Resveratrol Inhibits Zinc Deficiency-Induced Mitophagy and Exerts Cardiac Cytoprotective Effects.

Resveratrol (Res) possesses various beneficial effects, including cardioprotective, anti-inflammatory, anti-aging, and antioxidant properties. However, the precise mechanism underlying these effects remains unclear. Here we investigated the protective effects of resveratrol on cardiomyocytes, focusing on the role of Zn2+ and mitophagy. Using the MTT/lactate dehydrogenase assay, we found that addition of a zinc chelator TPEN for 4 h induced mitophagy and resulted in a significant reduction in cell viability, increased cytotoxicity, and apoptosis in H9c2 cells. Notably, resveratrol effectively mitigated these detrimental effects caused by TPEN. Similarly, Res inhibited the TPEN-induced expression of mitophagy-associated proteins, namely P62, LC3, NIX, TOM20, PINK1, and Parkin. The inhibitory action of resveratrol on mitophagy was abrogated by the mitophagy inhibitor 3-MA. Additionally, we discovered that silencing of the Mfn2 gene could reverse the inhibitory effects of resveratrol on mitophagy via the AMPK-Mfn2 axis, thereby preventing the opening of the mitochondrial permeability transition pore (mPTP). Collectively, our data suggest that Res can safeguard mitochondria protection by impeding mitophagy and averting mPTP opening through the AMPK-Mfn2 axis in myocardial cells.

Zinc Overload Induces Damage to H9c2 Cardiomyocyte Through Mitochondrial Dysfunction and ROS-Mediated Mitophagy.

Zinc homeostasis is essential for maintaining redox balance, cell proliferation, and apoptosis. However, excessive zinc exposure is toxic and leads to mitochondrial dysfunction. In this study, we established a zinc overload model by treating rat cardiomyocyte H9c2 cells with Zn2+ at different concentrations. Our results showed that zinc overload increased LDH and reactive oxygen species (ROS) levels, leading to cell death, mitochondrial membrane potential decrease and impaired mitochondrial function and dynamics. Furthermore, zinc overload activated the PINK1/Parkin signaling pathway and induced mitochondrial autophagy via ROS, while NAC inhibited mitophagy and weakened the activation of PINK1/Parkin pathway, thereby preserving mitochondrial biogenesis. In addition, our data also showed that Mfn2 deletion increased ROS production and exacerbated cytotoxicity induced by zinc overload. Our results therefore suggest that Zn2+-induced ROS generation causes mitochondrial autophagy and mitochondrial dysfunction, damaging H9c2 cardiomyocytes. Additionally, Mfn2 may play a key role in zinc ion-mediated endoplasmic reticulum and mitochondrial interactions. Our results provide a new perspective on zinc-induced toxicology.

Zn2+ protect cardiac H9c2 cells from endoplasmic reticulum stress by preventing mPTP opening through MCU.

Zn2+ regulates endoplasmic reticulum stress (ERS) and is essential for myocardial protection through gating the mitochondrial permeability transition pore (mPTP). However, the underlining mechanism of the mPTP opening remains uncertain. Cells under sustained ERS induce unfolded protein responses (UPR) and cell apoptosis. Glucose regulatory protein 78 (GRP 78) and glucose regulatory protein 94 (GRP 94) are marker proteins of ERS and regulate the onset of apoptosis through the endoplasmic reticulum signaling pathway. We found tunicamycin (TM) treatment activates ERS and significantly increases intracellular Ca2+ and mitochondrial reactive oxygen species (ROS) levels in H9c2 cardiomyocyte cells. Zn2+ markedly decreased protein level of GRP 78/94 and suppressed intracellular Ca2+ and ROS elevation. Mitochondrial calcium uniporter (MCU) is an important Ca2+ transporter protein, through which Zn2+ enter mitochondria. MCU inhibitor ruthenium red (RR) or siRNA significantly reversed the Zinc effect on GRP 78, mitochondrial Ca2+ and ROS. Additionally, Zn2+ prevented TM-induced mPTP opening and decreased mitochondrial Ca2+ concentration, which were blocked through inhibiting or knockdown MCU with siRNA. In summary, our study suggests that Zn2+ protected cardiac ERS by elevating Ca2+ and closing mPTP through MCU.

