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OBJECTIVE: To investigate the cardioprotective effect of exogenous zinc (Zn2+) on the mitochondrial pathway, and to explore its possible mechanism. METHODS: Rat heart tissue-derived H9c2 cardiac cells were cultured, and then randomly divided into control group, ZnCl2 group (1-20 μmol·L-1, 20 min), ZnCl2 plus inhibitor group [PI3K inhibitor LY294002, 10 μmol·L-1and mitochondrial ATP sensitive potassium channel (mKATP) inhibitor 5-HD, 0.5 mmol·L-1, inhibitors treated cells for 10 min and then ZnCl2 20 min] and inhibitor group (10 min). The mitochondrial permeability transition pore (mPTP) opening was evaluated by measuring mitochondrial membrane potential (ΔΨm). Tetramethylrhodamine ethyl ester (TMRE) diacetate fluorescence images were obtained with laser scanning confocal microscopy. GSK-3β and AKT phosphorylation were determined with Western blot. The cells were subjected to simulated ischemia/reperfusion injury, cell viability were determined with flow cytometry. Cells were transfected with constitutively active GSK-3β-S9A (GSK-3β-S9A) plasmid by Fugene 6 transfection kit, mPTP opening was evaluated by confocal microscopy. RESULTS: Compared with the normal, exposure of cells to H2O2 for 20 min caused a marked decrease in TMRE fluorescence, treatment of cells with different dose of Zn2+ prevented the loss of TMRE fluorescence caused by H2O2 with the peak at 10 μmol·L-1. Western blot showed that Zn2+ significantly enhanced the GSK-3β and AKT phosphorylation, the effect that was significantly reversed by LY294002, but not 5-HD. Compared with the normal, ischemia/reperfusion markedly reduced cell viability. Zn2+ applied at ischemia did not increase the cell viability, but significantly increased the cell viability when given at reperfusion. Zn2+ could mimic the specific mPTP inhibitor cyclosporin A (1 μmol·L-1) and prevent the mPTP opening, which was again reversed by LY294002 but not 5-HD. Zn2+ was not able to exert protection in cells transfected with the GSK-30-S9A. CONCLUSION: Zn2+ can induce myocardial mitochondrial protective effect by modulating the mPTP opening through the inactivation of GSK-3β via PI3K/AKT pathway. mKATP may not be involved in the action of Zn2+.
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OBJECTIVE: To investigate the cardioprotective effect of astragaloside IV on the mitochondrial pathway, and to explore its possible mechanism.
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<p><b>OBJECTIVE</b>To investigate the underlying mechanism of the protective effects of resveratrol on oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes.</p><p><b>METHODS</b>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-3β phosphorylation. The phosphorylation of GSK-3β 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.</p><p><b>RESULTS</b>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-3β Ser(9) phosphorylation, which could lead the inactivation of GSK-3β. These effects of resveratrol could be significantly abolished by protein kinase G inhibitor KT5823, while KT5823 alone did not affect GSK-3β 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.</p><p><b>CONCLUSIONS</b>Resveratrol could attenuate oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes by inactivating GSK-3β via cGMP/PKG signaling pathway independent of NO-related mechanism.</p>
Subject(s)
Animals , Rats , Carbazoles , Pharmacology , Cell Line , Cyclic GMP , Metabolism , Cyclic GMP-Dependent Protein Kinases , Metabolism , Glycogen Synthase Kinase 3 , Metabolism , Glycogen Synthase Kinase 3 beta , Hydrogen Peroxide , Metabolism , Mitochondria, Heart , Metabolism , Myocytes, Cardiac , Cell Biology , Oxidants , Metabolism , Signal Transduction , Stilbenes , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To define the effects of Astragalus membranaceus on the atrial dynamics and ANP secretion in the perfused beating rabbit atria.</p><p><b>METHOD</b>The experiments have been done in isolated perfused beating rabbit atria. ANP was measured by radioimmunoassay in the atrial perfusate in real-time base.</p><p><b>RESULT</b>A. membranaceus (2.0, 2.5, 3.0 g L(-1)) could increase atria stroke volume from (694.70 +/- 0.01) microL g(-1) (P<0.05) to (1,003.00 +/- 8.80) microL g(-1) (P<0.001); (1,120.00 +/- 17.71) microL g(-1) and (1,195.00 +/- 8.21) microL g(-1) (P<0.001), respectively, and its could difference increase atrial pulse pressure from (0.82 +/- 0.01) kPa to (0.86 +/- 0.01) kPa (P<0.01); (0.96 +/- 0.01) kPa (P<0.001) and (1.02 +/- 0.01) kPa (P<0.001), respectively; A. membranaceus obviously increased rabbit atrial dynamics with dose-dependent manner. Simultaneously, A. membranaceus inhibited ANP secretion. Nifedipine (1.0 micromol L(-1)), a L-type Ca2+ channel inhibitor, and KB-R 7943 (10.0 micromol L(-1)), an inhibitor of reversed Na+ -Ca2+ exchanger, blocked the effects of A. membranaceus-induced augmentation of atrial dynamics but failed to modulation the inhibition of A. membranaceus on ANP secretion.</p><p><b>CONCLUSION</b>A. membranaceus increases the atrial dynamics via Na+ -Ca2+ exchanger and L-type Ca2+ channel and negatively modulates ANP secretion in beating rabbit atria.</p>