Clinical Features In Patinets With Type 2 Myocardial Infarction And The Role And Mechanism Of MiR-542-5p/autophagy Pathway In H/R-induced Cardiomyocyte Injury

Objective:To analyze clinical features and to evaluate the validity of type 2 myocardial infarction(MI)diagnostic prediction score for distinguishing type 2 MI in patients with acute myocardial infarction in acute chest pain center.To investigate the incidence rate and risks of T2MI in critically ill elderly patients and further elucidate in-hospital prognostic factors.Based on the mechanism of type 2 myocardial infarction,we establish a myocardial hypoxia/reoxygenation H9C2 cell model to investigate how H/R injury effects cell autophagy and to explore the possible role of miR-542-5p in H/R injury.Methods:1.A total of 1629 patients with acute myocardial infarction who underwent coronary angiography in the emergency department of chest pain center of the hospital from January 2015 to June 2018 were enrolled.According to the myocardial infarction classification,patients were divided into type 2 MI group(T2MI)and type 1 MI group(T1MI,n=1001).Clinical information and laboratory data were obtained and follow-up.Data were analyzed by multivariate logistic regression analysis to identify the independent risk factors of T2MI.T2MI diagnostic prediction score was also calculated.Multivariate regression analysis was used to identify the independent risk factors of T2MI.The validity of the T2MI diagnostic prediction score was evaluated using the area under the curve of the receiver operating characteristic curve(ROC).2.A total of 223 critically ill elderly patients admitted to our hospital were recruited.Demographic variables were collected.The clinical data and the in-hospital mortality rate were compared between the T2MI and non-T2MI groups.Multivariate linear regression analysis was used to identify independent factors related to T2MI.The clinical data and incidence of T2MI were also compared between patients who survived and those who died;multivariate regression analysis was used to identify independent risk factors for in-hospital death and survival analysis was conducted.3.The hypoxic/reoxygenation model of H9c2 cardiomyocytes constructed,mir-542-5p overexpression,qPCR detection,cell viability test,immunofluorescence,detection of dual luciferase reporter gene,LDH release experiment,Western Blot,flow flow apoptosis detection.Results:1.The overall clinical diagnosis of myocardial infarction accounted for 33.8%(1862/5514)in the patients with chest pain.And the total in-hospital mortality was 2.8%(45/1629).The overall clinical diagnosis of type 2 myocardial infarction(T2MI)accounted for 11.4%(628/5514)in the patients with chest pain.The in-hospital mortality was 1.6%(10/628).Compared with type 1 myocardial infarction patients,the proportion of female(33.6%vs.24.9%,P<0.001),age(69.8±13.0 vs 66.8 ± 13.9,P<0.001),chronic heart failure(14.2%vs.6.1%),P<0.001),atrial fibrillation(18.2%vs.8.0%,P<0.001)and T2MI prediction score[2(1?2)vs.1(1?2),P<0.001]of T2MI patients were significantly higher;while proportion of smoker(4.9%vs.9.8%P<0.001),diabetes(23.1%vs.33.3%,P<0.001),history of MI(1.6%vs.4.2%,P=0.004),presenting with typical radiating chest pain(32.0%vs.57.7%,P<0.001),troponin T levels[0.03(0.02?0.08)vs.0.15(0.03?1.11),P<0.001]and eGFR levels[77.1(60.0?93.1)vs 84.8(64.5?97.9),P<0.001]were significantly reduced.Multivariate logistic regression analysis found that female(OR 1.365,95%CI 1.044?1.786,P=0.023),chronic heart failure(OR 1.735,95%CI 1.153?2.610,P=0.008),atrial fibrillation(OR 2.240,95%CI 1.547?3.244,P<0.001)and troponin T<40.8 ng/L(OR 1.881,95%CI 1.478?2.393,P<0.001)are the independent risk factors for T2MI;while patients with diabetes(OR 0.577,95%CI 0.442?0.753,P<0.001)and typical symptoms(OR 0.242,95%CI 0.186?0.315,P<0.001)has a significantly lower risk of T2MI.After adjusting other risk factors,patients scored 3 points(OR 10.898,95%CI 6.590?18.022,P<0.001),2 points(OR 4.301,95%CI 2.942?6.287P<0.001)and 1 point(OR 1.821,95%CI 1.253 to 2.648,P<0.001)in the T2MI prediction score has a significant increase in the risk of T2MI compared with patients who scored 0 point.The occurrence of T2MI increased with the score(17.8%vs.29.6%vs.48.8%vs.71.2%,P for trend<0.001).And the ROC curve of the incidence of T2MI was assessed by the T2MI diagnostic prediction score.The area under the curve was 0.682(95%CI 0.656-0.708,P<0.001),and the approximate index was 0.296.The corresponding optimal cutoff value was 2 points.The sensitivity is 0.640 and the specificity is 0.656.3.The most significantly upregulated miRNA,miR-542-5p,was selected to further functional study.the expression of miR-542-5p was increased in H/R-treated H92c cells at different time point.Cell viability was reduced after H/R treatment and miR-542-5p overexpression resulted in an aggravation of cell viability loss,whereas miR-542-5p inhibition exerted an