Study Of Morphine-induced Cardioprotection Against Ischemia/Reperfusion Injury

Part one Mitochondrial mechanism in morphine-induced cardioprotect- ion against ischemia/reperfusion injuryObjective: The purpose of this study was to investigate whether morphine protects rat hearts from myocardial ischemia/reperfusion injury through inhibiting the m PTP（mitochondrial permeability transition pore, m PTP） opening and to explore the potential intracellular signaling transduction mechanisms.Methods: Isolated rat hearts were perfused on a Langendorff apparatus and were subjected to 30 min ischemia followed by 2 h of and reperfusion. Rats were randomly divided into the control, morphine, and the m PTP opener/inhibitor groups. Powerlab biological signal acquisition system was used for the determination of cardiac function, includingheart heart rate（HR）, coronary flow（CF）, end diastolic pressure（EDP）, and left ventricular developed pressure（LVDP）. TTC staining was used to detect myocardial infarct size. Myocardial tissues were collected from the left ventricular risk zone 5 and 10 min after the onset of reperfusion. Mitochondria were isolated from cardiomyocytes by the differential centrifugation technique. Calcium-induced mitochondrial swelling was measured to assess the opening of the m PTP. Western blotting analysis was used to detect the phosphorylation of glycogen synthase kinase-3β（GSK-3β） and protein kinase B（PKB or AKT） with the myocardial tissue from the risk zone 5 and 10 minutes after the onset of reperfusion.Results:1 Effect of morphine on cardiac functionCompared to the control group, HR was not significantly different（P > 0.05） at reperfusion. The differences of CF, EDP and LVDP were statistically significant（P < 0.05）, implying that morphine can improve the myocardial contractile function.2 Effect of morphine on the myocardial infarctionCompared with the control group, morphine given at reperfusion significantly reduced the myocardial infarction. The anti-infarct effect of morphine was reversed by the m PTP opener Atractyloside（P < 0.05）, indicating that morphine protects the heart from reperfusion through inhibition of the m PTP opening.3 Effect of morphine on the m PTP openingThe absorbance of the myocardial mitochondria at 520 nm significantly decreased in the control group. Compared with the control group, morphine given at reperfusion could mimic the effect of the m PTP inhibitor cyclosporin A（Cs A） by inhibiting the decrease at 520 nm absorbance（P < 0.05）, indicating that morphine inhibits the myocardial m PTP opening.4 Effect of morphine on the GSK-3β phosphorylation at reperfusionCompared with the control group, morphine given at reperfusion significantly increased the phosphorylation of GSK-3β at 5 and 10 minutes from reperfusion（P < 0.05）, indicating that morphine protects the heart by inactivating GSK-3β.5 Effect of morphine on the phosphorylation of AKT at reperfusionCompared with the control group, morphine given at reperfusion significantly increased the phosphorylation of AKT at 5 and 10 minutes from reperfusion（P<0.05）, indicating that morphine protects the heart by activating the AKT signaling pathway.Conclusion: Morphine protects the heart from ischemia/reperfusion by modulating the m PTP opening through inactivation of GSK-3β via the AKT signaling pathway. Part two Role of endoplasmic reticulum stress in morphine-induced cardioprotection against ischemia/reperfusion injuryObjective: The purpose of this study was to investigate whether morphine protect rat hearts from myocardial ischemia/reperfusion injury through inhibiting of the ER stress（endoplasmic reticulum stress, ER stress）and to explore the potential intracellular signaling transduction mechanisms.Methods: Isolated rat hearts were subjected to 30 ischemia followed by 120 min of reperfusion on a Langendorff apparatus. Hearts were randomly divided into the sham, ischemia/reperfusion（I/R）, ER stress inhibitor tauroursodeoxycholic acid（TUDCA） and morphine groups. Myocardial tissues were taken from the left ventricular risk zone at different time points during ischemia and reperfusion. Western blot analysis was used to detect ER stress marker GRP78（chaperone glucose regulated protein 78） expression. ER stress inhibitor TUDCA and morphine were given at reperfusion. TTC staining was used to detect myocardial infarct size.Results:1 Effect of myocardial ischemia on ER stressThere was no significant change in the expression of ER stress chaperone GRP78 during myocardial ischemia for 30 minutes, implying that myocardial ischemia does not induce ER stress.2 Effect of myocardial reperfusion on ER stressCompared with the sham group, the expression of ER stress chaperone GRP78 during reperfusion was significantly increased during reperfusion（P < 0.05）, indicating that myocardial reperfusion causes ER stress.3 Effect of morphine on ER stressMorphine given at reperfusion could mimic the effect of the ER stress inhibitor TUDCA by significantly decreasing the expression of ER stress chaperone GRP78（P < 0.05）, indicating that morphine suppresses the reperfusion-induced ER stress.4 Effect of morphine on myocardial infarctionCompared with the control group, morphine given at reperfusion could mimic the effect of the ER stress inhibitor TUDCA by significantly reducing infarct size（P < 0.05）, indicating that morphine protects the heart from reperfusion through inhibition of ER stress.Conclusion: ER stress occurs upon reperfusion, not during ischemia. Morphine protects the heart from reperfusion injury by inhibiting ER stress. Part three Zinc mediates mitochondria-endoplasmic reticulum “CROSS TALK” in morphine-induced cardioprotection against is chemia/ reperfusion injuryObjective: The purpose of this study was to investigate whether zinc ion(Zn²⁺) was involved in morphine-induced cardioprotection against ischemia/ reperfusion