Remifentanil Protects The Heart From Ischemia/Reperfusion Injury By Maintaining Zinc Homeostasis

Part 1 Remifentanil induces cadioprotection against myocardiac ischemia/reperfusion injury by maintaining zinc homeostasis.Objective Although the impairment of zinc homeostasis characterized by a loss of zinc is associated with myocardial reperfusion injury,the role of zinc transporter in myocardial ischemia/reperfusion injury remains unclear.Remifentanil has been proven to be protective against myocardial ischemia/reperfusion(I/R)injury,but the underlying molecular mechanism has not been fully elucidated.The purpose of this part study is to test if remifentanil exerts cardioprotection by maintaining zinc homeostasis.Methods The model of regional cardiac I/R was established on Langendorff apparatus.Isolated rat hearts were subjected to 30 min of regional ischemia followed by 2 h of reperfusion.Remifentanil was given by 3 consecutive 5 min infusions and each infusion was followed by a 5 min drug-free perfusion prior to ischemia.The cardiac zinc concentration was detected by inductively coupled plasma emission spectrometer(ICPOES).The expression of metal responsive factor 1(MTF1)and zinc transporter 1(ZnT1)was determined by Western blotting.H9c2 cells were subjected to 6 h of hypoxia and 2 h reoxygenation.Remifentanil was given for 30 min before hypoxia.Cell viability was determined by CCK8 assay,and zinc level was detected by Newport Green DCF staining.MTF1 overexpression plasmids were constructed and transfected into H9c2 cells 48 h before hypoxia.ZnTl expression was detected by Western blotting.Intracellular zinc levels were detected with laser confocal microscopy.Results Remifentanil preconditioning improved the cardiac function and reduced infarct size in isolated rat hearts.Remifentanil also improved cell viability in H9c2 cells exposed to hypoxia/reoxygenation.The total zinc level of myocardium after cardiac ischemia/reperfusion was significantly reduced,which was reversed by the pretreatment with remifentanil.Western blotting analysis showed that I/R up-regulated the expression of MTF1 and ZnTl,which was inhibited by remifentanil.In addition,MTF1 over-expression blocked the action of remifentanil by increasing ZnT1 expression and the intracellular zinc level.Conclusion I/R causes zinc loss by enhancing ZnTl expression through up-regulation of MTF1.Remifentanil prevents zinc loss by inhibiting ZnTl expression through the suppression of MTF1 expression.Part 2 Zinc reduces mitochondrial damage by inhibiting zinc loss-mediated endoplasmic reticulum stressObjective It has been reported that exogenous zinc modulates the mitochondrial permeability transformation(mPTP)opening by inhibiting endoplasmic reticulum(ER)stress.We hypothesize that remifentanil protects mitochondria by inhibiting zinc deficiency-induced ER stress in the heart.Methods 20 min after stabilization,hearts were treated with Zn2+ chelator TPEN or the ER stress activator TG.Then the hearts were subjected to 30 min ischemia followed by 2h of reperfusion.Hemodynamic changes were recorded by Powerlab system and infarct size was determined by EB+TTC staining.The ER stress markers(CHOP,BIP)and apoptotic parameters were determined by Western blotting.In H9c2 cells,remifentanil was given for 30 min before hypoxia.MTF1 overexpression plasmid,TPEN or TG were administrated before the treatment with remifentanil.The Zn2+ level in ER,cell viability and mitochondrial injury parameters were determined as described above.Results The loss of intracellular zinc triggered by myocardial I/R induced ER stress,an effect that was prevented by remifentanil.TPEN and TG reserved the protective effects of remifentanil on infarct size and apoptosis.MTF1 overexpression decreased the zinc level in the ER,which enhanced the endoplasmic reticulum stress.Furthermore,the protective effects of remifentanil on mitochondrial reactive oxygen species(ROS)and mitochondrial membrane potential(ΔΨm)was abolished by TPEN and TGConclusion Zinc loss from cardiomyocytes by I/R triggered the ER stress and the subsequent mitochondrial damage,leading to myocardial damage.Remifentanil alleviated ER stress-mediated mitochondrial injury by maintaining zinc homeostasis.