| Objective:Heart failure,as a clinical syndrome of decompensation of heart function at the terminal stage of many cardiovascular diseases,has become a major cause of death worldwide.Although some progress has been made in the diagnosis and treatment of heart failure,the mortality rate remains high.Therefore,it is critical to find effective therapeutic targets and interventions to improve cardiovascular function.Moderate aerobic exercise training has many benefits for the cardiovascular system.Proper exercise training can reduce the incidence of many cardiovascular diseases and improve ventricular function.Previous studies have shown that moderate exercise training can enhance the exercise tolerance of heart failure patients,improve the endothelial function,and effectively suppress the sympathetic nerve excitability of patients,and improve patients' cardiac output,improve left ventricular function and neural hormone levels,so that exercise training on heart failure has certain prevention function,but the molecular mechanism of aerobic exercise to prevent heart failure is unclear.Therefore,in this study,through four weeks of aerobic exercise(in vivo experiment)combined with angiotensin intervention in myocardial cell lines(in vitro experiment),we explored the effect of aerobic exercise on the reduction of pathological cardiac hypertrophic through AMPK related signaling pathways and the molecular mechanism,providing a solid theoretical basis for the reduction of heart failure.Methods:1?Transverse aortic constriction was used for transverse aortic constriction of C57BL/6J mice and AMPK?2 knocked out mice,respectively.TAC to establish the pathological cardiac hypertrophy model,one week after the operation,exercise pre-conditioning for one week,aerobic exercise intervention for four weeks.Mouse ultrasound instrument was used to evaluate the heart function of mice.Masson staining was used to analyze Sirius red staining,and polarized light was used to observe and evaluate the myocardial fibrosis status of mice.2?In vitro experiment,the H9C2 cardiac muscle cell line was used in this research group,and Angiotensin ? was used after passage to establish a hypertrophic model of cardiomyocytes.NaHS were used to intervene hypertrophic cardiomyocytes,AMPK inhibitors(Compoundc dihydrochloride,CC)and autophagy inhibitors(3-methyladenine,3-MA)intervention were performed.After the intervention experiment,WGA immunofluorescence assay was used to evaluate the size of myocardial cells,and Western blot was used to detect the pro-fibrosis signaling pathway(NLRP3/IL-1?)and AMPK-mediated autophagy key proteins(p-AMPK?/AMPK?,p-mTOR/t-mTOR,LC3II/I,Beclin 1 and p62).Results1st part:Effect of aerobic exercise on pathological cardiac hypertrophy induced by TAC in C57BL/6J mice1)Compared with Sham group,TAC significantly increased heart MASS(HW)left ventricular MASS(LV MASS)left ventricular weight/weight ratio(LV/BW)left ventricular weight/tibia length ratio(LV/TL)and heart MASS/tibia length ratio(HW/BW)in C57BL/6J miceFive weeks of exercise training significantly reduced heart MASS(HW)left ventricular MASS(LV MASS)left ventricular weight/weight ratio(LV/BW)left ventricular weight/tibia length ratio(LV/TL)in TAC-induced C57BL/6J mice compared with TAC group.Increased the ratio of heart weight to body weight(HW/BW)and heart weight to tibia length(HW/TL),while there were no significant differences in the mass and tibia length of C57BL/6J mice between four groups.2)Compared with Sham group,left ventricular end-diastolic diameter(LVEDD)and left ventricular end-systolic diameter(LVESD)were significantly increased in TAC group,while shortener fraction(FS)and ejection fraction(EF)were significantly decreasedCompared with Sham+EX group,the left ventricular end-diastolic diameter(LVEDD)and end-systolic diameter(LVESD)of mice in TAC+EX group were increased,while the shorting fraction(FS)and ejection fraction(EF)were significantly decreasedCompared with TAC group,the left ventricular end-diastolic diameter(LVEDD)and left ventricular end-systolic diameter(LVESD)of mice in TAC+EX group were significantly decreased,while the shorting fraction(FS)and ejection fraction(EF)were significantly increased.3)Compared with the Sham group,the cross-sectional area of myocardial cells in the TAC group was significantly increased.The cross-sectional area of myocardial cells in the TAC+Ex group was larger than that in the Sham+Ex group,but the cross-sectional area of myocardial cells in the TAC+Ex group was significantly decreased compared with that in the TAC group.4)ANP protein was also significantly increased in the TAC+EX group compared with the SHAM+EX group,but was significantly decreased in the TAC+EX group after four weeks of aerobic exercise compared with the TAC group.5)Compared with Sham group,the myocardial collagen fibers of C57BL/6J mice in TAC group were significantly