Molecular Mechanism Of Circular Foxo3 In Cardiac Ischemia Reperfusion Injury In Heart Transplantation

BackgroundHeart transplantation is the last resort for the treatment of end-stage heart failure or severe coronary heart disease.However,ischemia-reperfusion injury is inevitable in the donor hearts,and can seriously worsen the clinical outcomes.Donor hearts preserved in organ preservation solutions can remain vital but they are not exempted from experiencing warm ischemia,cold ischemia and reperfusion injury,among which ischemia/reperfusion injury is the major cause of graft dysfunction and graft failure in the early stage of heart transplantation.Cold ischemia over 6 hours can lead to early dysfunction of heart transplantation,resulting in reduced 5-year survival rate and utilization rate of donor heart.Therefore,there is an urgent need for new mechanisms and therapeutic targets for effective prevention and treatment of ischemia-reperfusion injury after heart transplantation.As a new type of covalent closed-loop RNA,circ RNAs belong to a class of noncoding genes and become a spotlighted aspect in epigenetics in recent years for their roles in many diseases.In cardiovascular and circulatory diseases,circular RNAs has been reported to be associated with vascular endothelial cell proliferation,apoptosis,vascular smooth muscle cell phenotype conversion and myocardial cell necrosis/apoptosis.Meanwhile,their roles during ischemia-reperfusion injury are also suggested by some reports.However,research on circular RNAs in the pathogenesis of ischemia-reperfusion injury is still in its infancy.The specific mechanism of action in the disease remains unclear.ObjectiveTherefore,the present study is aiming at investigating the role of circ Foxo3 in myocardial ischemia-reperfusion injury.First,illustrating the involvement of circ Foxo3 in myocardial ischemia/reperfusion injury by in vitro cell experiments.Then gene binding with circ Foxo3 was verified by immunocoprecipitation to show how circ Foxo3 regulates myocardial ischemia-reperfusion injury.At last,ischemia-reperfusion animal model was established to verify the function of circ Foxo3 in myocardial ischemia-reperfusion injury the in vivo.The key pathogenesis of myocardial ischemia-reperfusion during heart transplantation is identified,and diagnosis markers and potential drug therapeutic targets can be proposed for the prevention,diagnosis and treatment of myocardial ischemia-reperfusion after heart transplantation.Method1.Cell model of ischemia-reperfusion injury was induced.Before the experiment,HL-1 was placed on a 6-well plate.After removing the culture medium,the cells were replaced with 2 ml UW solution,and then the cells were cultured in(0.5%O₂,5%CO₂,94.5%N₂,10?)for 24 hours,and then the normal culture was changed and cultured for 12 hours under normal culture conditions.The injury model of primary cardiomyocytes was induced by adding 0.5 ml UW solution,then cultured in hypoxia for 16 hours,then changing the normal culture and culturing for 6 hours under normal culture conditions.2.Mechanism of action of circ Foxo3 on myocardial injury during ischemia-reperfusion.q RT-PCR confirmed that circ Foxo3 was differentially expressed between hypoxia/reoxygenation injury cell model and normal cells.HL-1 cells were infected with circ Foxo3 by RNA interference technique.Apoptosis was detected by annexin V and mitochondrial membrane potential test,cell death was evaluated by Incyte system,apoptosis and inflammatory markers were detected by q RT-PCR and protein electrophoresis.3.Detection of downstream binding genes of circ Foxo3.q RT-PCR was used to confirm the expression of mi RNA combined with circ Foxo3 in the cell model of ischemia-reperfusion injury.To construct a circ Foxo3 biotinylated probe,and to confirm the target mi RNAs by RNA immunoprecipitation assay.Mi RNA binding to circ Foxo3 was detected.q RT-PCR was used to confirm the expression level of Foxo3and circ Foxo3 in the cell model of ischemia-reperfusion injury.Immunoprecipitation was used to verify the contaction between circ Foxo3 and Foxo3.4.Induction of ischemia-reperfusion injury animal model.Donor and recipient of heart transplantation animals were selected in 8-week-old C57BL/6ncrl mice.First,1ml UW preservation solution containing 50ug circfoxo3 interference or circfoxo3control were injected into donor heart.Then,different