Neuroprotection Effect of Astragaloside IV from 2-DG-Induced Endoplasmic Reticulum Stress.

OBJECTIVE: Astragaloside IV shows neuroprotective activity, but its mechanism remains unclear. To investigate whether astragaloside IV protects from endoplasmic reticulum stress (ERS), we focus on the regulation of glycogen synthase kinase-3ß (GSK-3ß) and mitochondrial permeability transition pore (mPTP) by astragaloside IV in neuronal cell PC12. METHODS AND RESULTS: PC12 cells treated with different concentrations of ERS inductor 2-deoxyglucose (2-DG) (25-500 µM) showed a significant increase of glucose-regulated protein 78 (GRP 78) and GRP 94 expressions and a decrease of tetramethylrhodamine ethyl ester (TMRE) fluorescence intensity and mitochondrial membrane potential (∆Ψm), with the peak effect seen at 50 µM, indicating that 2-DG induces ERS and the mPTP opening. Similarly, 50 µM of astragaloside IV increased the GSK-3ß phosphorylation at Ser9 most significantly. Next, we examined the neuroprotection of astragaloside IV by dividing the PC12 cells into control group, 2-DG treatment group, astragaloside IV plus 2-DG treatment group, and astragaloside IV only group. PC12 cells treated with 50 µM 2-DG for different time courses (0-36 hr) showed a significant increase of Cleaved-Caspase-3 with the peak at 6 hr. 2-DG significantly induced cell apoptosis and increased the green fluorescence intensity of Annexin V-FITC, and these effects were reversed by astragaloside IV. Such a result indicates that astragaloside IV protected neural cell survival from ERS. 2-DG treatment significantly increased the expressions of inositol-requiring ER-to-nucleus signal kinase 1 (IRE1), phosphor-protein kinase R-like ER kinase (p-PERK), but not affect the transcription factor 6 (ATF6) expression. 2-DG treatment significantly decreased the phosphorylation of GSK-3ß and significantly reduced the TMRE fluorescence intensity and ∆Ψm, following mPTP open. Astragaloside IV significantly inhibited the above effects caused by 2-DG, except the upregulation of ATF6 protein. Taken together, astragaloside IV significantly inhibited the ERS caused by 2-DG. CONCLUSION: Our data suggested that astragaloside IV protects PC12 cells from ERS by inactivation of GSK-3ß and preventing the mPTP opening. The GRP 78, GRP 94, IRE1, and PERK signaling pathways but not ATF6 are responsible for GSK-3ß inactivation and neuroprotection by astragaloside IV.

Zn2+ and mPTP mediate resveratrol-induced myocardial protection from endoplasmic reticulum stress.

Resveratrol displays cardioprotective activity; however, its mechanism of action remains unclear. In the current study, resveratrol-induced myocardial protection from endoplasmic reticulum stress (ERS) was investigated, focusing on the roles of Zn2+ and the mitochondrial permeability transition pore (mPTP). We found, using the MTT/LDH kit, that 2-DG-induced ERS significantly decreased H9c2 cell viability. Resveratrol markedly inhibited the expression of endoplasmic reticulum chaperone GRP 78/94 and ERS-related apoptosis proteins CHOP, Caspase12, and JNK induced by 2-DG. The zinc ion chelator TPEN, and ERK/GSK-3ß inhibitors PD98059 and SB216763 and their siRNAs blocked resveratrol function. The AKT inhibitor LY294002 and siRNA did not alter the action of resveratrol. In addition, resveratrol significantly increased the phosphorylation of ERK and GSK-3ß. Resveratrol prevented 2-DG-induced mPTP opening and increased intracellular Zn2+ concentration indicated by TMRE and Newport Green DCF fluorescence intensity, which were further abrogated by ERK/GSK-3ß inhibitors and siRNAs. Our data suggested that resveratrol protected cardiac cells from ERS by mobilizing intracellular Zn2+ and preventing mPTP opening through the ERK/GSK-3ß but not PI3K/AKT signaling pathway.