injury and to explore the potential endoplasmic reticulummitochondria "cross talk" mechanism.Methods: Isolated rat hearts were subjected to 30 min ischemia and 120 min reperfusion on a Langendorff device. Hearts were randomly divided into the sham, ischemia/reperfusion（I/R）, morphine and morphine plus the zinc chelator TPEN groups. Morphine and TPEN were given at reperfusion respectively. Western blot analysis was used to detect the expression of ER stress chaperone GRP78. Myocardial tissues were collected from the left ventricular risk zone at reperfusion. Immunofluorescence staining was performed to detect FITC（fluorescein isothiocyanate, FITC） labeled GRP78 and propidium iodide（PI） labeled nuclei. Green and red fluorescence intensity changes was observed with a laser scanning confocal microscope. Myocardial ultrastructure was observed with transmission electron microscope after 4% glutaraldehyde fixation. Myocardial tissues were taken and fixed in 4% formalin solution and stained with hematoxylin eosin（haematoxylin-eosin staning, HE staining） to observe morphological changes of myocardium. Embryonic rat heart tissue derived H9c2 cells were routinely cultured. Mitochondria was stained with the red fluorescent probe tetramethyl rhodamine ethyl ester（TMRE） to assess mitochondrial membrane potential（?Ψm） and the m PTP opening. H9c2 cells were stained with zinc ion specific fluorescent probe Newport Green DCF, and the changes of green fluorescence intensity were observed by laser scanning confocal microscope to detect intracellular free zinc.Results:1 Effect of morphine on reperfusion-induced ER stressCompared with the sham group, the expression of ER stress chaperone GRP78 was significantly increased during reperfusion period. Morphine given at reperfusion significantly decreased the expression of GRP78, which was reversed by the Znic chelator TPEN（P < 0.05）, indicating that morphine could suppress the reperfusion-induced ER stress through Zinc.2 Effect of morphine on the GRP78 fluorescence intensity during reperfusionCompared with the sham group, ischemia/reperfusion significantly increased green fluorescence intensity of the ER stress chaperone GRP78. Morphine given at reperfusion significantly inhibited the GRP78 green fluorescence intensity, which was reversed by the Zinc chelator TPEN（P < 0.05）. This result was consistent with the Western blotting sudy, implying that morphine inhibits the reperfusion-induced ER stress through Zinc.3 Effect of morphine on myocardial ultrastructureCompared with sham group, mitochondria and endoplasmic reticulum were injured by ischemia/reperfusion, including mitochondrial swelling, double membrane rupture, cristae broken, dissolved or disappeared, endoplasmic reticulum swelling and degranulation, etc. Morphine given at reperfusion significantly reduced the reperfusion-induced injury of mitochondria and endoplasmic reticulum. This protective effect of morphine was inhibited by the zinc chelator TPEN, indicating that morphine protects caardiac cells through zinc.4 Effect of morphine on morphology of cardiac muscleCompared with the sham group, myocardial fiber gap widened, arranged in disorder, fracture during ischemia/reperfusion. Morphine given at reperfusion significantly reduced the myocardial fiber damage, which was inhibited by the zinc chelator TPEN, further supporting that morphine protects the heart from reperfusion through zinc.5 Effect of morphine on the m PTP openingThere was no a significan change in the TMRE red fluorescence intensity for 20 minutes in H9c2 cardiac cells without the treatment. TMRE fluorescence intensity was significantly decreased 20 min after the treatment with H₂O₂, indicating that oxidative stress can cause the mitochondrial membrane potential decreased. Because the decrease of the mitochondrial membrane potential is a symbol of the m PTP opening, these results suggest that oxidative stress induces the m PTP opening. The ER stress inducer 2-deoxy-D-glucose（2-deoxy-D-glucose, 2-DG） could mimic H₂O₂, significantly reduced the TMRE red fluorescence intensity, indicating that ER stress induces the m PTP opening. ER stress inhibitor TUDCA significantly inhibited the decrease of the TMRE red fluorescence intensity, indicating inhibition of ER stress could prevent the m PTP opening. Morphine could mimic the effects of TUDCA, Zn Cl2 and Cyclosporin A by significantly inhibiting the decrease of the TMRE red fluorescence intensity. The effect of morphine was inhibited by the zinc chelator TPEN（P < 0.05）, further supporting that morphine prevents the m PTP opening through zinc.6 The signaling mechanism underlying the effect of momorphine on the m PTP openingCompared with the H₂O₂ treated group, morphine significantly inhibited the decrease of the TMRE red fluorescence intensity, which was reversed by the Akt inhibitor AKTI and constitutively acitve GSK-3β transfected plasmid mutant S9A（P<0.05）, indicating that morphine prevents the m PTP opening through through AKT/GSK-3β signaling pathway.7 Effect of morphine on the zinc releaseThere was no a significant changes in the Newport green DCF green fluorescence intensity for 10 minutes in H9c2 cardiac cells. Newport green DCF green fluorescence intensity significantly increased after treatment with Zn Cl2 for 10 minutes, indicating that intracellular free zinc ions was increased. ER stress inhibitor TUDCA and morphine could mimic Zn Cl2, by increasing the Newport green DCF fluorescence intensity. The effect of morphine was inhibited by znic chelator TPEN（P < 0.05）, indicating that morphine may inhibit ER stress by increasing the intracellular zinc.Conclusion: Morphine inhibits ER stress by increasing intracellular zinc and prevents the m PTP opening at reperfusion. The AKT/GSK-3β signaling pathway may be involved in the mitochondria-endoplasmic reticulum "cross talk" mechanism.