| BackgroundRestoring blood flow is essential for salvaging the ischemic myocardium,but myocardial revascularization also produces adverse effects known asmyocardial ischemia/reperfusion (MI/R) injury. MI/R may activate acuteinflammatory reactions, such as increasing tumor necrosis factor alpha (TNF-α)concentrations. Adiponectin (APN) is an adipocyte-derived cytokine with a highcirculatory concentration and exerts vascular-protective, anti-inflammatory, andanti-ischemic properties, but is typically downregulated by MI/R. Experimentaland clinical studies have found TNF-α markedly inhibits APN mRNAexpression in adipocytes, and elevated plasma TNF-α level is associated withlow plasma APN levels, whereas data showing prolonged TNF-α antagonisttreatment increase plasma APN levels in subjects with metabolic dysfunction.However, as such above experiments mainly utilized prolonged anti-TNF-αtherap, the direct evidence defining the causal relationship between TNF-αproduction and APN concentration during the myocardial acute ischemia reperfusion injury remained unclear.AIMSThe aims of the current study were:1) to determine whether a single injection of etanercept (a TNF-α neutralizingantibody) during MI/R may exert cardioprotective effect;2) to investigate whether TNF-α neutralization may upregulate APN levelsduring MI/R; and if so,3) to delineate the potential causative relationship between TNF-αneutralization-induced APN upregulation and the its cardioprotective effects.Methods1. MI/R model: C57BL/6mice were anesthetized with2%isoflurane. MI wasinduced by temporarily exteriorizing the heart via a left thoracic incision atthe fourth intercostal space, and a slipknot was tied around the left anteriordescending coronary artery.10minutes before reperfusion, mice wererandomized to receive either vehicle or etanercept. The slipknot was thenreleased after30minutes duration of ischemia.2. Small-interfering RNA-mediated AdipoR1+R2knock down mice model: Inbrief, C57BL/6mice were anesthetized with2%isoflurane. MI was inducedby temporarily exteriorizing the heart via a left thoracic incision at the fourthintercostal space, AdipoR1-, AdipoR2-specific siRNA or control nonspecificsiRNA oligos were diluted in5%glucose and mixed with in vivo jet PEI.SiRNA targeting AdipoR1and AdipoR2were combined and delivered viathree separate intra-myocardial injections to temporarily blanch the leftventricular free wall. Based upon our pilot experiments demonstratingAdipoR1and R2expression reaches nadir48hours after siRNA injection,mice were processed for further MI/R protocols after this exposure period. 3. After the creation of all these models:1) Cardiac function was determined via echocardiography at1d,7d and14dafter reperfusion by evaluating Left ventricular Ejection fraction (LVEF).Serum APN and TNF-α concentrations were determined by ELISA.Blood samples taken from orbital vein were taken at1,3, and8hours,and1,3,5,7, and14days after reperfusion.2) Mice in the other group were re-anesthetized at the conclusion of the24-hour reperfusion, and cardiac function was determined by invasivehemodynamic evaluation methods. Heart rate, LVEDP,+dp/dtmax and-dp/dtmax were obtained using computer software. After thedetermination of cardiac function, the ligature around the coronary arterywas re-tied, and then myocardial ischemia size was determined by Evansblue/TTC double staining.3) Cardiomyocytes apoptosis was determined by TUNEL labeling andcaspase-3activity assay3h after reperfusion.Results1. Administration of etanercept successfully neutralized serum TNF-α, andameliorated MI/R injury in mice, evidenced by consistently augmentedcardiac function, reduced infarct size and apoptosis.2. Intra-myocardial siRNA delivery successfully suppressed60-70%myocardial AdipoR1and R2protein expression in myocardium.3. Administration of etanercept elevated plasma APN concentration. Thecardioprotective effect of etanercept was significantly attenuated in AdipoR1and R2knockdown mice compared to WT, measured by infarct size andcardiomyocyte apoptosis. ConclusionThese data demonstrate that TNF-α is responsible for the decrease of APNlevel during MI/R, and the cardioprotective effect of anti-TNF-α neutralizingantibody is partially obtained by upregulation of APN level. |