Roles of Endoplasmic Reticulum Stress in NECA-Induced Cardioprotection against Ischemia/Reperfusion Injury.

OBJECTIVE: This study aimed to investigate whether the nonselective A2 adenosine receptor agonist NECA induces cardioprotection against myocardial ischemia/reperfusion (I/R) injury via glycogen synthase kinase 3ß (GSK-3ß) and the mitochondrial permeability transition pore (mPTP) through inhibition of endoplasmic reticulum stress (ERS). METHODS AND RESULTS: H9c2 cells were exposed to H2O2 for 20 minutes. NECA significantly prevented H2O2-induced TMRE fluorescence reduction, indicating that NECA inhibited the mPTP opening. NECA blocked H2O2-induced GSK-3ß phosphorylation and GRP94 expression. NECA increased GSK-3ß phosphorylation and decreased GRP94 expression, which were prevented by both ERS inductor 2-DG and PKG inhibitor KT5823, suggesting that NECA may induce cardioprotection through GSK-3ß and cGMP/PKG via ERS. In isolated rat hearts, both NECA and the ERS inhibitor TUDCA decreased myocardial infarction, increased GSK-3ß phosphorylation, and reversed GRP94 expression at reperfusion, suggesting that NECA protected the heart by inhibiting GSK-3ß and ERS. Transmission electron microscopy showed that NECA and TUDCA reduced mitochondrial swelling and endoplasmic reticulum expansion, further supporting that NECA protected the heart by preventing the mPTP opening and ERS. CONCLUSION: These data suggest that NECA prevents the mPTP opening through inactivation of GSK-3ß via ERS inhibition. The cGMP/PKG signaling pathway is responsible for GSK-3ß inactivation by NECA.

Tauroursodeoxycholic acid inhibits endoplasmic reticulum stress, blocks mitochondrial permeability transition pore opening, and suppresses reperfusion injury through GSK-3ß in cardiac H9c2 cells.

This study investigates whether inhibition of endoplasmic reticulum (ER) stress prevents opening of the mitochondrial permeability transition pore (mPTP) and evaluates the corresponding signaling pathways involved in this process. Exposure of cardiac H9c2 cells to 800 µM H2O2 for 20 min opened mPTP in response to oxidative stress, as demonstrated by quenching of tetramethylrhodamine ethyl ester (TMRE) fluorescence. Oxidative stress-induced mPTP opening was rescued by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) in a dose-dependent manner at low concentrations. The PI3K and PKG inhibitors LY294002 and KT5823 inhibited the effect of TUDCA on mPTP opening, suggesting the involvement of PI3K/Akt and PKG signaling pathways. TUDCA significantly increased glycogen synthase kinase 3 (GSK-3ß) phosphorylation at Ser-9, with peak effect at 30 µM TUDCA. The level of GRP78 (ER chaperone) expression was significantly upregulated by 30 µM TUDCA. TUDCA-induced increases in Akt and GSK-3ß phosphorylation were inhibited by LY294002, whereas KT5823 suppressed TUDCA-induced increases in VASP and GSK-3ß phosphorylation. Oxidative stress severely affected cell morphology and ultrastructure. TUDCA prevented H2O2-induced ER swelling and mitochondrial damage. TUDCA boosted the viability of cells disrupted by ischemia/reperfusion (I/R), indicating that TUDCA eased reperfusion injury. However, TUDCA did not improve the viability of cells expressing the constitutively active GSK-3ß mutant (GSK-3ß-S9A-HA) that were subjected to I/R, suggesting an essential role of GSK-3ß inactivation in TUDCA-mediated cardioprotection against reperfusion damage. These data indicate that ER stress inhibition prevents mPTP opening and attenuates reperfusion injury through GSK-3ß inactivation. The PI3K/Akt and PKG pathways may mediate GSK-3ß inactivation.

[Injurious effect of zinc deficiency on cardiomyocytes].

