| Cardiovascular disease is a kind of diseases with the highest morbidity and mortality in the world.Chronic heart failure refers to a heart disease in which the heart cannot adequately pump blood flow sufficient to maintain the metabolic needs of peripheral tissues.Heart failure is usually caused by ischemic heart disease,hypertension or valvular heart disease.Heart failure caused by hypertension and ischemic heart disease can only be slowed by treatments such as drugs,and cannot be reversed.As a result,heart failure has a high incidence,high medical expenditures and high mortality rates.It has been well established that exercise training not only reduces risk factors of cardiovascular diseases but also provides direct endogenous cardiovascular protection.The occurrence and development of heart failure are accompanied by oxidative stress and apoptosis,and miRNAs have also been proved to regulate important pathological processes in cardiovascular diseases such as myocardial infarction.The effects of exercise in preventing the development and progression of heart failure have been extensively studied,but the specific mechanisms by which exercise improves oxidative stress and regulates miRNAs are still unclear.In this study,we focused on the effects of exercise on the two important factors of heart failure--hypertension and ischemic heart disease--and their mechanisms.Starting from the two major factors affecting heart failure--oxidative stress and miRNA,We plan to provide new prevention and treatment strategies for the development of heart failure.Aims:(1)Aerobic exercise was performed in spontaneously hypertensive rats,and the effect and mechanism of exercise on oxidative stress in heart protection of hypertension were studied.(2)Aerobic exercise was performed in mice with myocardial infarction,and the effect and mechanism of exercise on miRNA in heart protection of myocardial infarction were explored.Methods:1.The spontaneous hypertensive rats and WKY rats were subjected to moderate-intensity exercise for 16 weeks and clarify the effect and mechanism of exercise on the cardiac protection of hypertensive rats.(1)Experimental animals and groups:8-week-old male spontaneously hypertensive rats and WKY rats in the control group were purchased from Weitong Lihua(China)and kept in the Animal Center of the Fourth Military Medical University of the Chinese People's Liberation Army.SHR and WKY rats were divided into two groups:exercise group(SHR+Exe group,WKY+Exe group)and sedentary group(SHR+Sed group,WKY+Sed group),with 8 rats in each group.The rats in the exercise group ran on a motor driven treadmill for 1 h,5d/wk,and 16wk at a speed of 20m/min every day.The rats in the sedentary group did not do any exercise.(2)Detection of blood pressure,hemodynamics and mesenteric vascular function:Tail arterial pressure was used to detect rat blood pressure every four weeks.At 24 h after the last exercise,the hemodynamics of the rats were measured by using a multi-channel physiologic recorder.The mesenteric arteries of rats were isolated and the endothelial-dependent and non-endothelial-dependent diastolic responses of the blood vessels were observed by adding acetylcholine(ACh)and sodium nitrate(SNP)at diferent concentrations.(3)Detection of myocardial mitochondria ultrastructure,ROS level,glutathione content and SOD2 activity:Transmission electron microscope was used to observe the treated myocardial samples,and Gatan digital microscope was used to take microphotographs of myocardial mitochondria.The oxidation sensitive fluorescent dye dihydroethyl(DHE)was used to evaluate ROS content in situ.Glutathione(GSH)and oxidized glutathione(GSSG)were determined using specialized kits.SOD2 activity was detected by water soluble tetrazolium salt method.(4)Detection of Grx-1 in mesenteric vessels,myocardial SIRT3,and SOD2 protein levels:Conventional western blot was used to detect Grx-1 in mesenteric vessels,myocardial SIRT3,and SOD2 protein levels.2.The myocardial infarction model of mice was established,and swimming exercise was performed for 4 weeks to study the cardiac protective effect and mechanism of exercise on myocardial infarction.