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Xi J, Tian W, Zhang L, Jin Y, Xu Z. Morphine prevents the mitochondrial permeability transition pore opening through NO/ cGMP/PKG/[Zn.sup.2+]/GSK-3[beta] signal pathway in cardiomyocytes. Am J Physiol Heart Circ Physiol 298: H601-H607, 2010. First published December 4, 2009; doi:10.1152/ajpheart.00453.2009.--The aim of this study was to test whether morphine prevents the mitochondrial permeability transition pore (mPTP) opening through [Zn.sup.2+] and glycogen synthase kinase 3[beta] (GSK-3[beta]). Fluorescence dyes including Newport Green Dichlorofluorescein (DCF), 4-amino-5-methylamino2',7'-difluorofluorescein (DAF-FM), and tetramethylrhodamine ethyl ester (TMRE) were used to image free [Zn.sup.2+], nitric oxide (NO), and mitochondrial membrane potential ([DELTA] [PHI] m), respectively. Fluorescence images were obtained with confocal microscopy. Cardiomyocytes treated with morphine for 10 rain showed a significant increase in Newport Green DCF fluorescence intensity, an effect that was reversed by the NO synthase inhibitor [N.sup.G]-nitro-L-arginine methyl ester (L-NAME), indicating that morphine mobilizes [Zn.sup.2+] via NO. Morphine rapidly produced NO. ODQ and NS2028, the inhibitors of guanylyl cyclase, prevented [Zn.sup.2+] release by morphine, implying that cGMP is involved in the action of morphine. The effect of morphine on [Zn.sup.2+] release was also abolished by KT5823, a specific inhibitor of protein kinase G (PKG). Morphine prevented oxidant-induced loss of [DELTA][PHI]m, indicating that morphine can modulate the mPTP opening. The effect of morphine on the mPTP was reversed by KT5823 and the [Zn.sup.2+] chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). The action of morphine on the mPTP was lost in cells transfected with the constitutively active GSK-3[beta] mutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK3[beta]. In support, morphine significantly enhanced phosphorylation of GSK-3[beta] at [Ser.sup.9], and this was blocked by TPEN. GSK-3[beta] small interfering RNA prevented the pore opening in the control cardiomyocytes but failed to enhance the effect of morphine on the mPTP opening. In conclusion, morphine mobilizes intracellular [Zn.sup.2+] through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3[beta] through [Zn.sup.2+]. nitric oxide; protein kinase G; guanosine 3',5'-cyclic monophosphate; glycogen synthase kinase 3[beta] doi: 10.1152/ajpheart.00453.2009

Our previous study demonstrated that zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via Akt and glycogen synthetase kinase 3[beta] (GSK-3[beta]). We aimed to address the mechanism by which zinc activates Akt. Treatment of H9c2 cells with Zn[Cl.sub.2] (10 [micro]M) in the presence of the zinc ionophore pyrithione (4 [micro]M) for 20 min enhanced Akt phosphorylation ([Ser.sup.473]), indicating that zinc can rapidly activate Akt. Zinc did not alter either phosphatase and tensin homolog deleted on chromosome 10 (PTEN) phosphorylation and total PTEN protein levels or PTEN oxidation, implying that PTEN may not play a role in the action of zinc. However, zinc-induced Akt phosphorylation was blocked by both the nonselective receptor tyrosine kinase (RTK) inhibitor genistein and the selective insulin-like growth factor-1 RTK (IGF-1RTK) inhibitor AG1024, indicating that zinc activates Akt via IGF-1RTK. Zinc-induced phosphorylation of protein tyrosine and Ser/Thr was also abolished by AG1024. In addition, zinc markedly enhanced phosphorylation of IGF-1 receptor (IGF-1R), which was again reversed by genistein and AG1024. A confocal imaging study revealed that AG1024 abolished the preventive effect of zinc on oxidant-induced mPTP opening, confirming that IGF-1RTK plays a role in zinc-induced cardioprotection. Furthermore, zinc decreased the activity of protein phosphatase 2A (PP2A), a major protein Ser/Thr phosphatase, implying that protein Ser/Thr phosphatases may also play a role in the action of zinc on Akt activity. Taken together, these findings demonstrate that exogenous zinc activates Akt via IGF-1RTK and prevents the mPTP opening in cardiac cells. Inactivation of Ser/Thr protein phosphatases may also contribute to zinc-induced Akt activation. insulin-like growth factor 1 receptor tyrosine kinase; protein phosphatase 2A; protein serine/threonine phosphatase