The aim of the present study was to investigate the effect of zinc deficiency on cardiomyocyte survival and the underlying mechanisms. Simulated zinc deficiency model was developed in H9c2 cardiac cells with zinc chelator N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). MTT assay was used to evaluate cell viability. Morphological changes of the cells were observed by optical microscope. Lacate dehydrogenase (LDH) levels of the cells were determined with LDH assay kit. Mitochondrial membrane potential (ΔΨ) was measured with confocal microscope using JC-1 dye. Intracellular reactive oxygen species (ROS) levels were determined by DCFH-DA staining. PD98059 (an inhibitor of ERK), SNAP, which can activate ERK, and the ROS scavenger, MPG, were respectively used to investigate mechanism of signal transduction. The phosphorylation of ERK was detected by Western blot. The results showed that TPEN significantly induced the cell morphological damage and the loss of ΔΨ, increased LDH leakage, and promoted ROS generation. In the H9c2 cells, TPEN significantly inhibited ERK phosphorylation and decreased cell viability, which was potentiated by PD98059, whereas both SNAP and MPG reversed the inhibitory effects of TPEN. These data suggest that zinc deficiency leads to the injury in H9c2 cardiac cells through down-regulating ERK pathway. Increased intracellular ROS may account for the effect of zinc deficiency.

Zn2+ and mPTP Mediate Endoplasmic Reticulum Stress Inhibition-Induced Cardioprotection Against Myocardial Ischemia/Reperfusion Injury.

The purpose of this study was to determine whether Zn2+ is involved in endoplasmic reticulum (ER) stress inhibition-induced cardioprotection against ischemia/reperfusion (I/R) injury by modulation of the mitochondrial permeability transition pore (mPTP) opening. Isolated rat hearts were subjected to 30-min regional ischemia followed by 2 h of reperfusion. Expression of glucose regulated protein 78 (GRP 78 or BIP), an ER homeostasis marker, was not increased during ischemia but was increased upon reperfusion, indicating that ER stress was initiated upon reperfusion but not during ischemia. The ER stress inhibitor tauroursodeoxycholic acid (TUDCA) given at reperfusion resulted in a significant reduction of GRP78 expression 30 and 60 min after the onset of reperfusion, an effect that was reversed by the zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN). The immunofluorescence study also showed that the effect of TUDCA on GRP78 expression was reversed by TPEN. TUDCA reduced infarct size and this was reversed by the mPTP opener atractyloside, indicating that ER stress inhibition may induce cardioprotection by modulating the mPTP opening. Experiments with transmission electron microscopy and hematoxylin-eosin staining also revealed that TUDCA prevented endoplasmic reticulum and mitochondrial damages at reperfusion, which was blocked by TPEN. Exposure of cardiac H9c2 cells to H2O2 increased GRP 78 and GRP 94 expressions, suggesting that oxidative stress can induce ER stress. Cells treated with H2O2 showed a significant decrease in tetramethylrhodamine ethyl ester (TMRE) fluorescence, indicating that H2O2 triggers the mPTP opening. In contrast, TUDCA prevented the loss of TMRE fluorescence, the effect that was blocked by TPEN, indicating a role of Zn in the preventive effect of ER stress inhibition on the mPTP opening. In support, TUDCA significantly increased intracellular free zinc. These data suggest that reperfusion but not ischemia initiates ER stress and inhibition of ER stress protects the heart from reperfusion injury through prevention of the mPTP opening. Increased intracellular free Zn accounts for the cardioprotective effect of ER stress inhibition.

Lipopolysaccharides may aggravate apoptosis through accumulation of autophagosomes in alveolar macrophages of human silicosis.

