The Mechanism Of Targeted Regulation Of HIF-1 To Attenuate I/R Injury In Atherosclerotic Myocardium

Objective:This study aimed to investigate（1）the mechanism and protective effect of ischemic postconditioning（IPo C）on myocardial ischemia/reperfusion（I/R）injury in atherosclerotic myocardium;（2）whether targeted upregulation of HIF-1 by AAV9-HIF-1αcould enhance the cardioprotective effect of IPo C;（3）the association and possible mechanism between hyperlipidemia and myocardial injury after noncardiac surgery（MINS）.Methods:PartⅠ:The Atherosclerosis mouse model was established by feeding APo E^-/-mice with a high-fat diet for 16 weeks.The age-matched healthy mice were randomly divided into three groups:（1）C-sham group:sham-operated treatment,（2）C-I/R group:I/R treatment,and（3）C-I/R+IPo C group:IPo C treatment.Atherosclerotic mice were divided into three groups:（1）AS-sham group:sham-operated treatment,（2）AS-I/R group:I/R treatment,（3）AS-I/R+IPo C group:IPo C treatment.Followed by（a）ELISA for markers of myocardial injury,（b）biochemical method for markers of oxidative stress,（c）TTC staining for myocardial infarct size,（d）echocardiography for cardiac function,（e）flow cytometry for mitochondrial function,（f）transmission electron microscopy for mitochondrial morphology,（g）TUNEL staining for myocardial apoptosis,（h）H&E staining for histopathology,（i）western blot for signalling protein.PartⅡ:Atherosclerotic mice were divided into six groups:（1）AS-sham group:sham-operated treatment,（2）AS-I/R group:I/R treatment,（3）AS-I/R+IPo C group:IPo C treatment,（4）AS-I/R+IPo C+AAV9-NC group:intravenous pretreatment with AAV9-e GFP,followed by IPo C treatment,（5）AS-I/R+IPo C+AAV9-HIF group:intravenous pretreatment with AAV9-HIF-1α,followed by IPo C treatment,and（6）AS-I/R+IPo C+AAV9-HIF+2ME2 group:intraperitoneal injection of 2ME2（a HIF-1inhibitor）,the remained procedure is the same as the group AS-I/R+IPo C+AAV9-HIF.The relevant indicators are measured in the same way as in Part I.PartⅢ:Data including medical history and levels of inflammatory cells and factors of patients who underwent noncardiac surgery were collected.Then（1）univariate analysis was used to screen the covariates related to the occurrence of MINS;（2）propensity scores were utilized to match the covariates between hyperlipidemia and normolipidemia groups;（3）logistic regression was used to analyse the different incidence of MINS between the hyperlipidemia and the normolipidemia group;（4）the levels of inflammatory cells and factors and the length of postoperative hospital stay were compared between the hyperlipidemia and the normolipidemia group.Results:PartⅠ:（1）Atherosclerosis increases the susceptibility of the myocardium to I/R injury,as evidenced by a significant decrease in cardiac function,a significant increase in oxidative stress,a significant increase in impaired mitochondrial morphology and function,and a significant increase in perioperative mortality in atherosclerotic mice subjected to myocardial I/R injury,as compared with healthy mice.（2）In healthy myocardium,IPo C favours a significant protective effect,as demonstrated by a significant increase in cardiac function and a prominent decrease in myocardial infarction following IPo C treatment.Moreover,IPo C activates the three pathways,namely HIF-1,AMPK and RISK pathway.（3）In atherosclerotic myocardium,IPo C still offers cardioprotection,which is weaker than that in healthy myocardium,as demonstrated by a still effective alleviation in I/R-induced cardiac dysfunction and myocardial infarction,but the degree of alleviation is not as pronounced as it is in healthy myocardium.Moreover,IPo C is able to only activate the HIF-1 pathway,not the AMPK and RISK pathways.PartⅡ:（1）HIF-1 upregulation by AAV9-HIF-1αtargeted transduction enhances the cardioprotective effect of IPo C,as evidenced by a significant alleviation in I/R-induced mitochondrial injury,cardiac dysfunction,and infarct size in mice subjected to AAV9-HIF-1 combined IPo C treatment,compared with which subjected to IPo C treatment alone.（2）The combined treatment of AAV9-HIF-1αand IPo C reactivated the AMPK and RISK pathways,as evidenced by increased expression of p-AMPK,p-Akt,and p-ERK1/2.PartⅢ:（1）Age,coronary artery disease,hypertension,diabetes mellitus,cerebral infarction,intraoperative hypotension,ASA classification,and duration of surgery were associated with the occurrence of MINS,so the above variables were used as covariates in the present study;（2）178 pairs of patients were successfully matched by propensity scores,and the above covariates were equitably distributed between the matched hyperlipidemia and normolipidemia group.（3）The incidence of MINS was higher in the matched hyperlipidemia group than in the matched normolipidemia group;blood inflammatory factor levels,inflammatory cell counts,and postoperative length of hospital stay were higher in the matched hyperlipidemia group than in the matched normolipidemia group.Conclusion:（1）In healthy myocardium I/R,IPo C favours cardioprotection by activating three pathways,namely HIF-1,AMPK and RISK pathways;in atherosclerotic myocardium I/R,IPo C offers cardioprotection by activating the HIF-1 pathway only;in atherosclerotic myocardium,the AMPK and RISK pathways failed to be activated by IPo C,so that they were not involved in the cardioprotection of IPo C.（2）In the atherosclerotic myocardium,HIF-1 upregulation by AAV9-HIF-1αenhanced the cardioprotection of IPo C;the promoted effect of AAV9-HIF-1αon the cardioprotection of IPo C was achieved by restoring the activation of AMPK and RISK pathway by IPo C.HIF-AMPK/RISK,acting as an endogenous signalling axis,plays an important role in IPo C′s cardioprotection.（3）Hyperlipidemia is positively associated with the incidence of MINS,and the mechanism by which hyperlipidemia promotes MINS is the higher inflammatory response.