The Role And Mechanisms Of MiR-323-3p Targeting Pim1 Regulated Autophagy And Apoptosis In Coronary Microembolization-induced Myocardial Injury

Coronary microembolizaiton?CME?comes from spontaneous rupture of vulnerable atherosclerotic plaques in acute coronary syndrome or from thrombus and plaque debris after interventional treatment.CME often leads to coronary microcirculation disturbance?MVO?.In STEMI emergencyPCI,MVO often manifests as no flow or slow flow,which is a very difficult problem for cardiovascular interventional physicians.The MVO reduces the clinical benefit of emergency PCI and is an important reason affecting the prognosis of patients with acute myocardial infarction.At present,there is no special and effective method to deal with myocardial injury caused by CME,including thrombus aspiration device and sodium nitroprusside vasodilator.Therefore,it is necessary to investigate the mechanism of myocardial injury caused by coronary microembolism.Recent studies have shown that in cardiovascular diseases,the activation of autophagy can reduce myocardial damage caused by acute myocardial ischemia,myocardial infarction and heart failure,also inhibit the progress of atherosclerosis,myocardial remodeling after myocardial infarction,and inhibit the aging of myocardial cells.Our previous studies have confirmed that there were multiple microinfarcts in the myocardium after rats or pigs CME,and there were apoptosis,necrosis,infiltration,and progressive decline in cardiac function.Cardiomyocytes autophagy was also foundat early stage after microembolization,and then progressively decreases.Whether promoting autophagy activation can improve cardiac function after coronary microembolization is still very little studied.MicroRNAs are small non-coding single-stranded RNA molecules,which control its post-transcriptional translation or promote its complete degradation by targeting target RNA.They are widely involved in the regulation of many cardiovascular diseases.It is significant benefical in many experimental models of cardiovascular diseases through regulating the expression of these microRNAs,including inhibition of myocardial remodeling after myocardial infarction and improvement of cardiac function.However,whether microRNAs play an important role in the treatment of myocardial injury caused by coronary microembolism by regulating autophagy is still very little studied.The expression of miRNA-323-3p in the heart tissues of pigs and rats after CME was significantly increased from our miRNA sequencing resuluts.The bioinformatics database predicts that Pim1 is a possible target gene.Does microRNA323-3p target Pim1 to regulate autophagy and apoptosis of cardiomyocytes after CME?We studied the primary cultures of cardiac myocytes in vitro and the model of coronary microembolism in vivo in order to provide a new treatment for myocardial injury caused by coronary microembolism.Part ? Dynamic changes of microRNA-323-3p and Pim1 mRNA in rat coronary microembolism modelsObjective:To observe the dynamic changes of miRNA-323-3p and Pim1mRNA expression in myocardial tissue by establishing rat CME models.Methods:Thirty-six SD rats were randomly divided into CME group?n=30?and sham operation group?Sham group,n=6?.CME group were randomly divided into 0h,3h,6h,9h,12h groups according to different observation time points.The CME model was constructed by injecting polystyrene microspheres into the left ventricle,and the Sham group was injected with normal saline.Cardiac function was detected by echocardiography.Serum cardiac troponin I?cTnI?level was detected by ELISA.HE staining and HBFP staining were used to observe and evaluate myocardial microinfarction area.RT-qPCR was used to detect the expression of miRNA-323-3p and Pim1mRNA.Results:1.Compared with the Sham group,the left ventricular ejection fraction?LVEF?,cardiac output?CO?,left ventricular short axis shortening rate?LVFS?were decreased,while left ventricular end diastolic diameter?LVIDd?,left ventricular end systolic diameter?LVIDs?were increased?P<0.05?in the CME 6h?9h?12h group.2.Compared with the Sham group,cTnI was significantly increased in each group of CME,and peaked at 9h,the difference was statistically significant?P<0.05?.3.Compared with the Sham group,HE staining of myocardial tissue in the CME group showed that the myocardial cell nucleus dissolved or disappeared in the microinfarction area,accompanied by inflammatory cell infiltration.The results of HBFP staining showed red staining of the myocardial area in the microinfarction area,however,yellow staining in normal heart.There are multiple microinfarctions in the focal area.4.Compared with the Sham group,the Pim1mRNAof