Effects Of Plasma Exosomes Derived From AMI Convalescent Patients On Cardiac Repair:the Role Of Mir-342-3p Dysregulation

BackgroundAcute myocardial infarction(AMI)is myocardial necrosis caused by acute,persistent ischemia and hypoxia of coronary arteries,and is an important cause of death in patients with cardiovascular diseases.Although percutaneous coronary intervention and other treatment methods can save patients’ lives in time,due to a large number of myocardial cells in the infarction area are lost and irreversible,patients develop myocardial remodeling and gradually progress to heart failure(HF),which seriously affects the long-term prognosis of patients with myocardial infarction.Therefore,it is of great significance to search for new intervention targets in the repair of heart injury and develop effective endogenous myocardial protective factors.Exosome is a cell secreted vesicle with a diameter of 30-100 nm that contains proteins,lipids,nucleic acids and other bioactive substances.Exosomes can regulate receptor cell function by releasing contents into receptor cells or by "receptor-ligand"mechanism.Therefore,exosomes play an important role in paracrine and information exchange between different tissues and cells.Because of the high exosome content in blood,the easy access and the convenience of sampling,the blood source exosomes have attracted extensive attention of researchers.In recent years,several studies have reported the role of plasma exosomes derived from healthy people in heart protection,but it is unclear whether plasma exosomes still have a protective effect and whether the protective efficacy changes in AMI convalescent patients.In-depth study on the biological efficacy of plasma exosomes in the recovery period of acute myocardial infarction under the pathological conditions is of great significance to understand the mechanism of self-repair after heart injury from multiple perspectives,and is also conducive to finding new intervention targets in the process of heart injury repair.Part Ⅰ Characterization of plasma exosomes derived from AMI convalescent patients and healthy personsPurpose:Blood samples of AMI convalescent patients and healthy people were collected to extract plasma exosomes,and the characterization of exosomes will be observed.Methods:1.Plasma exosomes were morphologically identified by transmission electron microscopy.2.Particle size distribution and number of plasma exosomes were analyzed by NTA.3.The labeled proteins of plasma exosome were detected by Western blot.4.Fluorescence microscopy was used to investigate the cellular uptake of PKH26-labeled plasma exosomes and their intercellular transmission.Results:1.There were no significant differences in the morphology,concentration,particle size distribution and expression of labeled proteins of plasma exosomes between the two groups.2.Plasma exosomes can be ingested by cardiomyocytes in vitro or in vivo,and the process of ingestion is dynamin-dependent.3.Plasma exosomes can be transferred between cells.Conclusion:There was no significant difference in the characterization of plasma exosomes between AMI convalescent patients and healthy persons.Part Ⅱ Effects of plasma exosomes derived from AMI convalescent patients and healthy persons on cardiac repair and the comparation between the two exosomesPurpose:The H2O2-induced myocardial cell oxidative damage model and rat myocardial infarction model were established to compare the repair effect of AMI-exo in convalescent patients and Nor-exo in healthy patients on cardiac injury,and to observe the effects of both on myocardial cell apoptosis and autophagy.Methods:1.In vitro,H9c2 cells were pretreated with Nor-Exo or AMI-Exo,then,H2O2 was added to induce cell oxidative damage.After treatment,CCK-8 method was used to detect cell vitality.Flow cytometry,Western blot and TUNEL immunofluorescence was used to detect cell apoptosis.Western blot,mRFP-GFP-LC3 fluorescence and electron microscopy were used to detect cell autophagy activity.2.In vivo,acute myocardial infarction model was established by ligation of the left anterior descending artery in rats.Then,Nor-Exo,AMI-Exo or PBS control was injected into the left ventricular myocardium of rats by point injection.In the sham group,only suture was performed without ligation.Echocardiography was performed on 28 days after myocardial infarction.After that,all rats were executed and cardiac tissue samples were collected.Masson staining,TUNEL immunofluorescence staining and electron microscopy were performed to determine the level of myocardial infarction,fibrosis,apoptosis and autophagy.Results:1.In vitro,Nor-Exo and AMI-Exo