The Role And Mechanisms Of MiR-30e-3p Regulated Autophagy In Cardiomyocyte Injury Induced By Ischemia And Hypoxia

Coronary microembolizaiton(CME)is resulted from the obstruction of microcirculatory vessels caused by the rupture of atherosclerotic vulnerable plaque and the entry of microthrombosis or atherosclerotic plaque debris into coronary microcirculation during percutaneous coronary intervention(PCI).Slow flow or no reflow induced by CME is a common complication in PCI,and is the main factor affecting the long-term prognosis of patients.A variety of medical imaging tests have shown that the extent of CME is associated with poor clinical outcomes,including adverse left ventricular remodeling and increased mortality rate.Although the research on CME has made some progress,there is still a lack of effective treatment for CME.Therefore,the prevention and treatment of CME is a difficult problem to be solved.The research shows that the pathophysiological basis of CME is continuous ischemia and hypoxia of cardiomyocytes.Therefor,It is necessary to study the molecular mechanism of CME at the cellular level.Autophagy is considered an indispensable self-digestive process for cardiomyocytes.Normal autophagy can provide energy and self-renewal of cardiomyocytes,which plays an important role in maintaining the normal physiological function of cardiomyocytes and reducing the damage of cardiomyocytes in the environment of hypoxia and energy deficiency.Insufficient or excessive autophagy can lead to various cardiovascular diseases.However,the activation level and mechanism of autophagy in ischemic and hypoxic(IH)environment remain limited.Micro RNAs(mi RNAs)are small non-coding RNA which primary function is to manipulate post-transcriptional gene expression,including degradation and translational repression.Mi RNAs have been shown to be involved in the development of cardiovascular diseases.However,there is still limited evidence suggesting mi RNAs playing a regulatory role in cardiac autophagy in an IH environment.In a previous study,we showed that mi R-30e-3p was notably decreased in a rat model of simulating coronary microembolization.Mi R-30e-3p is a member of the mi R-30 family.The mi R-30 family has been proved to protect the myocardium during myocardial hypertrophy and myocardial ischemia-reperfusion injury.However,whether mi R-30e-3p has a protective effect on cardiomyocytes exposed to the IH environment remains elusive.In this study,we established an model in-vitro study using Sprague Dawley(SD)rat cardiomyocytes which were exposed to IH environment to mimic CME-mediated myocardial injury.According to the model of cardiomyocytes,we are going to reveal the change of autophagy in the IH environment,discuss the expression changes and effects of mi R-30e-3p,elucidate the impacts of manipulated mi R-30e-3p expressions on autophagy of IH-exposed cardiomyocytes in a cellular level.Part 1 Changes of Mi R-30e-3p Expression and Autophagy in Cardiomyocytes in an Ischemia and Hypoxia EnvironmentObjective: To establish a model of ischemia and hypoxia(IH)in neonatal rat primary cardiomyocytes,and to observe the changes level of mi R-30e-3p expression and autophagy in cardiomyocytes.Methods:The neonatal primary cardiomyocytes were isolated and cultured from SD rats for an in-vitro study.According to the time point of IH,the cardiomyocytes were divided into five groups(0 h,3 h,6 h,9 h and 12 h groups).The serum-free medium was used to simulate the ischemic environment,and the anoxic chamber was used to simulate the hypoxic environment.Cell viability was determined by MTS;Lactate dehydrogenase(LDH)leakage was used to detect the injury of cardiomyocytes;RT-q PCR was used to measured the expression level of mi R-30e-3p;The expression of LC3 II,p62 and cleaved caspase 3 proteins were detected by Western blots.Autophagic vacuoles and autophagic flux were detected by transmission electron microscopy(TEM)and confocal microscope respectively.Flow cytometry was used to detect cardiomyocyte apoptosis in Annexin V-FITC / PI double staining.Results:1.Compared with the 0 h group,the activity