myocardial tissue in CME groups increased significantly in the 3h group and then decreased gradually?P<0.05?.The expression level of miRNA-323-3p increased in the 3h group and reached the peak at 6h,then gradually decreased,still is significantly higher than the Sham group at 12h.Conclusion:The CME rat model was successfully established,and the cardiac function showed a progressive decline,which was the most significant decrease in the 9-12h group.Pim1 mRNA increased significantly in the 3h group and then gradually decreased.The expression level of miRNA-323-3p was increased in the 3h group,peaked at 6h,and was significantly higher than the Sham group at12h.In the rat CME model,miRNA-323-3p may negatively regulate Pim1 and promote cardiac function decline.Part ? Inhibition of miRNA-323-3p expression targeting Pim1promotes autophagy,inhibits apoptosis,and improves cardiac function after coronary microembolization in ratsObjective:To investigate the effect of different miRNA-323-3p expression levels on the autophagy and apoptosis of cardiomyocytes after CME6h,and to elucidate whether inhibition of miRNA-323-3p targeting Pim1 promotes autophagy,inhibits apoptosis,and improves cardiac function after coronary microembolization in rats.Methods:Thirty SD rats were randomly divided into 5 groups:sham operation group?Sham6h group?,CME6h group,CME6h+miRNA-control?blank virus?group,CME6h+miRNA-323-3pgroup,CME6h+miRNA-323-3p-inhibitor group?n=6,in each group?.CME6h+miRNA-control group,CME6h+miRNA-323-3p group,CME6h+miRNA-323-3p-inhibitor group were transfected with blank vector,miRNA-323-3p overexpression rAAV9 and miRNA-323-3p-inhibitor rAAV9 via tail vein,respectively.Three weeks after transfection,along with the CME6h group,the CME models were established by intraventricular injection of microspheres after thoracotomy,and rats in the sham group received an injection of the same volume of normal saline only.The dual-luciferase reporter assay was performed to confirm whether Pim1 is the target gene of miRNA-323-3p.Six hours after the establishment of the microembolization model,cardiac function was detected by echocardiography.Serum cardiac troponin I?cTnI?levels were detected by ELISA.HE staining and HBFP staining were used to observe the myocardial microinfarct area.RT-qPCR was used to detect miRNA-323-3p and Pim1 mRNA,Western blot was used to detect autophagy-related proteins LC3II,p62,apoptosis-related proteins Caspase3,Cleaved-Caspase3,autophagy pathway-related proteins p-AMPK,AMPK,p-mTOR,mTOR,Parkin,ATG5 and Pim1.The cardiomyocytes autophagy was detected by transmission electron microscopy.Result:1.Bioinformatics database predictive analysis results suggest that Pim1 may be one of the potential target genes of miRNA-323-3p;dual-luciferase reporter gene detection results show Pim1 is the rno-miRNA-323-3p target gene.2.After three weeks of rAAV9 infection,the transfection efficiency reached 75%and the transfection was successful.3.Compared with the Sham6h group,the expression levels of miRNA-323-3p and Pim1 mRNA and protein in the CME6h group were significantly increased.Compared with the CME6h group,the expression level of miRNA-323-3p,Pim1mRNA and protein were significantly up-regulated in CME6h+miRNA-323-3p group.However,they were significantly decreased in CME6h+miRNA-323-3p-inhibitor group.Compared with CME6h group,There were no significant difference in CME6h+miRNA-control group.4.Compared with the Sham6h group,the level of cTnI was significantly increased in the CME6h group,and the cardiac function was significantly decreased in the rats.Compared with the CME6h group,the level of cTnI was significantly increased in the CME6h+miRNA-323-3p group,and the cardiac function of the rats was significantly decreased.However,compared with the CME6h group,the level of cTnI was significantly decreased and the cardiac function of the rats was significantly improved in the CME6h+miRNA-323-3p-inhibitor group.There was no significant difference in cTnI level and cardiac function in CME6h+miRNA-control group compared with the CME6h group.5.HE staining showed myocardial microinfarction around the microembolism in CME groups.The central of region showed myocardial cell lysis or disappearance,myocardial edema accompanied with inflammatory cells infiltration.HBFP staining showed that the microinfarction were focal,which were dyed red.The microinfarction area were?16.25±2.81?%,?16.52±3.14?%,?21.33±4.76?%,?7.88±3.25?