PLXND1-mediatedcalcium Dyshomeostasis Impairs Endocardialendothelial Cell Autophagy In Atrial Fibrillation

Objective:Left atrial appendage（LAA）thrombus detachment resulting in intracranial embolism is a primary complication of atrial fibrillation（AF）.Endocardial endothelial cell（EEC）injury leads to thrombosis,whereas autophagy protects against EEC dysfunction.However,the role and underlying mechanisms of autophagy in EECs during AF have not been elucidated.In the study,we constructed AF mice model and isolated EECs from AF mice model to observe the changes of autophagy flux and intracellular calcium concentration in EECs of AF mice model.At the same time,we observed the effect of the expression of the mechanical sensor protein PLXND1 in the cell membrane of the EECs of AF mice model on autophagy flux and intracellular calcium concentration,and preliminarily explored the role and mechanism of PLXND1 mediated calcium homeostasis imbalance in the endothelial damage of atrial fibrillation endocardium.Methods:1.Development of atrial fibrillation mouse models1.1 A combined intravenous injection of Ca Cl2（10 mg/m L）and acetylcholine（Ach;66μg/m L）was administered to the Tbx5+/-group（n=6/group）every 2 days for 30 consecutive days to induce AF.The control group（n=6/group）received saline simultaneously.1.2The wild type mice group（n=6/group）and atrial fibrillation mice model group（n=6/group）were continuously monitored by electrocardiogram,Judging by electrocardiogram results.1.3 The plasma levels of type B natriuretic peptide（BNP）,Cardiac troponin I（cTnI）and Creatine Kinase-MB（CK-MB）were detected by enzyme-linked immunosorbent assay in the wild type mice group and the AF mice model group.1.4 The two groups of mice were killed to obtain the heart samples,Histological analysis of the heart was performed after cell culture and drug treatment,including Masson trichrome staining and WGA staining.2、Isolation of primary mouse endocardial endothelial cell and Identification of autophagy phenotype2.1 Primary EECs were isolated from mice using a modified method,The expressions of NPR3 and CDH11 were detected by flow cytometry and confocal microscopy,and were compared with human umbilical vein endothelial cells（HUVEC）and human pulmonary microvascular endothelial cells（HPMEC）.2.2 To observe and evaluate the level of autophagy flux in the two models above,we treated EECs with BAFA1（a known inhibitor of the latter）stages of autophagy.3、To investigate the role and mechanism of PLXND1 in autophagy and dysfunction of endocardial endothelial cells in atrial fibrillation（AF）3.1 The expression of PLXND1 in wild-type mice combined with atrial fibrillation was compared by immunofluorescence assay.3.2 We first overexpressed PLXND1 in cells by transfecting GFP-mRFP-LC3adenovirus vector,and then knocked out PLXND1 gene to confirm the relationship between PLXND1 and autophagy.3.3 The association of Ca²⁺with autophagy was validated by the addition of a calmodulin inhibitor（STO-609）to measure the phosphorylation of calmodulin kinase 2（CAMK2）and by the addition of a calcium chelating agent（BAPTA）to measure Ca²⁺levels in endocardial endothelial cells.3.4 The Plxnd1-ORAI1-binding complex was analyzed by SDS-PAGE（sodium dodecyl sulfate polyacrylamide gel electrophoresis）using anti-Plxnd1 and anti-ORAI1.Results:1.Development of atrial fibrillation mouse models1.1 The ECG results of atrial fibrillation mice model were atrial fibrillation rhythm,and the ECG results of wild type mice were sinus rhythm.1.2 The values of BNP,cTnI and CK-MB in venous blood of atrial fibrillation mice model were increased,suggesting abnormal cardiac function in this group.1.3 WGA staining showed that the cross-sectional area of myocardial cells in the atrial fibrillation mice model was larger than that in the wild-type mice model,indicating myocardial hypertrophy in this group.Masson staining showed that The myocardial fibrosis of atrial fibrillation mice model was significantly increased than that of wild-type mice model.2.Isolation of primary mouse endocardial endothelial cell and Identification of autophagy phenotype2.1 Compared with HUVEC and HPMEC,NPR3 and CDH11 in the isolated endothelial cell group were highly expressed in WB assay and immunofluorescence assay,which confirmed that they were endocardial endothelial cells.2.2 LC3II/I expression decreased and SQSTM1 expression increased in atrial fibrillation mice model,indicating that autophagy of EECs was weakened in atrial fibrillation.However,after blocking autophagy flow with BAFA1,autophagy flux accumulated,LC3II/I expression increased and SQSTM1 expression decreased in atrial fibrillation mice model,which proved that the decrease of autophagosome is due to the increased degradation of lysosome.3.To investigate the role and mechanism of PLXND1 in autophagy and dysfunction of endocardial endothelial cells in atrial fibrillation（AF）3.1 In the WB experiment,PLXND1 expression in atrial fibrillation mice model was significantly higher than that in wild-type mice.3.2 In the WB experiment,PLXND1 expression in LV-Plxnd1 group was significantly taller than that in shPlxnd1 group.LC3II/I expression in LV-Plxnd1 group was lower than that in shPlxnd1 group,and SQSTM1 expression was taller than that in shPlxnd1 group.The results indicated that autophagy was activated after Plxnd1 gene knockout.3.3 After treatment with STO-609 and BAPTA,shPlxnd1 mice showed a decrease in CAMK2 phosphorylation levels,a decrease in LC3II/I expression,and an increase in SQSTM1 expression.The results showed that the decrease in CAMK2 phosphorylation levels caused a decrease in Ca²⁺influx level,Then could lead to inhibition of autophagy.3.4 The ZDOCK protein docking program found that PLXND1 and ORAI1 can be combinedConclusions:We found that the expression of PLXND1 increased on the endocardial endothelial cell membrane of the left atrial fibrillation mice model,and combined with the calcium channel（ORAI1）on the cell membrane,resulting in decreased Ca²⁺influx level,decreased calcium ion mediated CAMK2 phosphorylation,and thus inhibited autophagy,and ultimately caused the dysfunction of endocardial endothelial cells of the left atrial fibrillation model.Our study proposes a molecular mechanism of susceptibility to left atrial ear thrombosis during atrial fibrillation,providing a potential intervention target for the prevention of atrial fibrillation thrombosis and stroke.