increased(P<0.01).Compared with Sham+Ex group,the myocardial collagen fibers of TAC+Ex group were also significantly increased(P<0.01),but the myocardial collagen fibers of TAC+Ex group were significantly decreased compared with TAC group(P<0.01).Compared with Sham group,the levels of type ? collagen and type ? collagen in TAC group were significantly increased(P<0.01),while the levels of type ? collagen in TAC+EX group were significantly higher than those in Sham+EX group(P<0.05),and there was no significant difference in the levels of type ? collagen between the two groups.However,the levels of type ? collagen and type ? collagen in TAC+EX group were significantly lower than those in TAC group(P<0.01),MMP-9 protein levels in TAC group were significantly higher than those in Sham group(P<0.01).Compared with TAC group,MMP-9 protein levels were significantly lower after four weeks of aerobic exercise training(P<0.01).6)Compared with Sham group,TGF-? Smad2/3 phosphorylation and Smad4 protein expression were significantly increased in TAC group(P<0.01),while TGF-?Smad2/3 phosphorylation and Smad4 protein expression were significantly decreased in TAC+EX group compared with TAC mice after four weeks of aerobic exercise training.7)Compared with Sham group,the protein levels of NLRP3,c1.caspase-1 and IL-1?in TAC group were significantly increased(P<0.01),while the protein levels of Pro.caspase-1 were also increased,but there was no significant difference.Compared with Sham+EX group,the protein levels of c1.caspase-1 in TAC+EX group were significantly increased(P<0.01),and the protein levels of NLRP3,Pro.caspase-1 and IL-1? were also increased,but there was no significant difference.Compared with TAC group,the protein levels of NLRP3(P<0.01),cl.caspase-1(P<0.01)and IL-1?(P<0.05)were significantly decreased in TAC+EX group,while the protein levels of Pro.caspasse-1 were decreased,but there was no significant difference.8)Compared with the Sham group,TAC significantly decreased the expression ratio of LC3?/? protein and the expression level of Beclin-1 protein,and increased the expression level of p62 protein,indicating that stress load overload inhibited the increase of autophagy compared with the TAC group.LC3?/? protein expression ratio and Beclin-1 protein expression level were increased in TAC+Ex group,while the protein expression of p62 was decreased.The expression rates of p-AMPK?/AMPK? and p-mTOR/t-mTOR were significantly increased in TAC group compared with Sham group.Compared with TAC group,the expression rate of p-AMPK?/AMPK? was significantly increased in TAC+EX group,while the expression rate of p-mTOR/t-mTOR was significantly decreased.9)Compared with Sham group,the protein expressions of CBS(P<0.05)and CSE(P<0.01)in the left ventricular tissue of TAC group were significantly decreased,and the protein levels of CBS(P<0.05)and CSE(P<0.01)in TAC+EX group were significantly increased compared with TAC group after four weeks of exercise training.In addition,the endogenous H2S content of C57BL/6J mice induced by TAC was significantly lower than that of Sham group,while aerobic exercise significantly increased the endogenous H2S content of C57BL/6J mice induced by TAC.2nd part:AMPKa2 gene knockout blocks the effect of aerobic exercise on pathological cardiac hypertrophy1)Compared with KO+SHAM group,the ratio of heart MASS(HW)and left ventricular MASS(LV MASS)to left ventricular weight/body weight(LV/BW)to left ventricular weight/tibia length(LV/TL)in KO+TAC group was significantly higher than that in KO+SHAM group.Heart MASS/weight ratio(HW/BW)and heart MASS/tibia length ratio(HW/TL)were significantly increased in AMPK?2-/-mice induced by TAC compared with KO+SHAM+EX.Heart MASS(HW)and left ventricular MASS/weight ratio(LV/BW)were significantly increased in AMPK?2-/-mice induced by TAC compared with KO+SHAM+EX.Left ventricular weight/tibia length ratio(LV/TL),heart weight/weight ratio(HW/BW)and heart MASS/tibia length ratio(HW/TL)were significantly increased.Heart MASS(HW)and left ventricular MASS(LV MASS)of AMPK?2-/-mice in KO+TAC+EX group compared to KO+TAC group.Left ventricular weight/body weight ratio(LV/BW)left ventricular weight/tibia length ratio(LV/TL)heart weight/body weight ratio(HW/BW)and heart mass/tibia length ratio(HW/TL)did not significantly reduce the mass and tibia length of AMPK?2-/-mice.2)Compared with KO+SHAM group,left ventricular end-diastolic diameter(LVEDD)and left ventricular end-systolic diameter(LVESD)in KO+TAC group were significantly increased,while shorting fraction(FS)and ejection fraction(EF)were significantly decreased.Compared with KO+SHAM+EX group,left ventricular end-diastolic diameter(LVEDD)and end-systolic diameter in KO+TAC+EX group were significantly increased,while shorting fraction(FS)and ejection fraction(EF)were significantly decreased.Compared with KO+TAC group,left ventricular end-diastolic