%in CME6h group,CME6h+miRNA-control group,CME6h+miRNA-323-3p group,CME6h+miRNA-323-3p-inhibitor,respectively.Compared with the CME6h group,the myocardial microinfarction area was significantly increased in CME6h+miRNA-323-3p group?P<0.05?,however,it was significantly decreased in the CME6h+miRNA-323-3p-inhibitor group?P<0.01?.6.Compared with the sham6h group,the relative expression levels of LC3II/I,Cleaved-Caspase-3/Caspase-3 proteins in CME6h group were increased,and the relative expression level of p62 protein was decreased.Compared with CME6h group,the ratio of LC3II/LC3I was decreased,and the ratio of Cleaved-Caspase-3/Caspase-3 and p62 were significantly increased in CME6h+miRNA-323-3p.Compared with the CME6h group,the relative expression level of LC3II/I protein was significantly increased,and the relative expression levels of Cleaved-Caspase-3/Caspase-3 and p62 protein were significantly decreased in the CME6h+miRNA-323-3p-inhibitor group.Compared with CME6h group,there were no statistical difference in CME6h+miRNA-control group.7.Compared with the sham6h group,the relative expression levels of p-AMPK/AMPK,ATG5 and Parkin increased in the CME6h group,and the relative expression level of p-mTOR/mTOR protein was decreased.Compared with the CME6h group,the relative expression levels of p-AMPK/AMPK,ATG5 and Parkin were decreased in the CME6h+miRNA-323-3p group,the relative expression level of p-mTOR/mTOR was significantly increased.Compared with the CME6h group,the relative expression levels of p-AMPK/AMPK,ATG5 and Parkin proteins were significantly increased in the CME6h+miRNA-323-3p-inhibitor group,and the relative expression levels of p-mTOR/mTOR protein were significantly decreased.Compared with CME6h group,They was no significant difference in the CME6h+miRNA-control group.8.Transmission electron microscopy results showed that the myofiber was clear and the mitochondrial membrane was intact in the Sham6h group.Myocardial cell myofilament disorder,mitochondrial swelling and deformation after CME.Compared with CME6h,autophagy were significantly decreased in CME6h+miRNA-323-3pgroup;however,cardiomyocyteautophagywas significantly increased in the CME6h+miRNA-323-3p-inhibitor group.Conclusion:1.Pim1 is one of the target genes of miRNA-323-3p.2.Overexpression of microRNA-323-3p inhibits cardiomyocyte autophagy after CME6h,promotes apoptosis and necrosis of myocardial cells,aggravates cardiac function damage;however,inhibition of miRNA-323-3p,promotes cardiomyocyte autophagy,inhibits apoptosis and necrosis,and improves cardiac function.3.Inhibition of miRNA-323-3p can promote autophagy in cardiomyocytes,which may be partly through the promotion of Pim1 overexpression,then activate the AMPK/mTOR/ATG5 autophagy pathway after CME.Mitophagy is also activated after CME.Part ? Pim1 Overexpression Prevents Cardiomyocytes from Apoptosis after Hypoxia and Oxidative Stress via Up-Regulating Cell AutophagyObjective:Microvascular obstruction?MVO?,one of undesirable complications of percutaneous coronary intervention?PCI?,is independently associated with adverse left ventricle remodeling and prognosis after AMI.Hypoxia and oxidative stress play a major role in the pathophysiology of MVO.Pim1 serves an important protective role in ischemic myocardium,however,the specific mechanism remains poorly defined.Autophagy in the early hypoxia or moderate oxidative stress has been demonstrated to protecte myocardium.In this study we investigated the association between the protective effect of Pim1 and autophagy after hypoxia and oxidative stress.Methods:We isolated ventricular myocytes from neonatal rat heart?NRVMs?.NRVMs were exposed to hypoxia and H₂O₂.Rapamycin and 3-MA were used as activators and inhibitors for autophagy,respectively.pHBAd-Pim1 were transfected into NRVMs.We assessed cardiomyocytes apoptosis by Annexin V-FITC/PI flow cytometry.Autophagy was evaluated by mRFP-GFP-LC3adenovirus infectionby confocal microscope detection.Western blotting was used to quantify apoptosis or autophagy protein?Caspase3,LC3,P62,AMPK,mTOR,ATG5?concentrations.Results:We found that autophagy and apotosis in NRVMs significantly increased and reached the peak at 3h,6h,respectively,after hypoxia and H₂O₂.The mTOR inhibitor rapamycin induced autophagy,decreased apoptosis of cardiomyocytes,however,inhibition of autophagy,3-MA,decreased autophagy and increased apoptosis of cardiomyocytes at 3h after hypoxia and H₂O₂.Pim1in NRVMs increased at 3h and decreased gradually after hypoxia and H₂O_2.Pim1 overexpression enhanced autophagy and decreased apoptosis.The last,we found that the mechanism of Pim1 to promote autophagy is partly caused by the activation of AMPK/mTOR/ATG5 pathway after hypoxia and H₂O₂.Conclusions:Our results revealed Pim1 overexpression prevented NRVMs from apoptosis via up-regulating autophagy after hypoxia and oxidative stress,partly caused by the activation of AMPK/mTOR/ATG5 autophagy pathway.