Postconditioning protects the heart by targeting the mPTP through activation of &dgr;-opioid receptors. The nitric oxide–cyclic guanosine monophosphate–protein kinase G pathway may account for the effect of postconditioning on the mPTP opening.

Mechanism for resveratrol-induced cardioprotection against reperfusion injury involves glycogen synthase kinase 3β and mitochondrial permeability transition pore

Resveratrol pretreatment can protect the heart by inducing pharmacological preconditioning. Whether resveratrol protects the heart when applied at reperfusion remains unknown. 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 2 h 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 (A(520)). Resveratrol reduced infarct size and prevented cardiac mitochondrial swelling. Resveratrol enhanced GSK-3beta phosphorylation upon reperfusion, an effect that was mediated by the cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway. Resveratrol translocated GSK-3beta from cytosol to mitochondria via the cGMP/PKG pathway. Further studies showed that mitochondrial GSK-3beta was 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-3beta from cytosol to mitochondria. Translocated GSK-3beta may ultimately interact with cyclophilin D to modulate the mPTP opening.

Exogenous zinc protects cardiac cells from reperfusion injury by targeting mitochondrial permeability transition pore through inactivation of glycogen synthase kinase-3β

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.