The Protective Effect Of BIN1 Regulated By PRR On H9c2 Injury Induced By Hypoxia-reoxygenation

Myocardial ischemia-reperfusion injury is a common pathophysiological process in clinic,and its mechanism has been studied by a large number of scholars,but it has not been fully clarified.At present,it has been found that oxidative stress,calcium overload,disappearance of mitochondrial membrane potential and endothelial dysfunction are all involved in the process of myocardial ischemia-reperfusion injury,resulting in a significant increase in apoptosis and necrosis,followed by a deterioration of cardiac function,thus affecting the clinical prognosis of patients.Therefore,in order to reduce the degree of cell injury,we can reduce oxidative stress,calcium overload and stabilizing mitochondrial membrane potential,thereby reducing apoptosis and necrosis can relieve cardiomyocyte injury and then achieve the purpose of improving cardiac function.c BIN1(Bin1+13+17),a cardiac isoform of BIN1,is a cardiomyocyte membrane scaffold protein found in recent years.c BIN1 mainly affects the excitationcontraction coupling process and stable binary microdomain of cardiomyocytes by transporting LTCC to t-tubules,aggregating LTCC on t-tubules,recruiting Ry Rs to Dyads microdomains and forming extracellular ion diffusion barrier,thus affecting the contractile and diastolic function of cardiomyocytes.In patients with heart failure,the level of c BIN1 decreased significantly,and there was a correlation between the level of c BIN1 and the prognosis of patients,and in the mouse model of heart failure,the level of c BIN1 was also decreased,and c BIN1 gene therapy could improve the cardiac diastolic and systolic function of mice with heart failure by restoring T tubule microfold and the distribution of calcium transport proteins,and promoting SERCA2 a redistribution.At present,no one has studied the changes and the protective effect of BIN1 in cardiomyocytes under hypoxia-reoxygenation.In recent years,it has been found that the activation of renin-angiotensinaldosterone system(RAAS)is involved in the process of myocardial ischemiareperfusion injury,and its overactivation can not only mediate calcium overload in mitochondria through angiotensin II,but also regulate the expression and functional changes of calcium transport-related proteins in cardiomyocytes,and mediate the imbalance of calcium transport in sarcoplasmic reticulum.As an important new member of RAAS system,PRR also plays a vital role in a variety of cardiovascular diseases.PRR not only binds to renin and renin precursor,but also plays its pathophysiological role by increasing the activity of renin and renin proenzyme,activating RAAS system and increasing the level of angiotensin II;it can also act as a signal receptor to bind to renin precursor and activate other intracellular signal transduction pathways,which is independent of the production of angiotensin II.Our previous studies found that PRR mediates cardiomyocyte apoptosis,autophagy and inflammation induced by hypoxiareoxygenation by activating p38 MAPK,PI3K/AKT and Wnt/β-catenin signal pathways in cardiomyocytes.These results suggest that overactivation of PRR in cardiomyocytes can damage cell structure and function through β-catenin signal pathway.The activation of β-catenin signal pathway can regulate the expression of many downstream proteins.Some studies have shown that β-catenin can inhibit the expression of E2F1,while others have found that βcatenin can promote the expression of E2F1.At the same time,bioinformatics analysis predicted that E2F1 could be used as a potential transcription factor of BIN1,regulating the change of BIN1 protein expression.In this study,hypoxia and reoxygenation injury model was established at the cellular level to detect BIN1 expression in H9c2 cardiomyocytes,and BIN1 overexpression vector was used to explore the protective effect of BIN1 on cardiomyocytes.Meanwhile,PRR siRNA,E2F1 siRNA and DKK-1 were used to block the Wnt/β-catenin signaling pathway to study the upstream regulatory mechanism of BIN1 protein changes in H9c2 cardiomyocytes induced by hypoxia and reoxygenation.Methods:1.H9c2 cardiomyocytes were selected and treated with hypoxia for 24 hours and reoxygenation for 2 hours,so as to establish the model of hypoxia and reoxygenation successfully.Firstly,the changes of BIN1 protein expression and the relative m RNA expression in the hypoxic-reoxygenated group and the control group were detected by western blotting analysis and real-time quantitative PCR.Secondly,after the cells were treated with BIN1 overexpression vector,the protective effect of BIN1 overexpression on cardiomyocytes under hypoxia and reoxygenation conditions was verified by using MTT,CKK-8,ROS,LDH,calcium content and mitochondrial membrane potential detection kit.Annexin V-FITC/PI double dyeing method was applied.The apoptosis levels were compared by flow cytometry.2.Bioinformatics analysis was used to predict the relationship between BIN1 and E2F1.The cells were treated with PRR siRNA,DKK-1 and E2F1 siRNA,and the experiment was divided into seven groups: untransfected group,negative control group,PRR siRNA group,H/R group,H/R+DKK-1 group,H/R+PRR siRNA group,H/R+PRR siRNA+E2F1 siRNA group.The distribution and expression levels of PRR,β-catenin,E2F1 and BIN1 proteins in cells under different treatment conditions were detected by western blot analysis and protein western blot analysis.Results:1.Under hypoxia and reoxygenation stimulation,the expression of BIN1 protein in H9c2 cardiomyocytes was down-regulated,and the viability of cardiomyocytes was significantly decreased.2.Compared with H/R group,the apoptosis rate of H/R+BIN1 overexpression group was significantly decreased,and the cell activity was significantly increased;ROS accumulation decreased significantly;Intracellular calcium content decreased significantly;The number of cells with lost mitochondrial membrane potential decreased.These results indicate that BIN1 overexpression can alleviate H9c2 myocardial cell damage,oxidative stress and calcium overload caused by hypoxia and reoxygenation,reduce apoptosis and stabilize mitochondrial membrane potential.3.Bioinformatics data analysis by NCBI,UCSC and JASPAR suggests that E2F1 may be a transcription factor of BIN1.Binding the promoter sequence of BIN1 to regulate its transcription process.4.Hypoxia and reoxygenation lead to up-regulation of PRR protein expression,activation of β-catenin pathway,and then promote upregulation of E2F1 expression,and finally lead to down-regulation of BIN1 expression.The results of cellular immunofluorescence and western blot analysis of β-catenin indicated that the change of protein expression mainly occurred in the nucleus.Conclusion:1.The expression of BIN1 protein in H9c2 cardiomyocytes was downregulated by hypoxia and reoxygenation,and the viability of cardiomyocytes was significantly decreased.BIN overexpression can alleviate the damage,oxidative stress and calcium overload of H9c2 cardiomyocytes caused by hypoxia and reoxygenation,reduce apoptosis and stabilize mitochondrial membrane potential.2.The downregulation of BIN1 in H9c2 cardiomyocytes induced by hypoxia and reoxygenation is regulated by the PRR/β-catenin/E2F1 axis.