diameter(LVEDD)and left ventricular end-systolic diameter(LVESD)were not significantly decreased in KO+TAC+EX group,while the shortened fraction(FS)and ejection fraction(EF)were not significantly increased in KO+TAC+EX group.3)Compared with KO+Sham group,the cross-sectional area of myocardial cells in KO+TAC group was significantly increased.The cross-sectional area of myocardial cells in KO+TAC+EX group was larger than that in KO+SHAM+EX group.The myocardial cross-sectional area of mice in KO+TAC+EX group was not significantly reduced compared with that in KO+TAC group.4)ANP protein was significantly increased in KO+TAC group compared with KO+SHAM group,and significantly increased in KO+TAC+EX group compared with KO+SHAM+EX group.However,ANP protein level was not significantly decreased in KO+TAC+EX group compared with KO+SHAM+TAC group after four weeks of aerobic exercise.5)Compared with KO+SHAM group,the myocardial collagen fibers of AMPK?2-/-mice were significantly increased in KO+TAC+EX group compared with KO+SHAM+EX group,but there was no significant decrease in the myocardial collagen fibers of KO+TAC+EX group compared with KO+SHAM+EX group.Compared with KO+SHAM group,the levels of type ? and ? collagen in KO+TAC group were significantly increased,while the levels of type ? and ?collagen in KO+TAC+EX group were significantly higher than that in KO+SHAM+EX group.However,the levels of type ? and ? collagen in KO+TAC+EX group were not significantly higher than those in KO+SHAM+EX group.The MMP-9 protein level was significantly increased in the KO+TAC group.Compared with the KO+TAC group,the MMP-9 protein level was not significantly decreased after four weeks of aerobic training.6)Compared with KO+SHAM group,TGF-?/Smad2/3 phosphorylation and Smad4 protein expression were significantly increased in KO+TAC group,while TGF-?Smad2/3 phosphorylation and Smad4 protein expression were not significantly decreased in KO+TAC+EX group compared with KO+TAC mice after four weeks of aerobic exercise training.7)Compared with KO+SHAM group,the protein levels of NLRP3,c1.caspase-1,IL-1? were significantly increased in KO+TAC group,while the protein levels of Pro.caspasse-1 were also increased,but there was no significant difference.Compared with KO+SHAM+EX group,the protein levels of NLRP3,Pro.caspase-1,IL-1? in KO+TAC+EX group were significantly increased,and the protein levels of NLRP3,C1.caspase-1,IL-1? in KO+TAC+EX group were not significantly decreased compared with KO+TAC group.8)Compared with KO+SHAM group,KO+TAC significantly decreased LC3?/?protein expression ratio and Beclin-1 protein expression level,increased p62 protein expression level compared with KO+TAC group.LC3?/? protein expression ratio and Beclin-1 protein expression level in KO+TAC+EX group were not significantly increased,and the protein expression of p62 was not decreased.Compared with KO+SHAM group,the p-mTOR/t-mTOR protein expression rate in KO+TAC group was significantly increased.Compared with KO+TAC group,p-mTOR/t-mTOR protein expression rate was not significantly decreased in KO+TAC+EX group.9)Compared with KO+SHAM group,after four weeks of exercise training,CBS(P<0.01)and CSE(P<0.05)protein levels in KO+TAC+EX group were higher than those in KO+TAC group.In addition,compared with KO+SHAM group,the endogenous H2S content in KO+TAC group was significantly decreased,while aerobic exercise significantly increased the endogenous H2S content in the myocardium of AMPK?2 mice induced by TAC.3rd part:Study on the role and mechanism of H2S in pathological cardiac hypertrophy1)Compared with the control group,the area of cardiomyocytes induced by Ang?was significantly increased,while the hypertrophy area of cardiomyocytes was significantly reduced by the intervention of NaHS compared with the Ang? group.Compared with the control group,the expression ratio of LC3?/? protein and Beclin-1 protein were decreased,and the expression of p62 protein was increased(P<0.01).Compared with the Ang? group,exogenous H2S increased the expression rate of LC3II/I protein,increased the expression of Beclin-1 protein,while the expression of p62 protein was decreased.Compared with the control group,the expression rates of p-AMPK/AMPK and p-mTOR/mTOR were increased in the Ang? group.Compared with the Ang? group,the expression rates of p-AMPK/AMPK were further increased and the expression rates of p-mTOR/mTOR were decreased by H2S interventionIn contrast to control,the levels of NLRP3,c1.caspase-1,IL-1? protein were significantly increased in Ang?group(P<0.01),while there was no significant difference in Pro.caspase-1.Compared with Ang? group,exogenous H2S decreased NLRP3(P<0.01),c1.caspasse-1,IL-1? protein levels,and Pro.caspasse-1 protein levels were decreased,but there was no significant difference.2)Compared with the control group,the protein expression ratio of LC3II/I and the protein expression level of Beclin-1 were decreased in the Ang? group,while the protein expression of p62 was significantly increased.Compared with Ang?group,exogenous NaHS solution significantly increased the protein expression ratio of LC3II/I and the protein expression level of Beclin-1,while decreased the protein expression level of p62 as an autophagy inhibitor,3-MA can dose-dependently decrease the protein expression ratio of LC3?