of primary cardiomyocytes was decreased significantly in 6 h,9 h and 12 h after exposed to the IH environment.Compared with 0 h group,the level of LDH significantly was increased during IH.The injury of cardiomyocytes increased with the time-dependent of IH.2.RT-q PCR detected the expression of mi R-30e-3p.Compared with the 0h group,the expression of mi R-30e-3p was significantly reduced in 6 h,9 h and12 h after exposed to IH.3.The key proteins of autophagy detected by Western blots.Compared with the 0 h group,LC3 II expression was increased dramatically and p62 expression was decreased in 3 h and 6 h after exposed to IH;Meanwhile,LC3 II expression was decreased and p62 expression was increased significantly in 9 h and 12 h during IH.4.TEM was used to observe the ultrastructure in cardiomyocyte.Mitochondrial morphology was normal without swelling or vacuolation in 0 h group.After exposed to the IH environment,there were visible intracellular double-membrane autophagic vacuoles and mitochondrial swelling or vacuolation.Autophagic vacuoles were increased in the 3 h group and then decreased gradually after 6 h in the IH environment.5.Confocal microscope was used to evaluate autophagic flux after m RFP-GFP-LC3 adenovirus transfection.Autolysosomes were represented by red color and the autophagosomes were signified by yellow color(overlay).The results of autophagic flux demonstrated that autolysosomes(m RFP+dots)and autophagosomes(m RFP+GFP+dots)enhanced markedly in the 3 h group and reduced afterwards in the IH context.6.In the detection of myocardial apoptosis,the expression of cleaved caspase 3 protein was gradually increased after exposed to IH measured by Western blots.And the results of flow cytometry showed that apoptosis was gradually increased during IH.Conclution:The expression level of mi R-30e-3p was decreased significantly and autophagy was suppressed,while the cell injury and apoptosis increased in the IH-exposed cardiomyocytes.Part 2 The Protective Effect of Mi R-30e-3p Regulating Autophagy onCardiomyocytes Injury Induced by Ischemia and Hypoxia via Modulating Egr-1Objective: To study the relationship between the expression of mi R-30e-3p and autophagy level in the IH environment,and whether mi R-30e-3p can regulate autophagy to mediate IH induced cardiomyocyte injury via modulating Egr-1.Methods:The neonatal primary cardiomyocytes were isolated and cultured from SD rats for an in-vitro study,and the 9 h after exposed to IH was the time point for investigating.The lentiviral vectors were transfected to cells to suppress or increase the expression of mi R-30e-3p,including mi R-30e-3p mimic,mi R-30e-3p antagonist and mi R-30e-3p negative control(NC).The lentivirus were added directly to the complete medium at 50 multiplicity of infection(MOI).The cells were pretreated with 3-MA at the dose of 5m M for 2hours to inhibit autophagy before the lentivirus transfection.The cardiomyocytes were divided into six groups: normal control group(Con),IH group,IH+NC,IH+mi R-30e-3p mimic group(IH+MIR),IH+mi R-30e-3p antagonist group(IH+MIR Atagonist)and IH+mi R-30e-3p mimic group+3-MA group(IH+MIR+3-MA).The serum-free medium was used to simulate the ischemic environment,and the anoxic chamber was used to simulate the hypoxic environment.LDH leakage was used to detect the injury of cardiomyocytes;RT-q PCR was used to measure the expressions level of mi R-30e-3p and Egr-1 m RNA;The expressions of LC3 II,p62,Egr-1 and cleaved caspase 3 proteins were detected by Western blots.Autophagic vacuoles and autophagic flux were detected by TEM and confocal microscope respectively.Flow cytometry was used to measure cardiomyocyte apoptosis in Annexin V-FITC / PI double staining.Results:1.Compared with the control group,the level of LDH was increased significantly in IH group,IH+NC group,IH+MIR group,IH+MIR Antagonist group and IH+MIR+3-MA group in cardiomyocytes;Compared with the IH group,the level of LDH was decreased in IH+MIR group and was increased significantly in IH+MIR Antagonist group;Compared with the IH+MIR group,the level of LDH was increased significantly in IH+MIR Antagonist