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-3[beta] (GSK-3[beta]). The treatment of cardiac H9c2 cells with Zn[Cl.sub.2] (10 [micro]M) in the presence of zinc ionophore pyrithione for 20 min significantly enhanced GSK-3[beta] phosphorylation at [Ser.sup.9], indicating that exogenous zinc can inactivate GSK-3[beta] in H9c2 cells. The effect of zinc on GSK-3[beta] 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 roTOR phosphorylation. Further experiments found that zinc also increased mitochondrial GSK-3[beta] phosphorylation. This may indicate an involvement of the mitochondria in the action of zinc. The effect of zinc on mitochondrial GSK-3[beta] phosphorylation was not altered by the mitochondrial ATP-sensitive [K.sup.+] 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-3[beta] (GSK-3[beta]-S9A-HA) mutant, suggesting that zinc prevents reperfusion injury by inactivating GSK-3[beta]. Cells transfected with the catalytically inactive GSK-3[beta] (GSK-3[beta]-KM-HA) also revealed a significant decrease in cell death, strongly supporting the essential role of GSK-3[beta] inactivation in cardioprotection. Moreover, zinc prevented oxidant-induced mPTP opening through the inhibition of GSK-3[beta]. Taken together, these data suggest that zinc prevents reperfusion injury by modulating the mPTP opening through the inactivation of GSK-3[beta]. The PI3K/Akt signaling pathway is responsible for the inactivation of GSK-3[beta] by zinc. h9c2 cell; oxidant stress; simulated ischemia

Limitation of damage after ischemia and reperfusion injury to the myocardium remains an elusive clinical goal. Previous studies have suggested that molecular chaperones, which include members of the heat shock protein (Hsp) family, may have cardioprotective effects, although the protective role of endogenous chaperones has not been well documented. CHIP (carboxyl terminus of Hsp70-interacting protein) is a cochaperone/ubiquitin ligase that integrates the response to stress at multiple levels. We tested the response of CHIP(-/-) mice to in vivo ischemia and reperfusion injury induced by left anterior descending coronary artery ligation. Compared with wild-type littermates, CHIP(-/-) mice had decreased survival and increased incidence of arrhythmias during reperfusion. The size of myocardial infarction, as assessed by the ratio of infarct area to area at risk, was 50% greater in CHIP(-/-) mice. Increased infarct size was accompanied by impaired upregulation of the chaperone Hsp70 after ischemia-reperfusion injury. In situ analysis also indicated that hearts of CHIP(-/-) mice were more prone to develop apoptosis in cardiomyocytes and especially endothelial cells of intramural vessels. Previous studies have found that CHIP plays a central role in maintaining protein quality control and coordinating the response to stress. The present data indicate that these functions of CHIP provide a critical cardioprotective effect in the setting of ischemia-reperfusion injury due in part to increased apoptosis in cardiac cells. Quality control mechanisms therefore may be underappreciated clinical targets for maximizing myocardial protection after injury. chaperone; heat shock protein; apoptosis

