| Backgrounds:Cardiovascular disease is the leading cause of human "life loss",accounting for more than 44%of the total number of deaths in China.Despite great progress in clinical drugs and interventional therapy,myocardial infarction is still the main cause of cardiovascular disease onset and death.It is an important way to reduce the morbidity and mortality of cardiovascular diseases to pay attention to the early prevention,clinical treatment and later rehabilitation of cardiovascular diseases.Literature has shown that appropriate exercise can effectively prevent cardiovascular diseases and play an important role in cardiac rehabilitation of myocardial infarction,but the mechanism of rehabilitation has not been fully elucidated.The study on exercise and myocardial infarction cardiac rehabilitation and its mechanism will provide theoretical and experimental basis for screening the means and methods of ischemic heart exercise rehabilitation and their targets in the context of integration of sports and medicine.Intermittent hypoxic adaptation can significantly increase cardiac hypoxia tolerance and alleviate various clinical manifestations brought by myocardial ischemia injury.Intermittent hypoxia and exercise,as natural non-drug cardiac rehabilitation methods,have the advantages of simple,easy to apply and without obvious side effects.However,there has been no report on the comparative study in improving the cardiac function of myocardial infarction and mechanism of action.miRNAs play an important role in ischemic heart protection.In this paper,7 miRNAs related to exercise,myocardial infarction and angiogenesis have been screened by the literature method,including pro-angiogenesis miR-126,miR-130a,miR-17-5p and anti-angiogenesis miR-15b,miR-16,miR-221 and miR-222.However,there is a lack of experimental studies on the effects of these miRNAs on exercise,myocardial angiogenesis and cardiac function improvement in myocardial infarction.Under hypoxic conditions,HIF-1? regulates the expression of several genes,including miRNA,and exerts angiogenic effect.Exercise can significantly increase the expression of HIF-la in ischemic heart,but the role of HIF-la and miRNA in exercise-induced angiogenesis in myocardial infarction and its mechanism have not been reported yet.Objectives:(1)To investigate the expression of myocardial HIF-la and miR-126 after exercise and intermittent hypoxia intervention in myocardial infarction rats.(2)To reveal the role and main mechanism of HIF-la and miR-126 in promoting myocardial angiogenesis and improving myocardial infarction function by exercise.Methods:(1)In vivo experiments:3-month-old SD rats(purchased from the Experimental Animal Management Center of Xi 'anJiaotong University),weighing 180-220g,were prepared for myocardial infarction model by ligation of the left anterior descending coronary artery.The sham group was only thread threading without ligation.One week after the operation,exercise,hypoxia or drug injection intervention was performed.In the second part,rats were randomly divided into sham operation group(S),myocardial infarction group(MI),MI+interval exercise group(MIE)and MI+continuous exercise group(MCE).In the three part:MI+intermittent hypoxia group(MIH)was added;In the fourth part,MIE+2ME2 group(MIE+2ME2)and MIE+PBS group(MIE+PBS)were added.Cardiac function was measured the next day after training.Blood was taken from the abdominal aorta,and the head was cut off and sacrificed.(2)In vitro experiments:In the thrid part,human umbilical vein endothelial cells(HUVEC)were divided into 9 groups:Normoxia group,hypoxic group for 1h,hypoxic group for 2h,hypoxic group for 3h,hypoxic group for 4h,hypoxic group for 6h,hypoxic group for 8h,hypoxic group for 12h,and hypoxic group for 24h.In the fourth part:HUVEC cells were divided into 6 groups:exogenous intervention group,Normoxia control group,Normoxia+2ME2 group,Normoxia+DMOG group,Hypoxia control group,Hypoxia+2ME2 group,Hypoxia+DMOG group.Endogenous intervention:Normoxia control group,hypoxia control group,Hypoxia+miR-126 mimics group,Hypoxia+miR-126 inhibitor group,Hypoxia+mimics control group,Hypoxia+mimics control group and Hypoxia+inhibitor control group.The 293T promoter cells are divided into four groups,the PGL4.1-basic group,miR-126-promoter+DMOG group,miR-126-promoter+ HIF-l? group,and miR-126-promoter+HIF-1?