Silica dust mainly attacks alveolar macrophages (AMs) and increases the apoptosis of AMs in silicosis patients. However, it is still unclear whether autophagy is affected. Autophagy mainly has defensive functions in response to stress, contributing to cell survival in adverse conditions, and conversely it has also been implicated in cell death. Lipopolysaccharide (LPS) induces autophagy and apoptosis in macrophages. The role of LPS in autophagy and apoptosis in AMs of silicosis patients is unknown. In this study, we collected AMs from 53 male workers exposed to silica and divided them into an observer (control) group, and stage I, II and III patient groups. We found increased levels of LC3B, SQSTM1/p62 and BECN1，whereas the phosphorylation of MTOR，and levels of LAMP2, TLR4, MYD88, TICAM1, as well as the number of lysosomes decreased with the development of silicosis. LPS stimulation triggered autophagy and increased levels of SQSTM1 in AMs. The autophagy inhibitor, 3-methyladenine (3MA), inhibited LPS-induced apoptosis in the AMs of silicosis patients. Moreover, 3MA reversed the LPS-induced decrease in BCL2 and the increase in BAX and CASP3 levels in AMs. These results suggest that autophagosomes accumulate in AMs during silicosis progression. LPS can induce the formation of autophagosomes through a TLR4-dependent pathway, and LPS may exacerbate the apoptosis in AMs. Blockade of the formation of autophagosomes may inhibit LPS-induced apoptosis via the intrinsic apoptotic pathway in AMs. These findings describe novel mechanisms that may lead to new preventive and therapeutic strategies for pulmonary fibrosis.

[Effects of α1-PDX, a furin inhibitor, on growth, invasion, and tumorigenicity of cervical cancer HeLa cells].

OBJECTIVE: To investigate the effects of the furin inhibitor α1-PDX on the growth, invasion, and tumorigenicity of cervical cancer cells and explore the mechanisms. METHODS: The changes in the growth, migration and invasion of α1-PDX-transfected HeLa cells were observed using MTT assay, Boyden migration and invasion assay. The protein levels of furin and MT1-MMP were measured using Western blotting and furin activity was detected by enzyme activity assay in the transfected cells. HeLa cells were seeded subcutaneously in nude mice and the tumor volume changes were recorded. RESULTS: Compared with the control cells, α1-PDX-treated cells showed a significant growth inhibition by 18.4% at 24 h (P<0.01) with obviously lowered migration ability and cell invasiveness (P<0.01). Treatment with α1-PDX significantly reduced furin enzyme activity and MTI-MMP protein levels in HeLa cells. In nude mice, α1-PDX-treated HeLa cells exhibited a delayed and lowered tumorigenicity with reduced size of the tumors. CONCLUSION: α1-PDX can inhibit the growth, metastasis and tumorigenicity of HeLa cells, the mechanism of which may involve a decreased furin activity and MTI-MMP expression.

Astragaloside IV inhibits oxidative stress-induced mitochondrial permeability transition pore opening by inactivating GSK-3ß via nitric oxide in H9c2 cardiac cells.

OBJECTIVE: This study aimed to investigate whether astragaloside IV modulates the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3ß (GSK-3ß) in H9c2 cells. METHODS: H9c2 cells were exposed to astragaloside IV for 20 min. GSK-3ß (Ser(9)), Akt (Ser(473)), and VASP (Ser(239)) activities were determined with western blot. The mPTP opening was evaluated by measuring mitochondrial membrane potential (ΔΨ(m)). Nitric oxide (NO) generation was measured by 4-amino-5-methylamino-2', 7'-difluorofluorescein (DAF-FM) diacetate. Fluorescence images were obtained with confocal microscopy. RESULTS: Astragaloside IV significantly enhanced GSK-3ß phosphorylation and prevented H(2)O(2)-induced loss of ΔΨ(m). These effects of astragaloside IV were reversed by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, the NO sensitive guanylyl cyclase selective inhibitor ODQ, and the PKG inhibitor KT5823. Astragaloside IV activated Akt and PKG. Astragaloside IV was also shown to increase NO production, an effect that was reversed by L-NAME and LY294002. Astragaloside IV applied at reperfusion reduced cell death caused by simulated ischemia/reperfusion, indicating that astragaloside IV can prevent reperfusion injury. CONCLUSIONS: These data suggest that astragaloside IV prevents the mPTP opening and reperfusion injury by inactivating GSK-3ß through the NO/cGMP/PKG signaling pathway. NOS is responsible for NO generation and is activated by the PI3K/Akt pathway.