amelioration of H/R-induced cell viability loss.The LDH release was induced following H/R and miR-542-5p overexpression further facilitated LDH release,whereas the increase was markedly repressed by miR-542-5p inhibition.Meanwhile,miR-542-5p overexpression aggravated H/R-induced cell apoptosis,while miR-542-5p inhibition attenuated H/R-induced cell apoptosis.H9c2 cells were treated with H/R in the presence or absence of miR-542-5p mimics or inhibitor,and then the autophagy activation was assessed.Following miR-542-5p treatment,there was a significant upregulation of green puncta representing autophagic vacuoles in H9c2 cells exposed to H/R and miR-542-5p inhibition resulted in an aggravation of autophagy activation,whereas miR-542-5p overexpression exerted an amelioration of H/R-induced autophagy activation in H9c2 cells.The miR-542-5p reduced the ratio of LC3-?/?-actin,whereas miR-542-5p inhibition resulted in an increase of ratio of LC3-?/?-actin,indicating miR-542-5p negatively regulated autophagy activation in H9c2 cells exposed to H/R.p62/SQSTM1 protein is an autophagy substrate and then the autophagy flux was validated by measuring the change of p62 protein expression.The miR-542-5p repressed autophagy flux,whereas miR-542-5p inhibition resulted in an increase of autophagy flux.Cell viability was increased in H9c2 exposed to H/R after miR-542-5p inhibition,whereas autophagy inhibition partially alleviated the protective role of miR-542-5p inhibition in H/R injury.The LDH release was repressed following miR-542-5p inhibition,whereas the effect was partially destroyed because of autophagy inhibition.Furthermore,miR-542-5p inhibition alleviated H/R-induced cell apoptosis,while the effect was also partially repressed because of autophagy inhibition.The target genes of miR-542-5p were screened using Targetscan software(http://www.targetscan.org/vert₇1/).Four autophagy-related genes,ATG9B,ATG7,ATG4B,and ATG14,were the potential target genes of miR-542-5p.The luciferase reporter plasmids containing 3'-UTR of ATG9B,ATG7,ATG4B,and ATG14(ATG9B-3'UTR-LUC,ATG7-3' UTR-LUC,ATG4B-3'UTR-LUC,and ATG14-3'UTR-LUC)were constructed,respectively.The results from luciferase reporter assay showed that miR-542-5p specifically repressed the luciferase expression of ATG7-3'UTR-LUC,whereas mutation of 4 nucleotides in ATG7-3'-UTR resulted in complete abolition of the repressive role.Furthermore,miR-542-5p overexpression markedly inhibited ATG7 protein expression in H92c cells.Conclusion:1.More than 30%of the chest pain patients were diagnosed with myocardial infarction in the acute chest pain center.The in-hospital mortality was close to 3%.T2MI was diagnosed in nearly 40%of the MI patients who underwent coronary angiography.There were more than 60%patients presenting with no typical radiating chest pain and with low baseline level of troponin ?(hs-tni ?40.8 ng/l).And the majority of them were elderly(about 60%).The in-hospital motality was 1.6%in type 2 myocardial infarction.In addition to female,low troponin ? level and presenting with no typical radiating chest pain,the independent risk factors were chronic heart failure,atrial fibrillation and with no diabetes in patients with diagnosis of myocardial infarction.The incidence of type 2 myocardial infarction increased with the increase of T2MI diagnostic prediction score in the patients diagnosed with myocardial infarction.There was a potential value of screening T2MI patients using T2MI diagnostic prediction score.And it's still needs further verified.2.Lung disease was the major cause of admission into the ICU.Severe hypoxemia was the most common risk factor for oxygen supply and demand imbalances in the study population,followed by tachycardia and shock in the elderly critical illness patients.Type 2 MI is present occurring approximately 1 in 4 critically ill elderly patients at the ICU.Severe hypoxemia,bradycardia,shock,tachycardia and MODS were associated with type 2 MI.The motality was high in critically ill elderly patients,and it would be higher in the patients conbined T2MI.In addition to lower eGFR and requiring mechanical ventilation,the occurrence of type 2 MI increased the risk of in-hospital death and reduced the cumulative survival.Compared with survivors,in addition to increasing the proportion of chronic heart failure,severe hypoxemia and requiring mechanical ventilation,the incidence of type 2 MI was also increased in died patients.APACHE ? scores,contents of troponin T,hs-CRP and procalcitonin were significantly increased,meanwhile,eGFR was decreased in non-survivors.3.These data suggest that the mir-542-5p/autophagy pathway plays an important role in H/r-induced myocardial cell injury.Pharmacological intervention of the mir-542-5p/autophagy pathway may be an effective approach for the treatment of H/r-induced myocardial injury.This provides a new direction for the clinical treatment of type 2 myocardial infarction.