group.Cardiomyocytes injury was decreased significantly after up-regulating the expression of mi R-30e-3p.2.Compared with the control group,the expression of mi R-30e-3p was reduced significantly in IH group,IH+NC group and IH+MIR Antagonist group in cardiomyocytes,the expression of mi R-30e-3p was up-regulated dramatically in IH+MIR group and IH+MIR+3-MA group;The expression of Egr-1 m RNA was increased significantly in each group after exposed to IH;Compared with the IH group,the expression of mi R-30e-3p was increased dramatically in IH+MIR group and IH+MIR+3-MA group,and was decreased significantly in IH+MIR Antagonist group;The expression of Egr-1 m RNA increased in IH+MIR Antagonist group,and deceased in IH+MIR group;Compared with the IH+MIR group,the expression of mi R-30e-3p was reduced obviously and the expression of Egr-1 m RNA was increased in IH+MIR Antagonist group.3.Compared with the control group,the expression of LC3 II protein was reduced obviously and the expressions of Egr-1 and p62 were enhanced significantly in IH group,IH+NC group and IH+MIR Antagonist group;the expression of LC3 II protein was enhanced obviously and the Egr-1 expression was reduced significantly in IH+MIR group;Compared with the IH group,the expression of LC3 II protein was enhanced obviously and the Egr-1 expression was reduced significantly in IH+MIR group;Compared with the IH+MIR group,the expression of LC3 II protein was decreased obviously,while the expressions Egr-1 and p62 were increased significantly in IH+NC group and IH+MIR Antagonist group.4.TEM was used to observe the ultrastructure in cardiomyocytes.Mitochondrial morphology was normal without swelling or vacuolation in Con group.In IH group,IH+NC group and IH+MIR Antagonist group,TEM demonstrated mitochondrial swelling or vacuolation and intracellular double-membrane autophagic vacuoles were decreased;In IH+MIR group,intracellular double-membrane autophagic vacuoles were increased,and mitochondrial swelling was alleviated.5.Confocal microscope was used to evaluate autophagic flux after m RFP-GFP-LC3 adenovirus transfection.Compared with the control group,the autophagic flux was enhanced dramatically in IH+MIR group;Compared with the IH group,the autophagic flux was increased significantly in IH+MIR group;Compared with the IH+MIR group,the autophagic flux was reduced significantly in IH+NC group,IH+MIR Antagonist and IH+MIR+3-MA group.6.In the detection of myocardial apoptosis,compared with the control group,the results of the expression of cleaved caspase 3 protein and flow cytometry showed that the apoptosis was increased significantly in IH group,IH+NC group,IH+MIR Antagonist and IH+MIR+3-MA group;Compared with the IH group,the apoptosis reduced dramatically in IH+MIR group;Compared with the IH+MIR group,the apoptosis was increased significantly in IH+NC group,IH+MIR Antagonist and IH+MIR+3-MA group.Conclution:1.The autophagy changed with the expression level of mi R-30e-3p.Meanwhile,the level of cardiomyocyte autophagy was strongly linked to the mi R-30e-3p expression.2.mi R-30e-3p can promote autophagy and alleviate cardiomyocytes injury induced by IH exposure through regulating Egr-1.Part 3 The Mechanism of Egr-1/Bim/Beclin-1 Signal Pathway in Cardiomyocyte Injury Induced by Ischemia and HypoxiaObjective : To study the activation of Egr-1/Bim/Beclin-1 signaling pathway in the IH environment,and the mechanism of the pathway effecting on autophagy and injury in cardiomyocytes.Methods:The neonatal primary cardiomyocytes were isolated and cultured from SD rats for an in-vitro study,and the 9 h IH exposure was the time point for investigating.The lentiviral vectors were transfected to cardiomyocytes to suppress or over-express the expression of Egr-1,including Egr-1 mimic,Egr-1sh RNA and Egr-1 negative control(NC).The lentivirus were added directly to the complete medium at 50 MOI.The cells were pretreated with 3-MA at the dose of 5m M for 2 hours to inhibit autophagy before the lentivirus transfection.The cardiomyocytes were divided into six groups: normal control group(Con),IH group,IH+negative control