+DMOG group.(3)Test indicators:The cardiac function of rats was measured by carotid artery cannula;Masson staining was used to analyze the volume fraction of myocardial collagen(CFV).Expression of CD31 and ?-SMA were observed by immunohistochemistry.Immunofluorescence was used to observe PCNA+/vWF+positive particles.Expressions of miR-126,miR-130a,miR-17-5p,miR-15b?miR-16,miR-221,miR-222,egfl7?pik3r2 and spred1 were detected by RT-QPCR,and expressions of HIF-1?,PIK3R2,SPRES1,VEGF,P-PI3K/PI3K,P-Akt/Akt,P-eNOS/eNOS,Raf-1 and P-ERK/ERK were detected by Western Blotting.CCK-8 was used to detect cell activity.Fluorescence intensity test verified cell transfection efficiency.Cell scratch and tube formation were used to detect the ability of cell migration and differentiation and tubule formation.The dual luciferase reporter gene detected the binding of transcription factor and promoter.Results:(1)The expression of miR-222 was significantly upregulated(P<0.05)and the expression of miR-126 was significantly downregulated(P<0.05)in MI rats.After exercise intervention,the expressions of miR-126 and miR-17-5p in MI rats were significantly upregulated(P<0.05,P<0.01),among which the expression changes of miR-126 were the most significant,while the expressions of miR-15b,miR-16,miR-221,miR-222 and miR-130a showed no significant changes.(2)Exercise significantly downregulate the expression of pik3r2 mRNA and PIK3R2 protein of miR-126 target genes in MI rats(P<0.05,P<0.01),and downregulate the expression of spredl mRNA and SPRED1 protein(P<0.01,P<0.05)?and the inhibitory effect of IET was better than CET.(3)IET significantly upregulated the expression of host gene EGFL7 of miR-126 in MI rats(P<0.01),and egfl7 mRNA was closely related to the expression changes of miR-126(R=0.65,P<0.05).(4)CD31 was significantly increased in MI group compared with S group(P<0.05).Compared with MI group,CD31 in MIE group was significantly increased(P<0.05).The protein expression of VEGF was significantly upregulated in MI group compared with S group(P<0.05),and in MIE group(P<0.01)compared with MI group.These results indicated that compensatory angiogenesis occurred in the myocardial infarcted border zones after MI,and that exercise promoted angiogenesis in myocardial infarcted border zones,and IET was better.(5)?-SMA was significantly increased in MIE group compared with MI group(P<0.05).The results showed that IET reversed the phenotype of VSMC from synplastic to contractile in myocardial infarcted border zones.(6)Compared with MI group,LVSP was significantly increased in MIE group and MCE group(P<0.05),and LVEDP was significantly decreased(P<0.05).CVF in MIE group was significantly decreased compared with MI group(P<0.01).The results showed that IET inhibited the excessive proliferation of collagen fibers in the myocardial infarcted border zones of MI,significantly improved the cardiac performance,and the effect was superior to CET.(7)Compared with MIH group,HW,HW/TL and LVEDP were significantly reduced in MIE group(P<0.01,P<0.01,P<0.05),PCNA+/vWF+particle number and LVSP were significantly increased(P<0.01,P<0.05),and the expressions of HIF-1?and miR-126 in myocardium were significantly upregulated(P<0.01).The results showed that IET was more effective than IH in promoting myocardial angiogenesis and improving cardiac performance.These improvements may be related to the change expression of HIF-1? and miR-126.(8)In vitro HUVEC cell assay showed that the expression of HIF-1? increased at the beginning and then decreased with the prolongation of hypoxia,and showed a peak at hypoxia 2h.The expression of miR-126 was highly consistent with that of HIF-1?.This suggests that HIF-1 is likely upstream of miR-126.