Endocannabinoids Signaling In Cardioprotection By Propofol Preconditioning

Background:Perioperative myocardial ischemia is the leading cause of perioperative death.Reducing ischemia-reperfusion injury is becoming a major obstacle for better recovery with the introduction of immediate revascularization.Proper handling of perioperative injury by anesthesiologists helps improve prognosis.Propofol,a widely used intravenous anesthetic,has been found to be cardioprotective by preconditioning.The potential molecular mechanisms include antioxidation,regulating ROS/NO signaling,anti-inflammation,or activating RISK pathway.However,little is known about the upstreaming target or initiating molecule.Propofol is a competitive inhibitor of fatty acid amide hydrolase(FAAH),which catalyzes the degradation of endocannabinoids(ECs)such as anandamide(AEA)and 2-arachidonoylglycerol(2-AG).We explored the role of endocannabinoid signaling in cardioprotection by propofol preconditioning in current study.Section 1: Propofol preconditioning increased myocardial endocannabinoids levels in vitro and in vivoObjective: The endocannabinoid system comprises endocannabinoids,receptors(CB1R,CB2 R,TRPV1 and unidentified G-protein coupled receptors)and synthetic and degradative pathways.This section aims to investigate the roles of propofol preconditioning on cardiomyocyte hypoxia/reoxygenation injury in vitro,endocannabinoids release in vitro and in vivo,and CB1R/CB2 R transcription and translation levels in vitro.Methods: We first investigated the effects of propofol preconditioning on hypoxia/reoxygenation-induced cardiomyocyte apoptosis and endocannabinoids release.Neonatal rat ventricle cells were divided into four groups: control,propofol,H/R(hypoxia/reoxygenation)and propofol+H/R(propofol preconditioning by incubating cardiomyocytes with 50 μM of propofol 1 hour prior to hypoxia until the end of hypoxia).After reoxygenation,apoptosis was measured by flow cytometry with Annexin V/PI-staining.Endocannabinoid(2-AG and AEA)in cell culture media were detected by UPLC-MS/MS.We further measured mRNA and protein levels of CB1 R and CB2 R by Real-time PCR and Western Blot respectively.The effects of propofol preconditioning on serum 2-AG and AEA concentrations during myocardial ischemia reperfusion were further identified using an in vivo myocardial IRI model by ligaturing and loosing left anterior descending branch of coronary artery.In this model,propofol preconditioning was achieved by a bolus intravenous injection of 10mg/kg followed by one-hour continuous infusion at a rate of 39 mg/kg·h for one hour just before ischemia.The rats were randomized into four groups just like the experiment in vitro.Results: Propofol preconditioning helped reduce apoptosis compared with H/R group.Hypoxia/reoxygenation itself caused a transient release of 2-AG and AEA in vitro.Propofol preconditioning further increased 2-AG and AEA concentrations in cell culture media.Hypoxia/reoxygenation but not propofol enhanced the transcription and translation of CB1 R and CB2 R in cultured cardiomyocytes.Increased serum concentrations of 2-AG and AEA were also seen during rat myocardial IRI.Conclusion: Propofol preconditioning is cardioprotective and could increase 2-AG and AEA levels in cultured cardiomyocytes and in vivo.Section 2: Cardioprotection by propofol preconditioning was mediated by endocannabinoidsObjective: To investigate the role of endocannabinoids release in propofol preconditioning-induced cardiomyocyte protection from hypoxia/reoxygenation injury.Methods: An in vitro cardiomyocyte hypoxia/reoxygenation injury model was used.Neonatal rat ventricle cells were subjected to hypoxia/reoxygenation with or without propofol preconditioning.Selective FAAH inhibitor URB597 and endocannabinoids re-uptake inhibitor was further used to increase endocannabinoids levels before propofol preconditioning.The following indicators were measure to prove our hypothesis: Cell viability by using a commercial cell counting kit(CCK-8);apoptosis by flow cytometry using Annexin V/PI-staining;LDH,MDA and SOD concentrations by commercial kits and cell ROS and NO levels by measuring immunofluorescence intensity using flow cytometry.Results: Propofol preconditioning was useful in enhancing cell viability,decreasing apoptosis and reducing oxidation injury with a drop in MDA,ROS and NO concentrations and an elevation of SOD levels.Increasing endocannabinoids levels before reoxygenation by URB597 and VDM11 was cardiomyocyte protective.Cardioprotection by propofol preconditioning could be mimicked by URB597 and VDM11.No synergistic effect was found between propofol and URB597 or between propofol and VDM11.Conclusions: Increased endocannabinoids release protected cardiomyocytes against hypoxia/reoxygenation injury.Cardioprotection by propofol preconditioning depended on increased endocannabinoids release.Section 3: Propofol preconditioning reduced rat myocardial ischemia reperfusion injury through CB2RObjective: Both CB1 R and CB2 R were expressed in cardiomyocytes as well as other cells in myocardium.We explored the role of CB1 R and CB2 R signaling in propofol-induced cardioprotection in vivo.Methods: An in vivo rat myocardial ischemia reperfusion injury model was used.Adults rats received ligation of left anterior descending branch of coronary artery with or without propofol preconditioning.Moreover,we tested the role of CB1 R signaling in propofol preconditioning by intravenous injection of selective CB1 R antagonists AM251(1mg/kg)30 minutes before propofol exposure.The role of CB2 R receptor was also investigated using selective CB2 R antagonists AM630.After reperfusion,infarct area was evaluated using Evans blue plus TTC staining.Evidence of myocardial injury was observed after HE staining of myocardium.We further investigated the severity of myocardial injury by measuring serum cTnI levels,oxidation by measuring serum MPO and SOD and inflammation by measuring serum IL-1β,TNFα,sICAM-1 and IL-10.Results: Propofol preconditioning was useful in reducing infarction area,lowering serum levels of cTn I,MPO,IL-1β,TNFα and sICAM-1.Serum SOD and IL-10 concentrations were elevated by propofol preconditioning.Blocking of CB1 R signaling by AM251 provided some beneficial effects on alleviating myocardial reperfusion injury.CB2 receptor antagonists AM630 but not CB1 R antagonists AM251 could reverse the cardioprotective effects induced by propofol preconditioning.Conclusions: Cardioprotection by propofol preconditioning in vivo was mediated by CB2 R signaling.