The Role And Mechanism Of High-expression Wnt5a In Hypoxia/Reoxygenation Injury Of Cardiomyocytes Throughβ-catenin/TCF4/Cx43 Axis

Objective:Wnt5a,a non-classical Wnt ligand,is involved in the pathogenesis of cardiovascular diseases,but its role and specific mechanisms in ischemic cardiomyopathy and myocardial ischemia-reperfusion injury are not known.In this study,we aimed to investigate the role of Wnt5 a in myocardial myocardial H/R injury and its mechanism by establishing a hypoxia-reoxygenation(H/R)injury model,which could provide new insights and targets for clinical prevention and development of cardiovascular diseases.Methods:1.H9c2 cells were treated with hypoxia for 6h and reoxygenation for 3h.Cardiomyocyte viability was measured by CCK-8;protein expression of Bax,Bcl-2,cleaved caspase-3,Cx43 and Wnt5 a was detected by Western blot;intracellular ROS was probed by DCFH-DA probe;mitochondrial membrane potential was examined by JC-1 probe;intercellular gap junction communication was measured by Calcein/AM staining;Wnt5a m RNA expression was measured by RT-q PCR.The expression level of Wnt5 a m RNA was assessed by RT-q PCR.2.To investigate the effect of knockdown of Wnt5 a on H/R injury in cardiac myocytes.Firstly,cardiomyocytes were transfected with siWnt5 a to knock down the gene expression of Wnt5 a.western blot was performed to detect the protein expression of Wnt5 a in the cells.Secondly,cardiomyocytes were transfected with siWnt5 a for 24 h and then treated with H/R.Cell viability,protein expression of Bax,Bcl-2,cleaved caspase-3,Wnt5 a,β-catenin and Cx43,ROS levels and changes in mitochondrial membrane potential were examined in each group of cells.3.Prediction and validation of β-catenin-targeted regulation of Cx43.After knockdown of β-catenin,Western blot and cellular immunofluorescence assays were performed to observe changes in β-catenin expression distribution and effects on Cx43;bioinformatics predicted binding sites of TCF/LEF transcription factor family and Cx43 promoter,and Ch IP-PCR assays verified Bioinformatics to predict the binding sites of TCF/LEF transcription factors and Cx43 promoter,Ch IP-PCR to verify the binding and activation of Cx43 promoter transcription factors,COIP and immunolocalization assays to verify the binding and co-localization of the selected transcription factors with β-catenin.4.Probing the role of LF3,an antagonist of β-catenin interaction with TCF4,in H/R injury in cardiomyocytes subjected to knockdown of Wnt5 a.First,the Co IP assay was used to screen the concentration and duration of action of LF3;second,cardiomyocytes were transfected with siWnt5 a for 24 h and then treated with H/R,while 30 μmol/L LF3 was given as pretreatment at reoxygenation,and the cell viability,Bax,Bcl-2,cleaved caspase-3,Wnt5 a,β-catenin and Cx43,ROS levels and mitochondrial membrane potential.Results:1.An in vitro model of cardiomyocyte injury was successfully established in H9c2 cells treated with 6h of hypoxia/3h of reoxygenation.Cardiomyocyte viability was significantly reduced,expression of pro-apoptotic protein Bax and cleaved caspase-3 was significantly increased,expression of anti-apoptotic protein Bcl-2 and gap junction protein Cx43 was significantly reduced,intracellular ROS level was significantly increased,mitochondrial membrane potential was significantly reduced,intercellular gap junction communication function was weakened,while Wnt5 a was significantly increased in both gene and protein expression levels.The expression of Wnt5 a was significantly increased at both gene and protein levels.2.The knockdown of Wnt5 a by siWnt5 a was able to alleviate H/R-induced cardiomyocyte injury.The results showed a significant increase in cardiomyocyte viability,a positive decrease in Wnt5 a,Bax and cleaved caspase-3 protein expression,a dramatic increase in Bcl-2 and Cx43 protein expression,a significant decrease in intracellular ROS content and an increase in mitochondrial membrane potential in the siWnt5a+H/R group compared to the NC+H/R group,as well as an increase in the expression of its core response molecule β-catenin expression in whole cells and nuclei.3.When β-catenin was expressed at low levels,the expression of the gap junction protein Cx43 showed a significant positive correlation;the String database showed that β-catenin was predicted to bind to the TCF/LEF transcription factor family;the JASPAR database predicted that the TCF/LEF transcription factor family had a total of 10 binding sites to the Cx43 promoter;The Ch IP assay confirmed that TCF4 is a key transcription factor that binds to and activates the Cx43 promoter in hypoxic reoxygenation injury in cardiac myocytes,and the COIP assay and cellular immunofluorescence assay further demonstrated significant binding and co-localization of TCF4 with β-catenin.4.The results of Co IP assay showed that LF3 at 30 μmol/L could antagonize the interaction between β-catenin and TCF4.Immediately after,the changes of various indexes were examined after cardiomyocytes were treated with H/R after knockdown of Wnt5 a,while coincubation with LF3 was given during reoxygenation,and the results showed that: compared with the DMSO+siWnt5a+H/R group,The cardiomyocytes in the LF3+siWnt5a+H/R group showed a noticeable decrease in cell viability;a marked increase in protein expression of Bax and cleaved caspase-3and a considerable decrease in protein expression of Bcl-2;a significant increase in ROS levels;a distinct down-regulation of mitochondrial membrane potential;In addition,Cx43 protein expression was significantly reduced and intercellular gap junctional communication was diminished,but protein expression of Wnt5 a andβ-catenin was not significantly changed.Conclusions:1.Wnt5 a was highly expressed during H/R injury in cardiomyocytes,while knockdown of Wnt5 a alleviated H/R injury in cardiomyocytes as well as promotingβ-catenin nuclear translocation and upregulation of Cx43 expression.2.Nuclear β-catenin forms a complex with TCF4 and further binds to the activated Cx43 gene promoter,contributing to increased Cx43 expression.3.Downregulation of Wnt5 a regulates the effector molecule Cx43 via theβ-catenin/TCF4 cascade to enhance intercellular gap junctional communication and alleviate H/R injury in cardiomyocytes.