Exosome-Mediated Transfer Of MiR-30a Aggravates Cardiac Function By Regulating Autophagy After Acute Myocardial Infarction

Part? Mi R-30 a was transferred between cells via exosomes and affected cardiac function after AMIObjective: Studies have shown that many types of cells release micro RNA into exosomes and transmit them to other cells through blood or body fluid,which is involved in the development of various diseases.The level of mi R-30 a in serum is significantly elevated in patients after acute myocardial infarction.However,whether the mi R-30 a transferred by exosomes and its role in AMI are remains unclear.In this study,we explored the source and delivery of mi R-30 a and its effect on the heart after AMI.Method: Male C57BL/6 mice aged 8 to 10 weeks were randomly divided into groups of eight.Acute myocardial infarction(AMI)was induced by permanent ligation of the left anterior descending(LAD)artery.Mice in the sham-operated group underwent the same procedure,but without the LAD coronary artery ligation.Mice were euthanized by dislocating the neck at the designated times.Heart,liver,spleen,lung,kidney and blood of mice were excised.Mi R-30 a agomir or antagomir(200nmol)were suspended in PBS(in a volume of 20 ?L)was and administered intravenously by tail vein(200 nmol)at 1 day before surgery,while a negative control of agomir(agomir NC)or antagomir(antagomir NC)was given as controls accordingly.PBS-treated MI mice were used as controls.NCMs,NCFs and H9c2 cells were incubated and placed in a hypoxic incubator to mimic ischemic injury in vitro.Mi R-30 a in organs,serum and exosomes form serum or conditioned medium was detected by RT PCR.Mi R-30 a in cardiac tissue was localized and relatively quantified by in situ hybridization.Exosomes was identified by TEM,NTA analyses and western blot.Transwell co-culture system was applied to confirm that exosomes may be transferred among cells.PKH67 labeled exosomes from hypoxic H9c2 cells and incubated with normoxic H9c2 cells or RAW264.7 cells to indicate that exosomes from the hypoxic cells could be transferred efficiently to the normoxic cells.TTC staining,masson's trichrome staining and transthoracic echocardiography were used to assess infarct size and cardiac function after AMI.Result: The expression level of mi R-30 a in serum and heart of acute myocardial infarction mice was time-dependent,and the level of mi R-30 a in cardiomyocytes was significantly up-regulated after hypoxia.Mi R-30 a was derived from the myocardium and transferred between cells via exosomes.Compared with the control group,inhibition of mi R-30 a could decrease the infarct size,reduce the myocardial fibrosis and improve cardiac function.Increasing the level of mi R-30 a has the opposite effect.Conclusion: Cardiomyocytes release mi R-30a-enriched exosomes after ischemia or hypoxia,which can be transmitted between cells.Changes in mi R-30 a levels could affect infarct size and cardiac function.Part ? Exosome/mi R-30 a downregulates autophagy and affects cardiac function after AMIObjective: Recent studies have shown that the upregulation of autophagy has a protective effect in ischemic heart disease.The level of autophagy was rapidly up-regulated after AMI,which could rescue damaged cardiomyocytes.However,the elevated autophagic flux may be impaired and down-regulated during the later stages of MI.The regulatory mechanism of autophagy after MI is poorly understood.The purpose of this experiment is to explore the regulatory mechanism of exosome-mediated transfer of mi R-30 a on the cardiac autophagy,apoptosis and inflammatory response after AMI.Method: Male C57BL/6 mice aged 8 to 10 weeks were randomly divided into groups of eight.Acute myocardial infarction(AMI)was induced by permanent ligation of the left anterior descending(LAD)artery.Mice in the sham-operated group underwent the same procedure,but without the LAD coronary artery ligation.Mice were euthanized by dislocating the neck at the designated times.Heart and blood of mice were excised.Immediately after LAD artery ligation,exosomes from AMI mice at 4 h after surgery(Exo(AMI4h))or sham-operated mice(Exo(Sham))(80 ?g of protein in total)were suspended in PBS(in a volume of 20 ?L)and administered by intra-myocardial injection at four different sites of the border zone.For mice allocated to the PBS group,the same surgical procedure was performed,and only PBS was administered.For mice allocated to the sham-operated group,the same amount of PBS was injected into the myocardium at similar regions.Bioinformatic predictions and dual-Luciferase Reporter assay were used to determine the target genes BECN1 and ATG5 of mi R-30 a.The autophagy levels of the heart tissue from mice measured by real-time PCR and western blot and TEM.TTC staining,masson's trichrome staining and transthoracic echocardiography were used to assess infarct size and cardiac function after AMI.The apoptosis of myocardium was assessed by TUNEL staining and Real-time PCR.Immunofluorescence and Real-time PCR were used to detect the macrophage infiltration and differentiation in the heart from different treatment groups.H9c2 cells and RAW264.7 cells were cultured and then co-cultured with exosomes from hypoxic H9c2 cells.TUNEL staining and Real-time PCR were used to evaluate the apoptosis of H9c2 cells.The related markers of macrophage differentiation were detected by Western blotting.Result: Ischemic stress increased the autophagy in cardiomyocytes after AMI.Cardiomyocytes released mi R-30 a encapsulated in exosomes and inhibit autophagy by targeting BECN1 and ATG5 genes;Mi R-30a-containing exosomes increased infarct size,worsen cardiac function,aggravate fibrosis,induced apoptosis and promoted macrophage polarization to M1 type.Conclusion: The present study shows that mi R-30 a was transferred by exosomes in a paracrine manner to inhibit autophagy,induce apoptosis and promote macrophage polarization to M1 type,in turn affecting cardiac function.