Cardioprotective Effect Of CTRP9 Against Ischemia/Reperfusion Injury And The Underlying Mechanism

Background:Ischemic heart disease(IHD)has been the number one enemy against the healthy of human species,and obesity-related metabolic disorder,such as type 2 diabetes,is the most dangerous risk factor of IHD.The key to treat IHD is to restore blood supply to ischemic myocardium as soon as possible,however restoration of blood is always inevitably accompanied by further cardiac injury,widely acknowledged as ischemia/reperfusion(I/R)injury.I/R injury is the leading cause for poor prognosis of patients suffered from IHD and patients underwent by-pass cardiac surgery.Anti-I/R injury has been the hottest topic in the scientific field,the study of I/R injury and understanding of the underlying mechanism have valuable therapeutic potential.Apart from energy storage,the adipose tissue is capable of synthesis and secret multiple bio-active adipocytokines,including adiponectin.Adiponectin has been the focus of study for years,and its function in regulating metabolism,inflammation and I/R injury has been greatly revealed.Exogenous administrated adiponectin effectively reduces infarction and significantly improves cardiac function induced by I/R,however,this protective effect is blunted in diabetic condiction,known as diabetic resistance.In seek to solve this problem,the CTRP(C1q/TNF-related protein)family was discovered,which shares similar structure with adiponectin and consists of at least 15 newly discovered family members.CTRP9,one of the CTRP proteins,is expressed in cardiac tissue with abundance 100 times more than adiponectin.More importantly,circulating and cardiac CTRP9 level is closely related to obesity and type 2 diabetes.Our team previously reported that CTRP9 exerted vasorelaxant effect in isolated aortic vascular rings through eNOS/Nitric Oxide-mediated endothelium-dependent signaling pathway.We first found that CTRP9 improved post ischemia heart function and relieved post ischemia cardiac injury and prevented ventricular remolding.However,the molecular signal pathway underlying the cardioprotective effect of CTRP9 against cardiac I/R injury remains largely unknown.Endoplasmic reticulum stress(ERS)plays important part in the pathogenesis of atherosclerosis,insulin resistance,as well as type 2 diabetes and,is involved in cardiac I/R injury.However,the role of ERS in CTRP9 mediated cardioprotection was never reported.Aims:Protective effect of CTRP9 against cardiac ischemia/reperfusion injury in normal and type 2 diabetic condiction and the underlying mechanisms.Methods:Part1:Cardioprotective effect of different concentration of CTRP9 against myocardial I/R injury in normal rat.This part of experiment conducted in isolated SD rat hearts was designed to evaluate the cardioprotective effect of CTRP9 against I/R injury.Different concentrations of globular CTRP9 were added to the perfusate in the following setting.The isolated perfused rat hearts were randomly put into 4 groups: ?1 Vehicle;?2 CTRP9 0.3 ?g;?3 CTRP9 1 ?g;?4 CTRP9 3 ?g.Firstly,the hearts were perfused with KH buffer for 30 min.Secondly,they were perfused with KH buffer containing different concentration of CTRP9 or equal amount of vehicle.Different dosage of CTRP9 or KH buffer were add directly into the perfusate as 1 m L bolus injection.The drug or KHB were administrated at the end of 30-min-equilibration through an injection port above the heart.Thirdly,isolated hearts were subjected to ischemia by closing the valve positioned upstream of the heart for 30 min.Finally,isolated hearts were reperfused for 60 min by re-opening of the valve.The hemodynamic parameters were monitored and recorded in real time through a Biopac data collecting system,including HR,LVDP,+dP/dt,CF and RPP(calculated by heart×left vertrical pressure).LDH level in the coronary effluent(LDH Release Assay Kit),the myocardial infarct size(the hearts were frozen,sliced and stained with TTC),the apoptosis rate of cardiomyocytes(frozen