/? and Beclin-1 protein expression level,and meanwhile increase the protein level of P62,compared with NaHS+Ang?.Compared with the control group,the area of cardiomyocytes induced by Ang? increased significantly.Compared with the Ang? group,the hypertrophic area of cardiomyocytes significantly decreased after the intervention of NaHS+Ang? group.After the intervention of autophagy inhibitor,the reduction effect of exogenous hydrogen sulfide on hypertrophic cardiomyocytes was eliminated compared with the control group.The levels of NLRP3,c1.caspase-1,IL-1? protein were significantly increased in Ang? group,but there was no significant difference in Pro.caspase-1 protein.Compared with Ang? group,exogenous H2S decreased the protein levels of NLRP3,c1.caspase-1,IL-1?,and Pro.Caspasse-1,but there was no significant difference.Compared with the NaHS+Ang? group,the protein levels of NLRP3,C1.caspase-1,IL-1?were significantly increased in the 3-MA+NaHS+Ang? group.3)Compared with the control group,the expression rates of p-AMPK/AMPK and p-mTOR/mTOR increased in the Ang? group.Compared with the Ang? group,exogenous H2S intervention further increased the expression rates of p-AMPK/AMPK,while decreased the expression rates of p-mTOR/mTOR as an inhibitor of AMPK,CC can decrease the protein expression ratio of p-AMPK/AMPK in a concentration-dependent manner,and meanwhile increase the expression rate of p-mTOR/mTOR,indicating that 10UMCC can significantly inhibit the activity of AMPK protein compared with NaHS+Ang?.Compared with the control group,the area of cardiomyocytes induced by Ang? increased significantly,and compared with the Ang? group,the hypertrophic area of cardiomyocytes significantly decreased after the intervention of NaHS and Ang? group,and the reduction effect of exogenous hydrogen sulfide on hypertrophic cardiomyocytes was eliminated after the intervention of AMPK inhibitor.Compared with the control group,the protein expression ratio of LC3?/? and the protein expression level of Beclin-1 were decreased in the Ang? group,while the protein expression of p62 was significantly increased.Compared with Ang? group,exogenous NaHS solution significantly increased the protein expression ratio of LC3?/? and Beclin-1 protein expression level,and decreased the protein expression level of p62 compared with NaHS+Ang? group,CC+3-MANAHS+Ang? groupLC3?/?protein expression ratio and Beclin-1 protein expression level decreased significantly,while increasing P62 protein level increased compared with control.Compared with Ang? group,exogenous H2S decreased NLRP3(P<0.05)and NLRP3(P<0.05),while NLRP3,c1.caspase-1 and IL-1? protein levels in Ang?group increased significantly,while Pro.caspase-1 showed no significant difference.Compared with NaHS+Ang? group,NLRP3,c1.caspase-1,IL-1?protein levels in CC+NaHS+AngII group were significantly increased.Conclusion:1.This study confirmed that TAC induced stress overload in C57BL/6J mice resulted in increased heart and left ventricular weight,increased cross-sectional area of cardiomyocytes,accompanied by myocardial tissue fibrosis,and decreased systolic and diastolic functions of the heart in miceFive weeks of aerobic exercise intervention can improve the ejection fraction of C57BL/6J mice with heart failure induced by pressure overload,improve cardiac dysfunction,inhibit cardiomyocyte hypertrophy and reduce myocardial fibrosis,to a certain extent,prevent the development of heart failure in C57BL/6J mice,and thus play a certain protective role in the heart tissue of C57BL/6J mice.2.AMPK?2 knockout reduces TAC-induced myocardial injury,which may be due to the fact that AMPK?2 knockout blocks the activation of myocardial autophagy by aerobic exercise,and thus does not inhibit the NLRP3/IL-1? protein signaling pathwayAMPK?2 gene knockout unblocked aerobic exercise intervention increases endogenous H2S production in cardiac tissue.3.Exogenous H2S activates AMPK,reduces the active expression of mTOR,activates autophagy of cardiomyocytes,inhibates the expression of NLRP3/IL-1?signaling pathway protein level,and inhibates the myocardial hypertrophy induced by Ang?,thus mitigating myocardial hypertrophy.These results suggest that aerobic exercise prevents pathological hypertrophy mediated by AMPK?2 by increasing endogenous H2S production in cardiac tissue,activating autophagy,and inhibiting the activation of the NLRP3/IL-1? protein signaling pathway. |
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