donor hearts were stored at4?for 24 hours,and allogeneic heart transplantation was performed.5.The effect of circ Foxo3 on ischemia-reperfusion during mice heart transplantation.The cardiac ejection fraction was evaluated by echocardiography.After the mice were killed,the staining was used to evaluate the changes of heart tissue,Masson staining was used to evaluate the changes of myocardial fibrosis,and TUNEL staining was used to detect myocardial apoptosis,The activity and protein expression of Caspase-3 were detected.Resul1t.s The HL-1 cardiomyocytes hypoxia/reoxygenation injury model was successfully constructed.2.The expression of circfoxo3 in HL-1 cardiomyocytes and primary cultured cardiomyocytes was significantly higher than that in normal cultured cardiomyocytes.Annexin V assay showed that annexin-V+apoptotic cells were significantly increased in HL-1 cells after hypoxia/reoxygenation.The low expression of circfoxo3 in HL-1 cardiomyocytes significantly reduced the injury caused by ischemia-reperfusion,and the apoptotic cells of annexin-V+decreased significantly.The results of Incyte system showed that low expression of circfoxo3 significantly reduced the damage of mitochondria caused by ischemia-reperfusion.q RT-PCR showed that the expression levels of Bax,caspase 8,caspase 9 and caspase 3 were significantly decreased after low expression of circfoxo3.Western blot showed that the expression of Caspase-3 decreased significantly after low expression of circfoxo3.3.Circfoxo3 binds Foxo3 and inhibits Foxo3 phosphorylation.q RT-PCR showed that the expression of mir-433 and mir-136 decreased,while the expression of mir-22,mi R-138 and mir-149 increased after low expression of circfoxo3.RNA immunoprecipitation assay showed that mir-433 and mir-136 were not detected in the pull-down complex of circfoxo3 probe.The expression of p-foxo3 protein increased in HL-1 cardiomyocytes with low circfoxo3 expression,but decreased in HL-1cardiomyocytes with high circfoxo3 expression.Immunoprecipitation assay showed that Foxo3 protein expression was detected in the pull-down complex of circfoxo3probe.4.The animal model of orthotopic heart transplantation was successfully established by surgical method(all procedures were completed in Matthew Mailing Center of Western University,Canada).5.Circfoxo3 is involved in myocardial ischemia-reperfusion injury during heart transplantation mice.q RT-PCR analysis showed that the expression of circfoxo3 was higher in heart with 24 hour ischemia induce before transplantation donors than heart directly transplantation.Compared with the control group,the cardiac ejection fraction and left ventricular function were increased after heart transplantation.HE staining showed that the myocardial cells were arranged in order,the degree of myocardial injury was low,only watery degeneration,while the control group was disordered with a large number of necrosis and infarct substances.Masson staining showed that the degree of cardiac fibrosis decreased.Conclusion1.The expression of circfoxo3 in HL1 cells increased when the cells were challenged by hypoxia/reoxygenation was increased,and the expression of circfoxo3 in myocardium of mice increased when the mice were treated by heart transplantation.2.Low expression of circfoxo3 in HL-1 cells can significantly reduce the cell injury caused by ischemia-reperfusion,reduce the level of apoptosis and the degree of mitochondrial damage.3.The donor heart preserved with low expression of circfoxo3 can significantly improve cardiac function,reduce myocardial ischemia-reperfusion injury,and reduce the degree of myocardial fibrosis after myocardial injury.4.Circfoxo3 can induce myocardial ischemia-reperfusion injury by binding Foxo3 and inhibiting Foxo3 phosphorylation.In conclusion,high expression of circfoxo3 in myocardial tissue during myocardial ischemia-reperfusion injury can promote the adsorption of Foxo3 and inhibit the phosphorylation level of Foxo3,which leads to cardiomyocyte apoptosis,increased necrosis and mitochondrial damage.These results provide a new theoretical basis for the pathogenesis of circular RNA in myocardial ischemia-reperfusion injury after heart transplantation.It also provides early diagnostic serological markers and potential drug targets for the diagnosis,prevention and treatment of myocardial ischemia-reperfusion injury during heart transplantation.