Xu, Zhelong, Xiang Ji, and Philip G. Boysen. Exogenous nitric oxide generates ROS and induces cardioprotection: involvement of PKG, mitochondrial [K.sub.ATP] channels, and ERK. Am J Physiol Heart Circ Physiol 286: H1433-H1440, 2004. First published December 4, 2003; 10.1152/ajpheart.00882.2003.--We examined whether cGMP-dependent protein kinase (PKG) and mitochondrial ATP-sensitive potassium ([K.sub.ATP]) channels are involved in S-nitroso-N-acetyl penicillamine (SNAP)-induced reactive oxygen species (ROS) generation. SNAP significantly increased ROS generation in cardiomyocytes. This increase was suppressed by both 5-hydroxydecanoate (5-HD) and glibenclamide. Direct opening of mitochondrial [K.sub.ATP] channels with diazoxide led to ROS generation. The increased ROS generation was reversed by N-(2-mercaptopropionyl)glycine (MPG), a scavenger of ROS. Myxothiazol partially suppressed the ROS generation. KT-5823, an inhibitor of PKG, prevented ROS generation, indicating that PKG is required for ROS generation. In addition, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), an activator of PKG, induced ROS generation. The effect of 8-BrcGMP was reversed by either 5-HD or MPG. YC-1, an activator of guanylyl cyclase, also increased ROS production, which was reversed by 5-HD. Neither LY-294002 nor wortmannin, the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), affected SNAP's action. In a whole heart study, SNAP significantly reduced infarct size. The anti-infarct effect of SNAP was abrogated by either MPG or 5-HD. This effect was also blocked by PD-98059, an ERK inhibitor, but not by LY-294002. A Western blotting study showed that SNAP significantly enhanced phosphorylation of ERK, which was reversed by MPG. These results suggest that SNAP-induced ROS generation is mediated by activation of PKG and mitochondrial [K.sub.ATP] channels and that opening of mitochondrial [K.sub.ATP] channels is the downstream event of PKG activation. ROS and mitochondrial [K.sub.ATP] channels participate in the anti-infarct effect of SNAP. Moreover, phosphorylation of ERK is the downstream signaling event of ROS and plays a role in the cardioprotection of SNAP. reactive oxygen species; guanylyl cyclase: cGMP-dependent protein kinase; mitochondrial ATP-sensitive potassium channel; extracellular signal-regulated kinase: phosphatidylinositol 3-kinase

Zinc homeostasis has been known to play a role in myocardial ischemia/reperfusion (I/R) injury, but the precise molecular mechanisms regulating the expression of ZIP transporters during reperfusion are still unclear. The aim of this study was to determine whether ER Stress/CaMKII/STAT3 pathway plays a role in the regulation of cellular zinc homeostasis. Zinc deficiency increased mRNA and protein expressions of the ER stress relevant markers Chop and Bip, and STAT3 phosphorylation in H9c2 or HL-1 cells, an effect that was abolished by ZnCl2. ER calcium concentration [(Ca2+)ER] was decreased and cytosolic calcium concentration [(Ca2+)I] was increased at the condition of normoxia or ischemia/reperfusion, indicating that zinc deficiency triggers ER stress and Ca2+ leak. Further studies showed that upregulation of STAT3 phosphorylation was reversed by Ca2+ chelator, indicating that intracellular Ca2+ is important for zinc deficiency-induced STAT3 activation. In support, zinc deficiency enhanced ryanodine receptors (RyR), a channel in the ER that mediate Ca2+ release, and Ca2+-calmodulin-dependent protein kinase (CaMKII) phosphorylation, implying that zinc deficiency provoked Ca2+ leak from ER via RyR and p-CaMKII is involved in STAT3 activation. Moreover, inhibition of STAT3 activation blocked zinc deficiency induced ZIP9 expression, and resulted in increased Zn2+ loss in cardiomyocytes, further confirming that STAT3 activation during reperfusion promotes the expression of ZIP9 zinc transporter to correct the imbalance in zinc homeostasis. In addition, suppressed STAT3 activation aggravated reperfusion injury. These data suggest that the ER Stress/CaMKII/STAT3 axis may be an endogenous protective mechanism, which increases the resistance of the heart to I/R. [ABSTRACT FROM AUTHOR]

