Morphine Preconditioning Protects The Failing Rat Hearts Against Ischemia/reperfusion Injury By Regulating Cav-3/ERK1/2 Pathway And The Underlying Mechanisms

Background Chronic heart failure（CHF）is the end-stage of various heart diseases,with high morbidity,poor prognosis and heavy economic burden.Previous studies have demonstrated that cardiac sugery is one of the most effective ways to improve the long-term survival of patients with ischemia heart disease and valve heart disease.However,they will inevitably undergo myocardial ischemia and reperfusion（I/R）injury when undergo cardiac surgery.Studies show that failing hearts are more likely to suffer from I/R injury in cardiac surgery under cardiopulmonary bypass.Of note,even in patients subject to non-cardiac surgeries,undulated hemodynamics may also lead to myocardial injury or cardiac arrest resulting in poor perioperative outcomests and long-term survival.Ischemic preconditioning（IPC）is one of the powerful endogenous cardioprotective strategies,and opioid receptors are the main pharmacological target in IPC induced cardioprotection.Opioid preconditioning can mimic the myocardial protective effect of IPC through activing opioid receptors in the normal state.However,the cardioprotective effect of IPC or opioid preconditioning is weakened or abolished in some pathological conditions such as aging and diabetes.Nevertheless,serial clinical studies have shown that opioids are still effective in preventing post-ischemia heart injury and reducing the incidence of cardiovascular-related events in patients with chronic heart failure.Our previous studies found that opioid preconditioning alleviates I/R injury in failing heart is related to elevate mu-opioid receptor（MOR）and regulate ERK-GSK3βsignaling pathway.Studies have shown that the opioid receptor is mainly located in the microcapsule part of the cell membrane,and it especially is closely associated with the characteristic caveolin-3（Cav-3）on the microcapsule.Opioid receptors mediated cardioprotection depends on Cav-3.When Cav-3 is overexpressed in the heart,it can significantly enhance the cardioprotection induced by opioid preconditioning,while Cav-3 is knocked out,the opioid preconditioning can not effectively reduce the myocardial I/R injury by activating pro-survaival kinases such as ERK.As a classical opioid,morphine is widely used in cardiovascular surgery playing analgesic and sedative effects,and also reduce cardiac load and myocardial I/R injury.However,it is still unclear whether Cav-3 is involved in morphine preconditioning protecting the failing rat hearts against I/R injury and the underlying mechanism?To address these questions,post-infarcted failing rat hearts and global I/R injury model combined with intracardial adenovirus injection,histopathological and biological techniques were performed to explore the role of Cav-3/ERK1/2 pathway in morphine preconditioning protecting the failing rat hearts against I/R injury.Methods1.Preparation of a chronic heart failure model and the virus in situ injectionAdult male Sprague Dawley（S-D）rats（200-250 g）were used to establish post-infarcted failing heart by ligating the left anterior descending（LAD）coronary artery.In addition,the animals were subjected to AAV9-Cav3sh RNA-GFP or AAV9-GFP by intramyocardial injection in left ventricular after LAD ligation.All operated rats were monitored daily and used for the further study at the end of 4th weeks.2.Ischemia/reperfusion injury model in the islolated heartThe failing rat hearts were removed and suspended on the Langendorff apparatus and continuously retrograded K-H solution.All hearts were stabilized by 15 min,and ischemia/reperfusion（I/R）injury model was established by subjecting to 30 min global ischemia followed by 120 min reperfusion.3.Experimental protocolsFifty-five isolated failing rat hearts were divided into five groups（n=11/group）:Sham,I/R,morphine preconditioning（MPC）,MPC+methyl-β-cyclodextrin（MPC+MβCD）and MβCD group.K-H solution was continuously perfused in Sham group.MPC was performed with three