The Signaling Mechanism Responsible For Morphine-induced Cardioprotection

AMI (Acute Myocardial Infarction, AMI) is an acute myocardial ischemic disease. Over the past several decades it was seen mainly in Europe and the United States. However, because of the increase of people's living standards, the incidence of myocardial infarction in china has been increasing dramatically in recent years. Although the myocardial infarction caused by ischemia/reperfusion injury has been a critical clinical issue, there is no an effective therapy to successfully prevent ischemia/reperfusion injury. While experimental data show that opioid receptors are involved in pre-condition and post-condition-induced cardioprotection, the underlying mechanism remains elusive.Objective:To test whether morphine, which is a well-recognized cardioprotectant, prevents the mitochondrial permeability transition pore (mPTP) opening through Zn²⁺ and GSK-3β. Objects and Methods:1. Studies in isolated rat cardiomyocytes1.1 Intracellular free Zn2 + was measured in cells loaded with Newport Green DCF (specific fluorescent dye of Zn2 +);1.2 To detect intracellular NO, cells were loaded with DAF-FM (specific fluorescent dye of NO) and imaged with confocal microscopy;1.3 Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE) and the mPTP opening was evaluated with changes inΔΨm;1.4 Mitochondrial Ca2+ was determined by loading cells with Rhod-2 (specific fluorescent dye of Ca2+).The fluorescence images were obtained through the confocal microscopy.1.5 Phosphorylation of GSK-3β(Ser9) was determined with Western blot.2. Studies in H9c2. Cells were transfected with the constitutively active GSK-3βmutant plasmids and the role of GSK-3βin Zn²⁺ induced cardioprotection was evaluated.Results:1. Cardiomyocytes treated with 1μM morphine for 10 min showed a significant increase (155.9±26.1%) in Newport Green DCF fluorescence intensity compared to the control group (109.8±9.4%), indicating that morphine triggers release of Zn²⁺. L-NAME, an inhibitor of NOS, completely nullified the action of morphine on Zn²⁺ release (100.6±6.6%of baseline), suggesting that NO is involved in the action of morphine2 Compared to the control group (102.2±5.3%), morphine increased NO generation, which was indicated by the enhancement of DAF-FM signal (154.9±62.2% of baseline), suggesting that NO is involved in the action of morphine.3 The effect of morphine was abrogated by either ODQ (119.3±6.3 % of baseline) or NS2028 (108.0±5.9 % of baseline), the inhibitors of guanylyl cyclase, implying that cGMP is required for the morphine-induced Zn²⁺ release. The increased Zn²⁺ signal was also abolished by KT5823 (106.5±3.2 % of baseline), a specific inhibitor of PKG, suggesting that PKG is critical for the action of morphine on Zn²⁺ release.4 Both morphine and Zn²⁺ significantly enhanced phosphorylation of GSK-3βat Ser9, indicating that morphine inactivates GSK-3βthrough Zn²⁺.5 Compared to the control group (45.1±8.2%), morphine (1μM) prevented the loss of ?Ψm caused by oxidant stress (100μM H2O2) (90.0±2.0%), indicating that morphine can modulate the mPTP opening. The effect of morphine on ?Ψm was reversed by KT5823 (51.1±4.8%) and the Zn²⁺ chelator TPEN (57.5±2.0%), implying that morphine may prevent the mPTP opening through the PKG/Zn²⁺ pathway.6 Compared to the H2O2-treated control group (218.4±21.9%), cells treated with morphine showed a significant decrease (128.0±10.6%) in Rhod-2 fluorescence intensity, indicating that morphine prevents oxidant-induced mitochondrial Ca2+ overload.7 Exogenous Zn²⁺ inhibited oxidant-induced loss of ?Ψm, an effect that was lost in cells transfected with the constitutively active GSK-3βmutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK-3βthrough Zn²⁺.Conclusion:Morphine mobilizes intracellular Zn²⁺ through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3βthrough Zn²⁺.