section of the hearts were stained with TUNEL Cell Apoptosis Detection Kit and observed using fluorescence microscope),and protein expression levels(Caspase-3,DsbA-L,CHOP and GRP-78 determined by western blot)were determined after reperfusion.Part 2: Protective effect of CTRP9 against H/R injury in ER stressed H9c2 cells.The protective effect of globular CTRP9 against H/R injury in ER stressed H9c2 cells was explored.The cells were randomly put into 4 groups according to experiment design: ?1 H/R;?2 H/R +TG;?3 H/R +CTRP9;?4 H/R +TG+CTRP9.Firstly,the cells were cultured with medium containing TG(0.01 ?M/L)for 24 h.Secondly,the cells were cultured with medium containing CTRP9(1 ?g/ml)for 24 h.Thirdly,the cells were transfered to hypoxic environment for 2h and to oxygen-rich enviroment for 4h.The cell activity(MTT Cell Proliferation and Cytotoxicity Assay Kit),LDH release in the cluture medium(LDH Release Assay Kit),apoptosis rate of cardiomyocytes(TUNEL Cell Apoptosis Detection Kit)and protein expression levels(Caspase-3,DsbA-L,CHOP and GRP-78 determined by western blot)were determined after reoxygenation.Part 3: Effect of DsbA-L suppression on CTRP9 induced protective effect against H/R injury.The effect of DsbA-L suppression on CTRP9 induced protective effect against H/R injury was explored.The cells were randomly put into 4 groups according to experiment design: ?1 Scramble;?2 DsbA-L siRNA;?3 Scramble +CTRP9;?4 DsbA-L siRNA+CTRP9.The transfection efficiency was observed under fluorescence microscope 48 h after cells was transfected with Scramble or DsbA-L siRNA.Firstly,the cells were cultured with medium containing CTRP9(1 ?g/ml)for 24 h.Secondly,the cells were transfered to hypoxic environment for 2h and to oxygen-rich environment for 4h.The cell activity(MTT Cell Proliferation and Cytotoxicity Assay Kit),LDH release in the cluture medium(LDH Release Assay Kit),apoptosis rate of cardiomyocytes(TUNEL Cell Apoptosis Detection Kit)and protein expression levels(Caspase-3,DsbA-L,CHOP and GRP-78 determined by western blot)were determined after reoxygenation.Part4:Cardioprotective effect of CTRP9 against I/R injury in type 2 diabetic heart.The cardioprotective effect of globular CTRP9 against I/R injury in the isolated hearts from both normal and type 2 diabetic rats was explored.The hearts were randomly assigned into 4 groups as described: ?1 Normal;?2 Diabetic;?3 Normal +CTRP9;?4Diabetic +CTRP9.Firstly,the hearts were perfused with KH buffer for 30 min.Secondly,they were perfused with KH buffer containing different concentration of CTRP9 or equal amount of vehicle.Different dosage of CTRP9 or KH buffer were add directly into the perfusate as 1 mL bolus injection.The drug or KHB were administrated at the end of 30-min-equilibration through an injection port above the heart.Thirdly,isolated hearts were subjected to ischemia by closing the valve positioned upstream of the heart for 30 min.Finally,isolated hearts were reperfused for 60 min by re-opening of the valve.The hemodynamic parameters were monitored and recorded in real time through a Biopac data collecting system,including HR,LVDP,+d P/dt,CF and RPP(calculated by heart×left vertrical pressure).LDH level in the coronary effluent(LDH Release Assay Kit),the myocardial infarct size(the hearts were frozen,sliced and stained with TTC),the apoptosis rate of cardiomyocytes(frozen section of the hearts were stained with TUNEL Cell Apoptosis Detection Kit and observed using fluorescence microscope),and protein expression levels(Caspase-3,DsbA-L,CHOP and GRP-78 determined by western blot)were determined after reperfusion.Results:Part1:Cardioprotective effect of different concentration of CTRP9 against myocardial I/R injury.Langendorff perfused isolated heart experiment revealed that CTRP9(1 ?g/ml)exerted potent protective effect against I/R injury in isolated heart,as shown by significant improvement of cardiac hemodynamic parameters(HR,LVDP,+d P/dt,RPP and CF),dramatic reduction of myocardial infarct size,significant