cycles of 5 min of 1μmol/L morphine and 5 min drug-free perfusion followed by I/R model.200μmol/L MβCD was perfused for a period of 10 min before MPC until the beginning of ischemia in MPC+MβCD group.MβCD group was perfused 200μmol/L MβCD for a period of 40 min before I/R.In addition,total of 22 failing rat hearts injected with AAV9-Cav3sh RNA-GFP and AAV9-GFP were used to verify the role of Cav-3 in MPC induced cardioprotection（n=11/group）,and all hearts were administrated with MPC followed by by 30 min global ischemia and 120 min reperfusion.4.Detection of LDH activity,infarct size,apoptosis and proteinThe coronary effluent was collected to detect the activities of lactate dehydrogenase（LDH）by using chemical colorimetry at baseline,5 min and 10 min after reperfusion.The volume of infart size（IS）and area at risk（AAR）were measured by 2,3,5-triphenyltetrazolium chloride（TTC）at 120 min of reperfusion.IS/AAR was also calculated in the study.In addition,TUNEL staining was performed by detecting apoptosis,and the protein levels of Cav-3,ERK1/2 and p-EKR1/2 were detected by western blot at the end of reperfusion.Results1.Evaluation of post-infarcted failing rat heart and global I/R injury modelCompared with normal rat heart,echocardiography parameters showed that LVEDD and LVESD were significantly increased while LVEF were decreased by 45%in the failing heart at the end of 4^thweek.In the I/R injury model,the percentage of infarct size was remarkably elevated from 3%in sham group to 30%in I/R group.At the same time,the level of LDH activity in coronary effluent at 5 and 10 min of reperfusion significantly increased.Above results indicated that both post-infarcted failing rat heart and I/R injury model were successfully established in the study.2.MPC alleviated I/R injury in post-infarcted failing rat heartsCompared with the IR group,MPC significantly reduced the ratio of IS/AAR to13%and apoptosis（28%vs.12%）,as well as decreased the levels of LDH by approximately 100 IU/L at 5 min of reperfusion.The expression of Cav-3 protein was up-regulated and the ratio of p-ERK1/2/ERK1/2 increased by 2.6-fold in the MPC group（P<0.05）.These results demonstrated that MPC protecting the failing rat hearts against I/R injury is associated with the elevation of Cav-3 and phosphorylated ERK.3.MβCD blocked the cardioprotection induced by morphine preconditioning in failing heartTo explore the mechanism of MPC protecting failing heart against ischemia/reperfusion injury,MβCD,a non-specific caveolin destructor,was used in this study.As a result,MβCD abolished the effect of MPC by reversing the decreases in IS/AAR（13%vs.32%）and the activity of LDH,as well as the ratio of apoptosis（12%vs.25%）in failing rat hearts.What’s more,the expression of Cav-3 protein and the phosphorylation of ERK1/2 induced by MPC was strongly inhibited by MβCD.These results indicated that the protective effect of morphine preconditioning on failure heart was significantly weakened after non-specific destruction of pit protein,which was related to the failure of effectively increasing the levels of CAV-3 and ERK phosphorylation.4.Morphine preconditioning induced cardioprotection was blocked by AAV9-Cav3sh RNA-GFPTo further investigate the role of Cav-3 in morphine preconditioning,cardiac Cav-3was specifically knockout by injecting AAV9-Cav3sh RNA-GFP.The results showed that the ratio of IS/AAR and apoptosis were elevated to 27%and 30%in AAV9-Cav3sh RNA-GFP group,respectively.In addtion,the expression of Cav-3protein in AAV9-Cav3sh RNA-GFP group significantly decreased by around 50%,and the phosphorylation level of p-ERK1/2 decreased by 60%compared with AAV9-GFP group（P<0.05）.All the results strongly suggested that the protective effect of morphine preconditioning in the failing rat heart is dependent on Cav-3/ERK pathways.ConclusionMorphine preconditioning confers cardioprotection in post-infarcted failing rat hearts,and the mechanism is related to upregulate the expression of Cav-3 protein and elevate ERK1/2 phosphorylation resulting in reduction of apoptosis.