While the zinc transporter ZIP2 (Slc39a2) is upregulated via STAT3 as an adaptive response to protect the heart from ischemia/reperfusion (I/R) injury, the precise mechanism underlying its upregulation remains unclear. The purpose of this study was to investigate the role of NADPH oxidase (NOX) isoform NOX2-derived ROS in the regulation of ZIP2 expression, focusing on the role of the NOX2 cytosolic factor p67phox. Mouse hearts or H9c2 cells were subjected to I/R. Protein expression was detected with Western blotting. Infarct size was measured with TTC staining. The cardiac-specific p67phox conditional knockout mice (p67phox cKO) were generated by adopting the CRISPR/Cas9 system. I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression was reversed by the ROS scavenger N-acetylcysteine (NAC) and the NOX inhibitor diphenyleneiodonium (DPI). p67phox but not NOX2 expression was increased 30 min after the onset of reperfusion, and downregulation of p67phox by siRNA or cKO invalidated I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression. Both NAC and DPI prevented upregulation of STAT3 phosphorylation and ZIP2 expression induced by overexpression of p67phox, whereas the STAT3 inhibitor stattic abrogated upregulation ZIP2 expression, indicating that the increase of p67phox at reperfusion is an upstream signaling event responsible for ZIP2 upregulation via STAT3. Experiments also showed that chelation of Zn2+ markedly enhanced p67phox and ZIP2 expression as well as STAT3 phosphorylation, whereas supplementation of Zn2+ had the opposite effects, indicating that cardiac Zn2+ loss upon reperfusion triggers p67phox upregulation. Furthermore, ischemic preconditioning (IPC) upregulated ZIP2 via p67phox, and cKO of p67phox aggravated cardiac injury after I/R, indicating that p67phox upregulation is cardioprotective against I/R injury. In conclusion, an increase of p67phox expression in response to Zn2+ is an intrinsic adaptive response to I/R and leads to cardioprotection against I/R by upregulating ZIP2 via STAT3. [ABSTRACT FROM AUTHOR]

Xu, Zhelong, Michael V. Cohen, James M. Downey, Terry L. Vanden Hoek, and Zhenhai Yao. Attenuation of oxidant stress during reoxygenation by AMP 579 in cardiomyocytes. Am J Physiol Heart Circ Physiol 281: H2585-H2589, 2001.--AMP 579, an adenosine [A.sub.1]/[A.sub.2] receptor agonist, has a strong anti-infarct effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether AMP 579 can reduce the production of reactive oxidant species (ROS) during reoxygenation in cultured chick embryonic cardiomyocytes. The intracellular fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH) was used to detect ROS. The cells were subjected to 60 min of simulated ischemia, followed by either 15 min or 3 h of reoxygenation. AMP 579 (0.5 and 1 [micro]M), when started 10 min before reoxygenation, significantly reduced ROS generation from 4.86 [+ or -] 0.30 (arbitrary units) in untreated cells to 2.72 [+ or -] 0.31 and 1.85 [+ or -] 0.14, respectively (P < 0.05). Cell death that was assessed by propidium iodide uptake was markedly reduced by AMP 579 (49.6 [+ or -] 4.7% of control cells vs. 25.4 [+ or -] 2.4%, P < 0.05). In contrast, adenosine did not alter ROS generation or cell death. Attenuation of ROS production by AMP 579 was completely prevented by simultaneous exposure of cells to the selective adenosine [A.sub.2] antagonist 8-(13-chlorostyryl) caffeine. These results indicate that AMP 579 directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates intracellular oxidant stress. Furthermore, adenosine could not duplicate these effects. adenosine; cell viability; reperfusion injury; reactive oxygen species Received 16 April 2001; accepted in final form 31 August 2001