Vaspin Alleviates Myocardial Ischaemia/reperfusion Injury Through Activation Of Autophagic Flux

BackgroundVaspin(visceral adipose tissue-derived serine protease inhibitor)is an adipokine found in 2000,secreting from visceral adipose tissues and belonging to the family members of serine protease inhibitor.Vaspin has been verified to regulate the glucose and lipid metabolism and increase insulin sensitivity.Some researches in recent years indicate that the vaspin levels in peripheral blood have certain relations with the morbidity of coronary heart diseases(CHD)and the number of lesion vessels.Circulating vaspin levels in CHD patients are lower than that in healthy population,and levels in myocardial infarction(MI)patients are even lower.Low plasma vaspin levels are reported to associate with the poor prognosis of CHD patients,though the specific mechanism behind that is unclear.Therefore,we speculate that vaspin may offer protection on cardiomyocytes under pathologic conditions,with the mechanism remaining further exploration.In this research,we applied adeno-associated virus(AAV)expressing vaspin and recombinant human vaspin in vivo and vitro studies to investigate whether vaspin may show myocardial protection and reduce myocardial ischemia/reperfusion(I/R)injury.Objectives1.To study the influence of vaspin on myocardial I/R injury.2.To explore the specific molecular mechanism of vaspin’s effects on myocardial I/R injury and to provide further theoretical supports for improving prognosis of clinical MI patients.MethodsIn vivo study1.Recombinant adeno-associated virus(AAV)expressing vaspin and GFP were injected to C57BL/6 mice via caudal veins to establish vaspin overexpression or control in mice.Western blot(WB)and ELISA were performed to check vaspin levels in visceral adipose tissues and peripheral plasma.2.Establish myocardial ischemia/reperfusion(I/R)models in mice.Mice were divided into 6 groups:AAV-GFP sham,AAV-vaspin sham,AAV-GFP I/R,AAV-GFP+chloroquine(CQ)I/R,AAV-vaspin I/R and AAV,vaspin+CQ I/R.3.2%TTC dye was applied to measure myocardial infarction size in mice from each group.4.Cardiac function in mice was evaluated with echocardiography.5.DCFH-DA assay was performed to detect the ROS levels in mice.6.Immunohistochemical staining was performed to assess the expression of LC3 and LAMP2 in myocardium.7.WB was performed to determine the expressions of bcl-2,cleaved caspase-3,LC3-Ⅱ/Ⅰ,p62,LAMP2 and β-actin in myocardium.In vitro study1.Establish hypoxia/reoxygenation(H/R)models in H9C2 cells H9C2 cells were divided into groups as follows:1)dilution normoxia,vaspin normoxia,dilution H/R,vaspin H/R.2)dilution H/R,vaspin H/R,CQ H/R,vaspin+ CQ H/R.3)dilution H/R,vaspin H/R,Compound C H/R,vaspin+ Compound C H/R.2.Terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)assay was performed to detect apoptosis in H9C2 cells from each group.3.DCFH-DA assay was performed to measure the ROS levels in H9C2 cells.4.Immunofluorescent staining was performed to assess the expressions of LC3-Ⅱ/I and LAMP2 in H9C2 cells.5.Adenovirus carrying mRFP-GFP-LC3 was transfected to detect autophagosomes and autolysosomes in H9C2 cells.6.WB was performed to determine the expressions of bcl-2,bax,cleaved caspase-3,LC3,p62,LAMP2,p-AMPK/AMPK and β-actin in H9C2 cells.Results1.AAV-vaspin reduced myocardial infarct size and improved LVEF and FS in mice after I/R by decreasing the apoptosis and ROS levels in myocardium.2.AAV-vaspin upregulated myocardial autophagic flux in mice after I/R.With CQ application to block the autophagic flux,the myocardial protection from vaspin was weakened.CQ enlarged myocardial infarct size in mice with AAV-GFP and AAV-vaspin by aggravating the apoptosis and ROS levels in myocardium,and meanwhile lessened LVEF and FS.3.Recombinant human vaspin reduced the apoptosis and ROS levels in H9C2 cells after H/R.4.Recombinant human vaspin increased expressions of autophagy-associated proteins,LC3-Ⅱ/Ⅰ,p62,and LAMP2 and enhanced the generation of autophagosomes and autolysosomes to upregulate the autophagic flux in H9C2 cells.CQ aggravate the apoptosis and ROS levels in H9C2 cells with vaspin pretreatment by blocking the autophagic flux.5.Recombinant human vaspin played a protective role in H9C2 cells after H/R by activating AMPK-mTOR-autophagic flux.After inhibiting phosphorylation of AMPK with Compound C,the autophagic flux was suppressed in H9C2 cells and then the apoptosis and ROS levels augmented.Conclusions1.The adipokine,vaspin,can upregulate and restore the autophagic flux during I/R via activating AMPK/mTOR pathway.2.Vaspin can reduce the apoptosis and ROS levels in cardiomyocytes and then alleviate myocardial I/R injury through regulating the autophagic flux.