Atrial natriuretic peptide prevents the mitochondrial permeability transition pore opening by inactivating glycogen synthase kinase 3ß via PKG and PI3K in cardiac H9c2 cells.

The purpose of this study was to test if atrial natriuretic peptide (ANP) can prevent the mitochondrial permeability transition pore (mPTP) opening by inactivating glycogen synthase kinase 3ß (GSK-3ß). ANP prevented loss of mitochondrial membrane potential (ΔΨ(m)) caused by H(2)O(2) in a dose-dependent manner. Similarly, cyclosporin A, an inhibitor of the mPTP opening, could also preserve ΔΨ(m). ANP increased GSK-3ß phosphorylation at Ser(9), pointing to that ANP inactivates GSK-3ß. ANP could not prevent the loss of ΔΨ(m) in cells transfected with the constitutively active GSK-3ß (GSK-3ß-S9A) mutant. The effects of ANP on GSK-3ß phosphorylation and ΔΨ(m) were reversed by the selective PKG inhibitor KT5823 [2,3,9,10,11,12-hexahydro-10R-methoxy-2,9-dimethyl-1-oxo-9S,12R-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, methyl ester]. In support, PKG markedly enhanced GSK-3ß phosphorylation. ANP-induced GSK-3ß phosphorylation was also abolished by the PI3K inhibitor LY294002 [2-(4-morpholinyl-4H-1-benzopyran-4-one hydrochloride)] and ANP could not prevent H(2)O(2)-induced loss of ΔΨ(m) in the presence of LY294002. These data suggest that ANP modulates the mPTP opening by inactivating GSK-3ß through PKG and PI3K. GSK-3ß is a common downstream target of PKG and PI3K. Prevention of the mPTP opening may underlie the mechanism for ANP's protection against reperfusion injury.

[Resveratrol attenuates oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes via inactivating GSK-3ß].

OBJECTIVE: To investigate the underlying mechanism of the protective effects of resveratrol on oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes. METHODS: H9c2 cells, a permanent cell line derived from embryonic rat cardiac tissue, and then randomly divided into control group [PBS, cells exposed to H2O2 (600 µmol/L) for 20 min to induce mitochondrial oxidant damage], resveratrol group (0.01, 0.1, 1, 5, 10 and 20 µmol/L for 20 min at 20 min before exposing to H2O2), resveratrol plus inhibitor group (1 µmol/L KT5823 for 10 min at 10 min before 5 µmol/L resveratrol treatment) and inhibitor group (1 µmol/L KT5823 for 10 min). Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE) and the mitochondrial permeability transition pore (mPTP) opening was evaluated by measuring the decrease of TMRE fluorescence intensity. Immunofluorescence assay was used to observe GSK-3ß phosphorylation. The phosphorylation of GSK-3ß and VASP were determined by Western blot. To detect intracellular NO, cells were loaded with DAF-FM DA (specific fluorescent dye of NO) and imaged with confocal microscopy. RESULTS: Compared to the control group, resveratrol (0.01-5 µmol/L) attenuated H2O2-induced mitochondrial damage reflected by attenuating the H2O2-induced TMRE fluorescence intensity decrease in a dose-dependent manner and the efficacy of 10 and 20 µmol/L resveratrol was significantly lower than that of 5 µmol/L resveratrol. Resveratrol also significantly upregulated the protein expression of VASP and increased GSK-3ß Ser(9) phosphorylation, which could lead the inactivation of GSK-3ß. These effects of resveratrol could be significantly abolished by protein kinase G inhibitor KT5823, while KT5823 alone did not affect GSK-3ß and VASP phosphorylation. Confocal microscopy showed that DAF-FM (specific NO indicator) was similar between resveratrol and control group, suggesting that resveratrol did not produce NO. CONCLUSIONS: Resveratrol could attenuate oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes by inactivating GSK-3ß via cGMP/PKG signaling pathway independent of NO-related mechanism.

The molecular mechanism underlying morphine-induced Akt activation: roles of protein phosphatases and reactive oxygen species.

