| Acute myocardial infarction (AMI) is one of the most important cause of mobidityand motality worldwide. Reperfusion injury is believed to be a major contributor ofcardiomyocytes loss in MI. Reducing reperfusion injury should be able to producelong-term benefit in the chronic phase of heart failure post MI. Others and our previousstudies have demonstrated that activation of cardiomyocyte κ-opioid receptors withexogenous κ-opioid agonist is protective against MI/R injury. However, Whether shortterm activation of cardiomyocyte κ-opioid receptors at the beginning of reperfusionproduces long-term cardiac benefits has never been investigated.The mechanism of by which κ-opioid activation mediates its cardioprotection is notclear. Evidence it might involve activation of endogenous cell survival signaling. HO-1isa well-documented cytoprotective enzyme. Ample evidences have implicated a criticalrole for HO-1in protection against ischemia/reperfusion (I/R) injury. Opioids has beenshown to increase HO-1expression in some pathological conditions. Nevertheless,whether HO-1plays a role in the cardioprotective effects of κ-opioid receptor activationhas not been established. The expression of HO-1is largely controlled by Nrf2, a gene transcription activator,whose nuclear translocation was in turn, controlled by protein kinases such as PI3K/Akt.In our previous study, PI3K/Aktsignaling is shown to be activated by κ-opioid receptoractivation. Based on this we speculate that1) HO-1mediates the cardioprotection ofκ-opioid activation2) κ-opioid activation induced cardioprotection has long-term benefits.3) PI3K/Akt pathway activation and Nrf2nuclear translocation mediates κ-opioidactivation induced HO-1expression. Based on the background above, this study will focuson the following:Objectives1. To investigate the role of HO-1in κ-opioid activation induced cardioprotection.2. To investigate the potential long-term benefit of κ-opioid activation inducedcardioprotection.3. To investigate the mechanism by which κ-opioid activation induced HO-1geneexpression.MethodsExperiment11. Male adult SD rats were randomly assigned to the following groups:1) Sham group,2)I/R group,3) U50,488H treated (I/R+U50) group,4) U50,488H and Nor-BNIco-treated (I/R+U50+Nor-BNI) group,5) U50,488H and LY294002co-treated(I/R+U50+LY) group,6) U50,488H+zinc protoprophyrin-IX co-treated (I/R+U50+ZnPP-IX) group.Cardiac damage was evaluated by measuring cardiomyocytes apoptosis, inflammation,infarct size and cardiac function. HO-1gene transcription and expression wasevaluated by real-time PCR and western blot respectively.2. Cultured cardiomyocytes were subjected to viral infection for HO-1gene interference. Cultured cardiomyocytes were randomly divided into four groups:1) control virus+SI/R,2) HO-1silencing virus+SI/R,3) control virus+SI/R+U50,488H,4) HO-1silencing virus+SI/R+U50,488H. Cardiomyocytes were treated with viral infectionbefore subjected to SI/R. Cardiomyocytes death was evaluated by apoptosismeasurement. HO-1gene transcription and expression was evaluated by real-timePCR and western blot respectively.Results:1. U50,488H treated group have a significantly lower TUNEL positive rate（I/R,27.45±2.23%; I/R+U50,488H,14.84±1.99%, P<0.05）, myocardium caspase-3activity（I/R,89.90±5.37nmol/pNA/h/mg protein; I/R+U50,488H,68.83±4.86nmol/pNA/h/mg protein, P<0.05）, myocardial MPO activity（I/R,22.71±2.32u/gprotein; I/R+U50,488H,14.82±1.06u/g protein, P<0.05） and infarct size（I/R,43.71±2.94%; I/R+U50,488H,27.62±2.92%, P<0.05） compared to I/R group,indicating protection by κ-opioid activation. ZnPP-IX+U50,488H group have ahigher TUNEL positive rate （I/R+U50,488H,14.84±1.99%; I/R+U50,488H+ZnPP-IX,24.67±1.43%; P<0.05）, myocardium caspase-3activity（I/R+U50,488H,68.83±4.86%nmol/pNA/h/mg protein; I/R+U50,488H+ZnPP-IX,85.70±6.907nmol/pNA/h/mg protein; P<0.05）, myocardial MPO activity（I/R+U50,488H,14.82±1.06u/g protein; I/R+U50,488H+ZnPP-IX,24.96±1.624u/g protein;P<0.01） and infarct size （I/R+U50,488H,27.62±2.92%; I/R+U50,488H+ZnPP-IX,40.17±3.912%; P<0.05）compared to U50,488H group. These results suggestthat HO-1inhibition alleviates the κ-opioid cardioprotection.2. U50,488H treated group have a significantly higher HO-1mRNA（I/R,1.000±0.08607; I/R+U50,488H,3.987±0.5504, P<0.01）and protein level （I/R,0.2261±0.04975; I/R+U50,488H,1.089±0.1082, P<0.01）compared to I/R group,indicating increased transcription and expression of HO-1gene by κ-opioidactivation.3. In cultured cardiomyocytes, HO-1gene interference abolished the anti-apoptoticeffect of κ-opioid activation. U50,488H treated cardiomyocytes have a