Adiponectin By Adipor1 - Appl1 Signaling Pathway To Protect Myocardial Ischemia Reperfusion

Background and ObjectiveObesity related diseases have an important impact on the occurrence,severity and outcome of ischemic heart disease. Low plasma concentration ofAPN under the condition of obesity or insulin resistance increases the risk ofcardiovascular disease. Patients with obesity, hyperlipidemia or diabetesmellitus （metabolic syndrome） are more likely to develop myocardial infarction.APN level in these patients is negatively related with morbidity of myocardialinfarction.Adiponectin （APN）, synthesized and secreted by white/brown adiposetissue, is an adipose tissue-derived hormone abundant in plasma, functioning asmultiple protection. Numerous studies reveal that APN is implicated inmodulation of insulin sensitization, anti-inflammation, anti-atherosclerosis aswell as glucose/lipid metabolism. It is evidenced that exogenous APN treatmentmitigates ischemia/reperfusion injury （IRI） of normal myocardium throughAMPK signaling pathway and reduction of oxidative/nitrative stress. APN reduces myocardial IRI by activation of AMPK signaling pathway. It isconfirmed that heart protection of APN displays as enhanced survival ofmyocardial cells and less production of TNF-α. APN might exert anti-apoptosiseffect through direct activation of AMPK signaling pathway in myocardial cells.Large amount of in vivo studies have evidenced that down-regulation of AMPKincreases myocardial cell apoptosis; Meanwhile, APN reduces apoptosis ofcardiac fibroblasts through AMPK pathway. Therefore, it is of great importanceto clarify the mechanism of APN-AMPK signaling pathway for more efficiencyin reducing myocardial IRI. APN protects myocardium from IRI throughextenuation of oxidative/nitrative stress. It is documented that APN not onlymarkedly inhibits·O₂-release in endothelial cells, but also eliminates incrementof·O₂-release induced by LDL, leading to extenuation of oxidative stress inmyocardial cells. NO is nontoxic even in a higher concentration. Whereas NOcauses oxidative/nitrative stress through reaction with localized superoxide,which produces cytotoxic peroxynitrite, resulting in oxidative/nitrative stress.Cytotoxic products as ONOO-destroy DNA and protein through nitration.Protein nitration is also crucial in physiological signaling pathway. APNincreases NO level through eNOS activation, attaining vascular/heart protectionunder physiological condition. When in pathological condition, APN inhibitsiNOS to reduce excessive production of NO, which mitigates nitrative stressinjury in tissues. Hence identifying the NO pathway in APN reducingmyocardial IRI is beneficial for the application of APN in clinical practice.APPL1, a signaling protein consisting of multiple functioning domains,plays an important role in APN signaling pathway. APN receptors contains twosubtypes, AdipoR1, mainly distributed in skeletal muscles, and AdipoR2predominantly in liver. APN modulates glucose/lipid metabolism throughAdipoR1-APPL1pathway in myocardium. It is revealed that APN exerts heartprotection by APPL1activating AMPK. APPL1-AMPK axis mediates thebeneficial metabolic effect of APN in heart. Whereas the conclusive signaling molecules involved in APPL1-dependent pathway remains unclear. whether ornot APN prevents myocardium from IRI through AdipoR1-APPL1pathwayremains elusive. Unraveling the downstream of APN implicated in reduction ofmyocardial ischemia/reperfusion injury promises novel method to treat IRI bytargeting APN.Therefore, the aim of this study was to investigate:1. WhetherAdipoR1-APPL1combination activates AMPK to protect myocardial IRI;2.Whether AdipoR1-APPL1reduces myocardial IRI through modulation ofoxidative/intrative stress.MethodExperiment1: Mimicking myocardial IRI in primary SD neonatal ratmyocardial cells: Primary SD neonatal rat myocardial cells was isolated andincubated in humidified atmosphere with95%CO₂at37℃for3～4days.After treatment with ischemic medium for24h in5%CO₂at37℃, cells wererefreshed with normal medium for6h. Then apoptotic index of myocardial cellswere determined by TUNEL assay and caspase-3activity. Remarkable apoptosisin control group suggested that MI/R model was successfully established bymyocardial cells experiencing H/R.Experiment2: Effects and mechanisms of down-regulation of APPL1inmyocardial cells to MI/R: SiRNA was synthesized by Genepharma corporationaccording to SD rat APPL1DNA sequence provided in GeneBank. Myocardialcells were transfected with siRNA. APPL1protein was evaluated by WesternBlot analysis. After cells experiencing H/R, apoptotic index was assessed byTUNEL assay and caspase-3activity; Expressions of AMPK, eNOS and iNOSprotein were evaluated by Western Blot analysis.Results1. Mimicking H/R in primary SD neonatal rat myocardial cells after isolation and incubation.Cell attachment4～6h, apparent proliferation12～24h and confluence ofcells3～4days after incubation were observed. Round shaped myocardial cellswere converted into fusiform, star shaped and polygon with spontaneousrhythmic contraction.2. APN inhibited apoptosis of myocardial cells induced by H/RApoptotic index was evaluated by TUNEL assay. Compared with controlgroup, AI in H/R group was markedly increased （n=6, P<0.01）. AI andcaspase-3activity in H/R+APN group were lower than those in H/R group （n=6,P<0.01）, while increased superoxide anion in cardiac tissue was found inH/R+APN group. All these confirmed that APN inhibited apoptosis ofmyocardial cells induced by H/R.3. Possible mechanisms of APN extenuating MI/R throughAdipoR1-APPL1pathway.1) Knock-down of APPL1exacerbated MI/R injury.Compared with control group, the expression of APPL1in H/R group wassignificantly lower（n=3, P<0.05）; APPL1expression in H/R+gAd group wasmarkedly higher compared with H/R group（n=3, P<0.05）. After inhibition ofAPPL1expression in cadiocytes by RNA interference, AI in H/R groupincreased（P<0.01）, which could not be reversed by gAd treatment, indicatingthat APN exerts anti-apoptotic effect through APPL1activation.2) APN might mitigate MI/R injury through AdipoR1-APPL1-AMPKpathwayAI and p-AMPK level in H/R group were higher than those in controlgroup（P<0.05）; decreased AI, caspase-3activity and increased p-AMPK levelwere found in H/R+gAd group（P<0.05）. APPL1siRNA application increased AI,caspase-3activity and reduced p-AMPK level（P<0.05）. These suggested down-regulation of APPL1lead to less AMPK phosphorylation.3) APN reduced oxidative/nitrative stress to mitigate MI/R injury throughAdipoR1-APPL1signaling pathway.Determinations of iNOS and eNOS revealed increase of eNOSphosphorylation and decrease of AI, caspase-3activity and iNOS concentrationin H/R+gAd group compared with H/R group（P<0.05）. Moreover, lower eNOSphosphorylation in APPL1siRNA group was found. These suggested reductionof oxidative/nitrative stress through AdipoR1-APPL1pathway.Conclusion:This study documented that APN mitigates MI/R injury throughAipoR1-APPL1pathway for the first time, whose downstream signalingpathways were related with mechanisms of AMPK-dependent anti-apoptosis andAMPK-independent anti-oxidative/nitrative stress. These results provideexperimental evidences for further investigation on the cardiovascular protectionof APN.