Endothelial-expressed Ubiquitin-like Protein FAT10 Stabilizes NLRP3 Expression And Aggravates Hypoxic-induced Myocardial Injury

Background:Disruption of normal homeostasis of vascular endothelial cells,changes in vascular structure and abnormal vascular functions are the necessary factors for the occurrence and progression of acute myocardial infarction(AMI).Existing research reports that FAT 10 is expressed in myocardial tissue and can effectively reduce myocardial ischemic injury.However,the mechanism of vascular endothelialexpressed FAT 10 on myocardial injury remains unknown.Aseptic inflammatory response of AMI can increase myocardial infarction size and lead to further myocardial injury,which is mainly mediated by NLRP3 inflammasome.Therefore,NLRP3 inflammasome is expected to be a therapeutic target for MI.Meanwhile,protein interaction prediction indicated that NLRP3 might interact with FAT10,so it is speculated that endothelial-expressed FAT10 may mediate myocardial ischemia and hypoxia injury through direct regulating the expression of NLRP3.To verify this hypothesis,vascular endothelium specific FAT10 gene knockout(FAT10flox/flox:Cre+)mice(eFat10-/-)were used as animal experimental subjects,and a series of in vitro experiments were conducted in human umbilical vein endothelial cells(HUVECs)to investigate the mechanism of endothelial-expressed FAT 10 regulating myocardial hypoxia injury.Objective:This study attempted to reveal the regulatory effect of endothelial-expressed FAT10 on myocardial hypoxia injury and its mechanism,and to determine whether NLRP3 is a substrate protein of FAT10 in the regulation of myocardial injury.Methods:In this study,homologous recombination was used to construct vascular endothelium specific FAT10 gene knockout(FAT10flox/flox:Cre+)mice(eFat10-/-).Together with wild-type mice(WT),myocardial infarction model was established respectively.HUVECs were used to investigate the regulation and mechanism of endothelial-expressed FAT10 on myocardial hypoxia injury,as follows:1.To explore the effect of endothelial-expressed FAT10 on myocardial hypoxia injury in vivoMyocardial infarction models were established in eFat10-/-mice and WT mice,and the aorta endothelia of WT mice were extracted before and after the operation respectively to detect FAT 10 expression.Tetrazolium chloride(TTC)staining was used to detect the myocardial infarction area in both groups.2.To determine whether FAT10 could regulate the expression of NLRP3 in vitroTo regulate FAT 10 expression level,HUVECs were transfected with overexpression and interference adenovirus vectors of FAT 10 respectively.Western Blot was used to detect the effect of FAT 10 on NLRP3 expression.3.To clarify the specific mechanism of FAT10 regulating NLRP3 expressionProtein synthesis inhibitor CHX and proteasome inhibitor MG132 were used to determine whether NLRP3 was degraded by proteasome.Western Blot was used to detect whether FAT 10 regulated the expression of NLRP3 by affecting its degradation.At last,co-immunoprecipitation(Co-IP)was used to verify the interaction between FAT 10 and NLRP3.Results:1.The endothelial-expressed FAT10 can aggravate myocardial ischemia and hypoxia injury.Western Blot indicated that the expression of FAT 10 in aortic endothelium of WT mice increased after myocardial infarction.TTC staining indicated that the myocardial infarction area of eFat10-/-group was decreased compared with WT group.2.FAT10 stabilizes NLRP3 expression.Western Blot showed that NLRP3 expression could be increased by FAT 10 overexpression and be decreased by FAT 10 interference.3.FAT10 can regulate the degradation of NLRP3.Western Blot showed that NLRP3 expression gradually decreased in HUVECs after treated with CHX,while NLRP3 expression did not change significantly after treated with CHX and proteasome inhibitor MG132 simultaneously.In addition,MG132 can block the regulatory effect of FAT 10 on NLRP3.4.FAT10 can combine with NLRP3.Co-IP showed that FAT 10 could combine with NLRP3.Conclusion:1.The endothelial-expressed FAT 10 aggravates myocardial ischemia and hypoxia injury.2.FAT10 is involved in the regulation of NLRP3 protein expression.3.FAT 10 regulates the degradation of NLRP3 to stabilize its expression.