Although Akt is reported to play a role in morphine's cardioprotection, little is known about the mechanism underlying morphine-induced Akt activation. This study aimed to define the molecular mechanism underlying morphine-induced Akt activation and to determine if the mechanism contributes to the protective effect of morphine on ischemia/reperfusion injury. In cardiac H9c2 cells, morphine increased Akt phosphorylation at Ser(473), indicating that morphine upregulates Akt activity. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling, was not involved in the action of morphine on Akt activity. Morphine decreased the activity of PP2A, a major protein Ser/Thr phosphatase, and inhibition of PP2A with okadaic acid (OA) mimicked the effect of morphine on Akt activity. The effects of morphine on PP2A and Akt activities were inhibited by the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)glycine (MPG) and the mitochondrial K(ATP) channel closer 5-hydroxydecanoate (5HD). In support, morphine could produce ROS and this was reversed by 5HD. Finally, the cardioprotective effect of morphine on ischemia-reperfusion injury was mimicked by OA but was suppressed by 5HD or MPG, indicating that protein phosphatases and ROS are involved in morphine's protection. In conclusion, morphine upregulates Akt activity by inactivating protein Ser/Thr phosphatases via ROS, which may contribute to the cardioprotective effect of morphine.

The critical role of intracellular zinc in adenosine A(2) receptor activation induced cardioprotection against reperfusion injury.

Exogenous zinc can protect cardiac cells from reperfusion injury, but the exact roles of endogenous zinc in the pathogenesis of reperfusion injury and in adenosine A(2) receptor activation-induced cardioprotection against reperfusion injury remain unknown. Adenosine A(1)/A(2) receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size in isolated rat hearts subjected to 30min ischemia followed by 2h of reperfusion. This effect of NECA was partially but significantly blocked by the zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and ZnCl(2) given at reperfusion mimicked the effect of NECA by reducing infarct size. Total tissue zinc concentrations measured with inductively coupled plasma optical emission spectroscopy (ICPOES) were decreased upon reperfusion in rat hearts and this was reversed by NECA. NECA increased intracellular free zinc during reperfusion in the heart. Confocal imaging study showed a rapid increase in intracellular free zinc in isolated rat cardiomyocytes treated with NECA. Further experiments revealed that NECA increased total zinc levels upon reperfusion in mitochondria isolated from isolated hearts. NECA attenuated mitochondrial swelling upon reperfusion in isolated hearts and this was inhibited by TPEN. Similarly, NECA prevented the loss of mitochondrial membrane potential (DeltaPsim) caused by oxidant stress in cardiomyocytes. Finally, both NECA and ZnCl(2) inhibited the mitochondrial metabolic activity. NECA-induced cardioprotection against reperfusion injury is mediated by intracellular zinc. NECA prevents reperfusion-induced zinc loss and relocates zinc to mitochondria. The inhibitory effects of zinc on both the mPTP opening and the mitochondrial metabolic activity may account for the cardioprotective effect of NECA.

Morphine prevents the mitochondrial permeability transition pore opening through NO/cGMP/PKG/Zn2+/GSK-3beta signal pathway in cardiomyocytes.

The aim of this study was to test whether morphine prevents the mitochondrial permeability transition pore (mPTP) opening through Zn(2+) and glycogen synthase kinase 3beta (GSK-3beta). Fluorescence dyes including Newport Green Dichlorofluorescein (DCF), 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), and tetramethylrhodamine ethyl ester (TMRE) were used to image free Zn(2+), nitric oxide (NO), and mitochondrial membrane potential (DeltaPsi(m)), respectively. Fluorescence images were obtained with confocal microscopy. Cardiomyocytes treated with morphine for 10 min showed a significant increase in Newport Green DCF fluorescence intensity, an effect that was reversed by the NO synthase inhibitor N (G)-nitro-L-arginine methyl ester (L-NAME), indicating that morphine mobilizes Zn(2+) via NO. Morphine rapidly produced NO. ODQ and NS2028, the inhibitors of guanylyl cyclase, prevented Zn(2+) release by morphine, implying that cGMP is involved in the action of morphine. The effect of morphine on Zn(2+) release was also abolished by KT5823, a specific inhibitor of protein kinase G (PKG). Morphine prevented oxidant-induced loss of DeltaPsi(m), indicating that morphine can modulate the mPTP opening. The effect of morphine on the mPTP was reversed by KT5823 and the Zn(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-3beta mutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK-3beta. In support, morphine significantly enhanced phosphorylation of GSK-3beta at Ser(9), and this was blocked by TPEN. GSK-3beta 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(2+) through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3beta through Zn(2+).