(1)Establishment of experimental animal model and exercise intervention:Male C57/BL6 mice(8 weeks old)were purchased from the Experimental Animal Center of the Fourth Military Medical University of the People's Liberation Army of China.All surgical procedures and animal care protocols are approved by the Animal Care and Use Committee.The myocardial infarction model was prepared by ligation of the anterior descending branch of the left coronary artery.24h after surgery,the exercise group mice were given adaptive swimming training for 5min every day,which was gradually increased to 60min in the following week,and the exercise duration was 4wk,5d/wk.(2)Detection of cardiac function and apoptosis of myocardial tissue:Miller's catheter was used to obtain hemodynamic data,and small animal cardiac ultrasound was used to determine the morphology and function of the heart in mice.Apoptosis of myocardial cells was evaluated by TUNEL staining.(3)Determination of miRNAs differential expression in myocardial tissue and detection of specific miRNAs levels:Illumina Hiseq 2500 platform was used for miRNA high-throughput sequencing to analyze miRNAs differential expression in myocardial tissue of mice in the exercise group and the quiet group;Real-time quantitative PCR(QRT-PCR)was used to detect the level of specific mirnas in mouse myocardial tissue.(4)Up-regulation or down-regulation of specific miRNA levels at the in vivo and cell levels:In vivo validation was performed by intramocardial injection of Agomir and Antagomir of specific miRNA to simulate the up-regulation and down-regulation of specific miRNA.At the cell level,mimics of specific miRNA were used to further determine the target of specific target genes.(5)Identification of specific miRNA target genes:Dual luciferase reporter system and western blot technique were used to determine specific miRNA target genes.Results:1.16-week moderate intensity exercise improved cardiac function in spontaneously hypertensive rats through REDOX homeostasis and SIRT3/SOD2 signaling.(1)After 16 weeks of moderate intensity exercise,the diastolic and systolic blood pressures of the hypertensive rats were significantly reduced,while LVSP of the hypertensive rats was significantly decreased(p<0.01),+LVdp/dtmax(p<0.01)and-LVdp/dtmax(p<0.05)were significantly increased.This indicated that exercise significantly improved the systolic and diastolic functions of SHRs.(2)After 16 weeks of moderate intensity exercise,the endothelium dependent diastolic effect induced by ACh in the exercise group was significantly higher than that in the quiet group(p<0.01),and the non-endothelium dependent diastolic effect induced by SNP was not significantly decreased.It indicates that exercise can improve the vascular endothelial dysfunction caused by hypertension.(3)After 16 weeks of moderate intensity exercise,the disorder of mitochondrial arrangement,morphological changes and swelling of myocardial mitochondria in hypertensive rats were significantly improved,indicating that exercise could improve the damage of myocardial mitochondria caused by hypertension.16 weeks of moderate intensity exercise significantly reduced the ros levels in the myocardium of the control group and the hypertensive rats(p<0.01).This suggests that moderate exercise can significantly reduce the level of reactive oxygen species in myocardial tissue in both normal and spontaneously hypertensive rats.(4)16 weeks of moderate intensity exercise reduced reactive oxygen species level and increased Grx-1 expression in mesenteric vascular tissue,indicating that exercise could improve the REDOX state in mesenteric vascular tissue.In addition,the expression of GSH was increased and the content of GSSG was decreased,indicating that exercise could enhance the ability of eliminating reactive oxygen species by increasing the content of reducing glutathione in myocardial tissue.(5)Western blot analysis showed that SIRT3 protein levels were significantly increased in the SHR+Exe group compared with the WKY+Exe(p<0.0001)and SHR+Sed groups(p<0.0001).SOD2 protein levels were also elevated in the SHR+Exe group compared with the WKY+Exe(p<0.05)and SHR+Sed groups(p<0.001).SOD2 activity was also increased.This indicates that exercise can improve the cardiac function of spontaneous hypertension rats by upregitating SIRT3/SOD2 signaling pathway.2.4-week swimming exercise showed a protective effect on the heart after myocardial infarction by increasing circulating miR-1192.