(9)Compared with MIE group,CVF and LVEDP in MIE+HIF-1? inhibitor(2ME2)group were significantly increased(P<0.05),PCNA+/vWF+particle number and LVSP were significantly decreased(P<0.01,P<0.05),the expressions of HIF-la and miR-126 were significantly downregulated(P<0.01),and the protein expressions of PIK3R2 and SPRED1 were significantly upregulated(P<0.01).In animals experiment,HIF-la has been shown to inhibit miR-126 expression and promote upregulation of its target protein,and to play an important role in myocardial fibrosis,angiogenesis and improvement of cardiac function in MI.(10)Bioinformatics analysis showed that HIF-la binding sites existed in the upstream promoter region of miR-126.The dual luciferase promoter results show that the fluorescence intensity of the miR-126-promoter+HIF-1? and the miR-126-promoter+HIF-1?+DMOG group is significantly higher than that of the miR-126-promoter+DMOG group(P<0.01).In vitro HUVEC cell assay showed that DMOG significantly upregulated the expression of HIF-1? and miR-126 in a concentration dependent manner,and 2ME2 also significantly downregulated HIF-1? and miR-126 in a concentration dependent manner,compared with the control group(P<0.05).It was shown that HIF-la can bind to the upstream promoter region of miR-126 and promote its transcriptional expression.(11)Compared with mimics control group,tube formation length of miR-126 mimics group was significantly higher(P<0.01).Compared with the inhibitor control group,tube formation length of miR-126 inhibitor group was significantly reduced(P<0.05).Compared with mimics control group,the mobility of miR-126 mimics group was significantly increased(P<0.01).Compared with the inhibitor control group,the mobility of miR-126 inhibitor group was significantly reduced(P<0.05).These results suggest that miR-126 plays an important role in hypoxia-induced tubule formation and cell migration in HUVEC cells.(12)Compared with the mimics control group,the protein expression of PIK3R2 and phosphorylation of PI3K,Akt and eNOS in the miR-126 mimics group were significantly decreased(P<0.01).Compared with the inhibitor control group,the protein expression of PIK3R2 in the miR-126 inhibitor group was significantly increased(P<0.01),and the phosphorylation levels of PI3K,Akt and eNOS were significantly decreased(P<0.01),indicating that miR-126 may activate the PI3K/Akt/eNOS angiogenesis signaling pathway by downregulating the expression of PIK3R2 under hypoxia conditions.(13)Compared with the mimics control group,SPRED1 protein expression in the miR-126 mimics group was significantly decreased(P<0.01),Raf-1 protein expression was significantly increased(P<0.01),and ERK phosphorylation level was significantly increased(P<0.05).Compared with the inhibitor control group,the expression of SPRED1 protein in the miR-126 inhibitor group was significantly increased(P<0.01)and the phosphorylation level of ERK was significantly decreased(P<0.01).This suggests that miR-126 may activate the MAPK angiogenesis signaling pathway by downregulating SPRED1 expression under hypoxic conditions.Conclusion:(1)Intermittent exercise,continuous exercise and intermittent hypoxia can promote myocardial angiogenesis of myocardial infarction to different degrees and improve cardiac function of MI:The improvement effect of intermittent exercise was significantly better than that of continuous exercise and intermittent hypoxia.(2)Intermittent exercise significantly upregulated HIF-1?/miR-126 expression in myocardial infarction,down-regulated PIK3R2 and SPRED1 protein expression,increased PCNA+/vWF+particle number in myocardial infarction,and promoted cardiac repair,and this protective effect was inhibited by HIF-lainhibitor 2ME2.(3)HIF-1? can bind to the upstream promoter region of miR-126 and promote its transcriptional expression.miR-126 may activate the PI3K/Akt/eNOS and MAPK angiogenesis signaling pathways by downregulating the expression of its target protein PIK3R2/SPRED1,and play an important role in hypoxia-induced tubule formation and cell migration in HUVEC cells. |
PDF Full Text Request