Adenosine A2A and A2B receptors work in concert to induce a strong protection against reperfusion injury in rat hearts.

We aimed to test if stimulation of both adenosine A2A and A2B receptors is required to produce an effective cardioprotection against reperfusion injury. Isolated rat hearts were subjected to 30-min regional ischemia followed by 2 h of reperfusion. The adenosine A1/A2 receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size, an effect that was reversed by both the adenosine A2A antagonist SCH58261 and the A2B antagonist MRS1706. The A2B agonist BAY 60-6583 but not the selective A2A agonist CGS21680 reduced infarct size. Interestingly, a combination of BAY 60-6583 and CGS21680 further reduced infarct size. These results suggest that both A2A and A2B receptors are involved in NECA's anti-infarct effect at reperfusion. NECA attenuated mitochondrial swelling upon reperfusion and this was blocked by both SCH58261 and MRS1706, indicating that activation of A2 receptors with NECA can modulate reperfusion-induced mitochondrial permeability transition pore (mPTP) opening. In support, NECA also prevented oxidant-induced loss of mitochondrial membrane potential (DeltaPsi(m)) and matrix Ca2+ overload in cardiomyocytes via both the A2 receptors. In addition, NECA increased mitochondrial glycogen synthase kinase-3beta (GSK-3beta) phosphorylation upon reperfusion and this was again blocked by SCH58261 and MRS1706. In conclusion, A2A and A2B receptors work in concert to prevent reperfusion injury in rat hearts treated with NECA. NECA may protect the heart by modulating the mPTP opening through inactivating mitochondrial GSK-3beta. A simultaneous stimulation of A2A and A2B receptors at reperfusion is required to produce a strong cardioprotection against reperfusion injury.

Mechanism for resveratrol-induced cardioprotection against reperfusion injury involves glycogen synthase kinase 3beta 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.

Ethanol prevents oxidant-induced mitochondrial permeability transition pore opening in cardiac cells.

AIMS: The purpose of this study was to determine if ethanol prevents the mitochondrial permeability transition pore (mPTP) opening via glycogen synthase kinase 3beta (GSK-3beta). METHODS: Cardiac H9c2 cells were exposed to ethanol (10-1000 microM) for 20 min. GSK-3beta activity was determined by measuring its phosphorylation at Ser(9). Mitochondrial membrane potential (DeltaPsi(m)) was assessed by imaging (confocal microscopy) H9c2 cells loaded with tetramethylrhodamine ethyl ester (TMRE). To activate GSK-3beta, cells were transfected with constitutively active GSK-3beta (GSK-3beta-S9A-HA) mutant plasmid. RESULTS: Treatment of cardiac cells with low doses of ethanol (10-500 microM) significantly enhanced GSK-3beta phosphorylation, indicating that ethanol can inactivate GSK-3beta in H9c2 cells. The effect of ethanol on GSK-3beta activity was reversed by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and ethanol could enhance Akt phosphorylation, implying that the PI3K/Akt pathway accounts for the action of ethanol. Ethanol prevented oxidant (H(2)O(2))-induced loss DeltaPsi(m), an effect that was reversed by LY294002, indicating that ethanol can modulate the mPTP opening caused by oxidant stress through the PI3K/Akt pathway. Ethanol failed to preserve DeltaPsi(m) in cells transfected with the constitutively active GSK-3beta (GSK-3beta-S9A-HA) mutant, suggesting that ethanol prevents the mPTP opening by inactivating GSK-3beta. CONCLUSIONS: These data suggest that ethanol prevents the mPTP opening through inactivation of GSK-3beta. The PI3K/Akt signaling pathway is responsible for inactivation of GSK-3beta by ethanol.