(1)4-week swimming exercise improved the post-MI survival rate.Compared with MI group,swimming exercise decreased the heart weight/body weight and lung weight/bodyweight.Furthermore,swimming exercise after MI increased dp/dtmax,and decreased LV end diastolic pressure.The left ventricular systolic function of the mice was also improved.These results indicated that swimming exercise protected the heart against MI in mice.(2)After 4 weeks swimming exercise,the proportion of cardiac apoptosis was reduced and the interstitial fibrosis was suppressed by 30.0%.Results from western blot showed that the expression of the anti-apoptotic protein Bcl-2 was upregulated in post-exercise MI mice.The apoptotic proteins,Bax and cleaved caspase 3,and the fibrosis protein,TGF-?1,were all downregulated after swim training compared with the non-exercise group.These results indicated that swim training after MI attenuated MI-induced(3)A total of 10 differentially expressed miRNAs(fold change>2.0;p<0.05)were identified using Illumina Hi Seq 2500 high-throughput sequencing and 5 of them upregulated after exercise were further confirmed by qRT-PCR analysis.All 5 upregulated miRNAs were evaluated in vitro using NRVMs.The mimics of these miRNAs have no significant impact on the survival and apoptosis of cardiomyocytes subjected to hypoxia except for miR-1192.miR-1192 mimic protected neonatal cardiomyocytes against hypoxia as evidenced by increased survival and decreased apoptosis.(4)Agomir of miR1192 was used by intramyocardially injection to upregulate the level of miRNA-1192.Agomir treatment increased LVFS and LVEF by 56.3%and 37.7%respectively,increased dp/dtmax by 31.97%,and decreased LV end diastolic pressure by 22.91%.Furthermore,the proportion of cardiac apoptosis was reduced(42.2%reduction versus MI group)and the interstitial fibrosis was suppressed by 40.2%.The expression of the anti-apoptotic protein Bcl-2 was upregulated in agomiR-1192-treated MI mice.The apoptotic proteins,Bax and cleaved caspase 3,and the fibrosis protein,TGF-?1,were all downregulated in agomiR-1192-treated MI mice.These results indicated that miR-1192 exerted cardioprotective effects against MI in vivo.(5)After antagomir administrated,miRNA level of miR-1192 was down-regulated.Index of cardiac function,including LVEF,LVFS,dp/dtmax and LVEDP showed substantial improvement after exercise but these were abolished by administration of miR-1192 antagomir.Further study showed that administration of miR-1192 antagomir abolished the anti-fibrotic effect of exercise after MI.Results of western blot also showed downregulation of Bcl-2 and upregulation of Bax,cleaved caspase 3 and TGF-?1 after administration of miR-1192 antagomir.These results suggested that inhibition of miR-1192 abolished the cardiac pro-tective effect of exercise in post-ischemic heart and indicated that exercise exerted cardioprotection against MI via upregulation of miR-1192.(6)Target genes of mo-miR-1192 were predicted and functionally classified by the DAVID gene functional classification tool.The miR-1192 mimic decreased caspase 3 mRNA levels,while mRNA levels of Caspase 9 and CREB1 showed no difference.Protein levels in cultured cardiomyocytes detected by western blot showed the same trends consistent with the results of mRNA level,indicating caspase 3 as a potential target of miR-1192 in cardiomyocytes.The binding sites for miR-1192 in the 3'-untrans-lated regions(3'UTRs)of caspase3 were further examined using a luciferase reporter assay.Then the myocardial infarction was simulated by hypoxia cardiomyocytes and found that the expression level of Caspase 3 could be significantly changed by giving miR-1192 mimics.Conclusion:(1)Aerobic exercise improves the redox balance in the heart of spontaneously hypertensive rats by increasing the expression of SIRT3/SOD2.The enhancement of anti-oxidant defense ability caused by long-term exercise training improves mitochondrial function.(2)Aerobic exercise increases the circulating level of miR-1192,and miR-1192 mediates the protective effect of exercise on ischemic hearts by down-regulating the expression of Caspase 3.Inhibition of miR-1192 expression inhibits the protective effect of exercise.These findings indicates that miR-1192 is a new exerkine in exercise-induced cardioprotection and is expected to be used in treatment of ischemic hearts. |
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