group(IH+NC),IH+Egr-1 group,IH+Egr-1sh RNA group(IH+sh RNA)and IH+Egr-1 sh RNA+3-MA group(IH+sh RNA+3-MA).The serum-free medium was used to simulate the ischemic environment,and the anoxic chamber was used to simulate the hypoxic environment.LDH leakage was used to detect the injury of cardiomyocytes;RT-q PCR was used to measured the expressions level of Egr-1m RNA,Bim m RNA and Beclin-1 m RNA;The protein expressions of LC3 II,p62,Egr-1,Bim,Beclin-1 and cleaved caspase 3 were detected by Western blots.Autophagic vacuoles and autophagic flux were detected by TEM and confocal microscope,respectively.Flow cytometry was used to detect cardiomyocyte apoptosis in Annexin V-FITC / PI double staining.Results:1.Compared with the control group,the level of LDH was increased significantly in IH group,IH+NC group,IH+Egr-1 group,IH+sh RNA group and IH+sh RNA+3-MA group in cardiomyocytes;Compared with the IH group,the level of LDH was decreased in IH+sh RNA group,and was increased significantly in IH+Egr-1 group;Compared with the IH+sh RNA group,the level of LDH was increased significantly in IH+Egr-1 group.Cardiomyocytes injury was decreased significantly after suppressing the expression of Egr-1.2.Compared with the control group,the expression of Egr-1 m RNA and Bim m RNA were increased significantly,and the expression of Beclin-1 m RNA was reduced dramatically in IH group,IH+NC group and IH+Egr-1 group in cardiomyocytes.Compared with the IH group,the expression of Egr-1 m RNA and Bim m RNA were increased dramatically,and Beclin-1 m RNA was reduced obviously in IH+Egr-1 group;Meanwhile,the expression of Egr-1 m RNA and Bim m RNA were decreased significantly and Beclin-1 m RNA expression was enhanced dramatically in IH+sh RNA and IH+sh RNA+3-MA group;Compared with the IH+sh RNA group,the expression of Egr-1 m RNA and Bim m RNA were increased obviously and the expression of Beclin-1 m RNA was decreased significantly in IH+NC group and IH+Egr-1 group.3.Compared with the control group,the expression of Egr-1,Bim and P62 proteins were enhanced significantly,and the expression of LC3 II and Beclin-1proteins were reduced obviously in IH group,IH+NC group and IH+Egr-1group;Compared with the IH group,the expression of Egr-1,Bim and P62 proteins was increased significantly and the expression of LC3 II and Beclin-1proteins were decreased dramatically in IH+MIR group;Compared with the IH+sh RNA group,the expression of Egr-1,Bim and P62 proteins were increased significantly,and the expression of LC3 II and Beclin-1 protein was decreased obviously in IH+NC group and IH+Egr-1 group.4.TEM was used to observe the ultrastructure in cardiomyocyte.Mitochondrial morphology was normal without swelling or vacuolation in Con group.In IH group,IH+NC group and IH+Egr-1 group,TEM demonstrated mitochondrial swelling or vacuolation and intracellular double-membrane autophagic vacuoles were decreased;In IH+sh RNA group,intracellular double-membrane autophagic vacuoles were increased,and mitochondrial swelling was alleviated.5.Confocal microscope was used to measure autophagic flux after m RFP-GFP-LC3 adenovirus transfection.Compared with the control group,the autophagic flux was enhanced significantly in IH+sh RNA group;Compared with the IH group,the autophagic flux was increased dramatically in IH+sh RNA group;Compared with the IH+sh RNA group,the autophagic flux was reduced obviously in IH+NC group,IH+Egr-1 and IH+sh RNA+3-MA group.6.In the detection of myocardial apoptosis,compared with the control group,the results of the expression of cleaved caspase 3 protein and flow cytometry showed that the apoptosis was increased significantly in IH group,IH+NC group,IH+Egr-1 and IH+sh RNA+3-MA group;Compared with the IH group,the apoptosis was reduced dramatically in IH+sh RNA group;Compared with the IH+sh RNA group,the apoptosis was increased obviously in IH+NC group,IH+Egr-1 and IH+sh RNA+3-MA group.Conclution:1.Egr-1/Bim/Beclin-1 signal pathway is involved in and activated after exposed to IH.2.Egr-1/Bim/Beclin-1 signaling pathway is involved in IH induced cardiomyocyte injury by regulating cardiomyocyte autophagy.