significantly lower TUNEL positive rate（SI/R,48.58±4.55%; SI/R+U50,488H,24.24±2.6%,P<0.05）and caspase-3activity （SI/R,121.9±6.739nmol/pNA/h/mg protein;SI/R+U50,488H,79.41±3.56nmol/pNA/h/mg protein, P<0.05） compared to SI/Rgroup, indicating protection by κ-opioid activation. HO-1interference virustransfected cardiomyocytes have a higher TUNEL positive rate（SI/R+U50+HO-1virus,56.50±4.95%; SI/R++U50+control virus,30.34±4.88%, P<0.05） andcaspase-3activity （SI/R+U50+HO-1virus,134.2±12.38nmol/pNA/h/mg protein;SI/R++U50+control virus,86.33±9.54nmol/pNA/h/mg protein, P<0.05）aftersubjected to SI/R+U50,488H compared with control virus transfectedcardiomyocytes.Experiment2Male adult SD rats were randomly assigned to the same groups as in Experiment1.Heart function were monitored by echocardiography sequentially for28d beforeanimals were sacrificed for histological examination of the hearts. Cardiac remodelingand functional change were evaluated by measuring cardiac hypertrophy, cardiacmorphology, cardiac fibrosis and ejection fraction.Results:After28d, U50,488H treated group have a significantly higher LV ejection fraction（I/R,42.23±2.82%; I/R+U50,488H,58.74±3.718%, P<0.05）, lower LVend-systolic diamete（rI/R,0.5575±0.02194cm; I/R+U50,488H,0.3890±0.02580cm,P<0.05）, LV end-diastolic diamete（rI/R,0.6929±0.05406cm; I/R+U50,488H,0.5244±0.03469cm, P<0.05）, HW/BW（I/R,3.562±0.1506mg/g; I/R+U50,488H,2.655±0.1030mg/g, P<0.05）and Masson staining positive rate （I/R,40.64±3.482%;I/R+U50,488H,29.92±2.194%, P<0.05）compared to I/R group, indicating long-termbenefit by κ-opioid activation cardioprotection. ZnPP-IX+U50,488H group have alower LV ejection fraction（I/R+U50,488H+ZnPP-IX,45.72±3.803%; I/R+U50,488H,58.74±3.718%, P<0.05）, higher LV end-systolic diameter（I/R+U50,488H+ZnPP-IX, 0.5786±0.03589cm; I/R+U50,488H,0.3890±0.02580cm, P<0.05）, LVend-diastolic diameter （I/R+U50,488H+ZnPP-IX,0.7017±0.03439cm;I/R+U50,488H,0.5244±0.03469cm, P<0.05）, HW/BW（I/R+U50,488H+ZnPP-IX,3.70±0.1619mg/g; I/R+U50,488H,2.655±0.1030mg/g, P<0.05） Masson stainingpositive rate （I/R+U50,488H+ZnPP-IX,39.89±2.925%; I/R+U50,488H,29.92±2.194%, P<0.05）compared to I/R group compared to U50,488H group. These resultssuggest that HO-1inhibition alleviates the long-term benefit by κ-opioid activationcardioprotection.Experiment3Male adult SD rats were randomly assigned to the same groups as in Experiment1.P3IK/Akt activation was evaluated by western blot of phos-Akt. Nrf2nucleartranslocation was evaluated by nuclear protein isolation and western blot. HO-1genetranscription and expression was evaluated by real-time PCR and western blotrespectively.Results:1. κ-opioid activation increased PI3K/Akt activation. U50,488H treated group have asignificantly higher level of phos-Akt compared to I/R group（I/R,0.2511±0.09309;I/R+U50,488H,0.9992±0.08677, P<0.05）. LY294002+U50,488H group have alower level of phos-Akt compared to U50,488H group（I/R+U50,488H+LY294002,0.4037±0.03916; I/R+U50,488H,0.9992±0.08677, P<0.05）.2. κ-opioid activation increased Nrf2nuclear translocation. U50,488H treated grouphave a significantly higher level of nuclear Nrf2compared to I/R group（I/R,0.1126±0.0168; I/R+U50,488H,0.3376±0.03206, P<0.05）. LY294002+U50,488Hgroup have a lower level of nuclear Nrf2compared to U50,488H group（I/R+U50,488H+LY294002,0.0921±0.01172; I/R+U50,488H,0.3376±0.03206,P<0.05）.3. PI3K/Akt inhibition blocked κ-opioid activation induced HO-1gene transcription and expression. U50,488H treated group have a significantly higher level of HO-1mRNA（I/R,0.9602±0.08607; I/R+U50,488H,3.987±0.5504, P<0.01） andprotein （I/R,0.2261±0.04975; I/R+U50,488H,1.089±0.1082, P<0.01）comparedto I/R group. LY294002+U50,488H group have a lower level of HO-1mRNA（I/R+U50,488H+LY294002,1.782±0.2795; I/R+U50,488H,3.987±0.5504,P<0.05）and protein（I/R+U50,488H+LY294002,0.2636±0.06386; I/R+U50,488H,1.089±0.1082, P<0.01）compared to U50,488H group.Conclusion1. κ-opioid activation increased HO-1transcription and expression, HO-1mediatesthe cardioprotection by κ-opioid activation in a rat model of myocardialischemia-reperfusion injury and a cultured neonatal rat cardiomyocytes simulatedischemia-reperfusion model.2. κ-opioid activation induced cardioprotection have long-term benefit in cardiacfunction and remodeling.3. Inhibition of PI3K/Akt abolished the increased transcription and translation ofHO-1by κ-opioid activation in a rat model of myocardial ischemia-reperfusioninjury. κ-opioid activation induced HO-1gene transcription and translationthrough PI3K/Akt activation and Nrf2nuclear translocation. |
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