reduction of percentage of apoptotic cells,significant reduction of of Caspase-3 expression,and significant reduced LDH activity in the coronary effluent.ERS was significantly suppressed in CTRP9 1 ?g and CTRP9 3 ?g group,as shown by significantly reduced CHOP and GRP-78 expression.Part 2: Protective effect of CTRP9 against H/R injury in H9c2 cells.TG down-regulated DsbA-L exprrssion and increased ERS in H9c2 cells,as evidenced by significantly decreased DsbA-L and increased expression level of ER stress markers CHOP and GRP-78.CTRP9 increased DsbA-L expression and reduced ERS in cells treated with TG,as shown by significantly increased DsbA-L and decreased expression of ER stress markers CHOP and GRP-78.CTRP9 also increased Dsb A-L expression and reduced ERS in cells treated without TG,as demonstrated by significantly increased DsbA-L and decreased expression of ER stress markers CHOP and GRP-78.TG treatment aggravated H/R induced injury as shown by significantly increased expression level of apoptosis protein Caspase-3,increased cell apoptosis rate,elevated LDH activity in the culture medium and,reduced cell activity.CTRP9 was competent in protecting cells treated with TG against H/R injury,as depicted by significantly reduced percentage of cell apoptosis,Caspase-3 expression,LDH activity in the culture medium and significantly increased cell activity.Part 3: Protective effect of CTRP9 agaisnt H/R injury was attenuated by suppressing Dsb A-L expression.DsbA-L suppression by siRNA increased ERS in H9c2 cells,as evidenced by significantly increased expression of ERS markers CHOP and GRP-78.Suppressing DsbA-L protein expression by siRNA mediated gene knockdown increased H/R induced injury in H9c2 cells,as shown by significantly increased expression of apoptosis protein Caspase-3,increased percentage of apoptotic cells,elevated LDH release in the culture medium and,reduced cell activity.CTRP9 increased Dsb A-L expression and reduced ERS in cells treated with DsbA-L siRNA and reversed H/R injury as depicted by significantly reduced expression level of ERS markers CHOP and GRP-78,significantly decreased expression level of apoptosis protein Caspase-3,reduced percentage of apoptotic cells,decreased LDH activity in the culture medium and,increased cell activity.Part4:CTRP9 exert potent protective effect against I/R injury in isolated diabetic rat heart.Isolated perfused heart experiment revealed that type 2 diabetes decreased DsbA-L expression in the heart,increased ERS and aggravated I/R injury,as shown by significantly increased ERS marker CHOP and GRP-78 expression,significantly decreased LVDP,+dP/dt,RPP and CF,significantly increased myocardial infarction,significantly increased rate of apoptotic cells,significantly increased expression of apoptosis protein Caspase-3 and,significantly increased LDH activity in the coronary effluent.Administration of CTRP9 significantly increased DsbA-L expression in diabetic heart,reduced type 2 diabetes induced ERS and alleviated I/R injury,as shown by significantly decreased ERS marker CHOP and GRP-78 expression,significantly improved LVDP,+dP/dt,RPP and CF,dramatically decreased myocardial infarction,significantly decreased cell apoptosis,significantly decreased expression of apoptosis protein Caspase-3 and,significantly decreased LDH activity in the coronary effluent.Conclusions:1.CTRP9,at the concentration of 1 and 3 ?g/ml,was capable of protecting isolated rat heart against I/R injury.2.TG decreased ER chaperone DsbA-L expression,intensified ERS and aggravated H/R injury.CTRP9 reversed TG induced DsbA-L down regulation and mitigated ERS and alleviated H/R injury.DsbA-L siRNA intensified ERS,aggravated I/R injury and nullified the cardioprotective effect of CTRP9,indicating that DsbA-L was involved in CTRP9 cardioprotection.3.Tpye 2 diabetes decreased ER chaperone DsbA-L expression in the heart,intensified ERS and aggravated I/R injury,while CTRP9 increased DsbA-L in diabetic heart,mitigated ERS and alleviated I/R injury.