could both enhance cell vitality,reduce cell apoptosis and inhibit excessive autophagy under H2O2 treatment,but the above effects of AMI-Exo were weaker than Nor-Exo.2.In vivo,intramyocardial injection of Nor-Exo and AMI-Exo both reduced the infarct size,improved cardiac function,and inhibited myocardial apoptosis and autophagy in rats after AMI,but the effects of AMI-Exo were also weaker than that of Nor-Exo.Conclusion:The repair effect of plasma exosomes in AMI convalescent patients on cardiac injury is weaker than that in healthy patients,and the mechanism of the repair effect may be related to the inhibition of cardiomyocyte apoptosis and excessive autophagy.Part Ⅲ Study on effects of miR-342-3p dysregulation in plasma exosomes derived from AMI convalescent patients on cardiac repairPurpose:MiRNAs sequencing,qRT-PCR and preliminary functional screening were used to identify the key miRNA with dysregulation in AMI-exo.Subsequently,the effect of the key miRNA on cardiac repair was determined in H2O2-induced cell oxidative damage model and rat myocardial infarction model.Methods:1.High-throughput sequencing technology was used to sequence the miRNAs contained in Nor-exo and AMI-exo.Then,qRT-PCR was conducted to verify the miRNAs with large differences and changes according to the sequ encing results.After that,CCK-8 method was used to screen the miRNAs with clear differences to determine the key miRNA.2.In vitro,the key miRNA mimic or negative control was transfected into H9c2 cells,then,H2O2 was added to induce cell oxidative damage.After treatment,CCK-8 method was used to detect cell vitality.Flow cytometry,Western blot and TUNEL immunofluorescence was used to detect cell apoptosis.Western blot,mRFP-GFP-LC3 fluorescence and electron microscopy were used to detect cell autophagy activity.3.In vivo,key miRNA mimic or negative control was transfected into AMI-Exo by electroporation.Thereafter,rat acute myocardial infarction model was established,key miRNA over-expressed AMI-Exo or negative control AMI-Exo was injected into the left ventricular myocardium of rats by point injection.Echocardiography was performed on 28 days after myocardial infarction.After that,all rats were executed and cardiac tissue samples were collected.Masson staining,TUNEL immunofluorescence staining and electron microscopy were performed to determine the level of myocardial infarction,fibrosis,apoptosis and autophagy.Results:1.Compared with Nor-exo,miR-342-3p level in AMI-Exo was decreased.2.In vitro,compared with negative control,transfection of miR-342-3p mimic into cardiomyocytes enhanced cell viability,reduced apoptosis and inhibited excessive autophagy under H2O2 treatment.3.In vivo,compared with negative control,AMI-exo transfected with miR-342-3p mimic significantly reduced the infarct size,improved cardiac function,and inhibited myocardial apoptosis and autophagy in rats after AMI.Conclusion:The low expression of miR-342-3p in plasma exosomes derived from AMI convalescent patients impairs therapeutic potential.Part Ⅳ Study on the role of target genes regulated by miR-342-3p in cardiomyocyte apoptosis and autophagyPurpose:Target genes of miR-342-3p were predicted by bioinformatics,and genes related to apoptosis and autophagy pathway were selected for targeted validation and functional validation based on previous literature reports.Methods:1.MiRanda,miRDB and TargetScan were used to predict the potential target genes of miR-342-3p.According to the predicted results and literature analysis,SOX6 and TFEB were selected as candidate target genes.2.Western blot and dual luciferase reporter system were used to verify the targeting relationship between miR-342-3p and the above two genes.3.SOX6 overexpressed plasmid(pcDNA3.1-SOX6)or no-load control plasmid(pcDNA3.1)was co-transfected with miR-342-3p mimic or its negative control into H9c2 cardiomyocytes.Then,H2O2 was added to induce cell damage.After treatment,SOX6 protein was detected by Western blot,cell activity was detected by CCK-8.4.TFEB small interfering RNA(TFEB SiRNA)or its negative control(NC SiRNA)was transfected into H9c2 cardiomyocytes.Then,H2O2 was added to induce cell damage.After treatment,the expression levels of TFEB and autophagy related protein LC3 were detected by Western blot.Results:1.MiR-342-3p directly targeted SOX6,and SOX6 overexpression impaired the cellular protective effect of miR-342-3p under H2O2 treatment.2.MiR-342-3p directly targeted TFEB,and transfection of TFEB SiRNA into cardiomyocytes significantly inhibited H2O2-induced autophagy.Conclusion:MiR-342-3p resists H2O2-induced apoptosis and autophagy by targeting SOX6 and TFEB,respectively.