| Background:Myocardial Infarction(MI)has become one of the main cardiovascular diseases that results in myocyte necrosis and apoptosis,and in alternative myocardial fibrosis,which severely impair cardiac function.In addition to effective clinical treatment,it is significative to improve cardiac function and improve the quality of life of patients with MI by using rehabilitation therapy in the later period.Studies have shown that exercise can significantly improve the cardiac function in MI,but the mechanism has not fully directed.It has been found that exercise significantly inhibited oxidative stress and inflammation in myocardial infarction,decreased apoptosis and improved cardiac function,but theaccurate mechanism still need to be futher studied.Insulin-like factor 1(IGF-1)is a well-known insulin-like peptide secreted mainly by the liver and skeletal muscle,which promotes cell differentiation and inhibits oxidative stress and inflammation-induced apoptosis.Exercise significantly increase the IGF-1 levels in skeletal muscle and myocardium,ultimately contributing to skeletal muscle and cardiac hypertrophy.Previous studies in our laboratory had found that the expression of IGF-1 is significantly higher after interval exercise than that of resistance exercise in the myocardium of rats with MI,and interval exercise has a significant effect on improving cardiac function,but its specific molecular mechanism has not been clarified.Studies had shown that SET and MYND domain-containing protein 1(Smyd1),which is the first Bacteriorhodopsin(Bop)gene specifically expressed in the heart and muscle,plays a key role in myomatogenesis and muscle fiber assembly during cardiomyocyte differentiation,maturation and heart formation.Exercise has a cardioprotective effect through the activation of the IGF-1-PI3K-Akt pathway,but it is still unclear whether Smyd1 is involved in exercise-stimulated endogenous IGF-1-induced cardiac structural and functional remodeling.Whether exercise can regulate the expression of Smyd1 and the sarcomere assembly of cardiomyocytes to achieve cardiac protection has not been reported,and it is worthy of further study.In this study,in order to investigate the role of IGF-1 in the improvement of cardiac function in MI by interval exercise and its molecular mechanism,we planned to use the intervention of Tempol(oxygen free radical scavenger),Etanercept(TNF-?inhibitor),NVP-AEW541(IGF-1R inhibitor),rhIGF-1,LY294002(PI3K inhibitor),Smyd1 interference and overexpression lentivirus vector to carry out the related experiments.Objective:(1)To investigate the effect of interval exercise on improving cardiac function of myocardial infarction function by inhibiting oxidative stress and inflammation induced myocardial apoptosis through IGF-1.(2)To reveal the molecular mechanism of whether interval exercise can improve myocardial function by activating IGF-1/IGF-1R-PI3K-Akt pathway,regulating the expression of Smydl and skeleton protein,inhibiting inflammation and cell apoptosis.Methods:1 Animal experiments:(1)3-month-old healthy male Sprague Dawley(SD)rats were selected to prepare myocardial infarction model and treated with interval exercise or intervention with Tempol and Etanercept.Interval exercise or Tempol/Etanercept interventions were administrated post-operation.Groups:normal control group(C),interval group(CE),sham operation group(S),myocardial infarction group(MI),MI+interval exercise group(ME),MI+Tempol group(MT),MI+Etanercept group(MIT),MI+saline injection group(MIS),ME+ Etanercept group(MET),ME+saline injection group(MES),10 rats per group.(2)8-week-old healthy male C57BL/6J mice were selected to make a model of MI,and then were treated with interval exercise or NVP-AEW541 intervention.Groups:sham operation group(S),S +exercise group(SE),myocardial infarction group(MI),MI+NVP-AEW541 group(MIN),MI+interval exercise group(ME)and ME+NVP-AEW541 group(MEN),10 rats per group.(3)MI model preparation:Rats and mice models of MI were prepared by left coronary artery(LAD)coronary artery ligation.Elevation of the dorsal arch of the S-T segment or inversion of the T-wave was detected by electrocardiogram,which was used as a sign of successful operation.In the S group,only thoracotomy was performed and suture was performed at the same position of the heart without ligation.(4)Intermission exercise program:our laboratory program was selected for rats.The ME group and the CE group started adaptive step-by-step platform running at 1wk after surgery.Formal training:CE and ME group warmed up at 10m/min(40%-5 0%V O2max)for 10min/d,and then alternated with 25m/min×7min(85%-90%VO2max)and 15m/min×3min(50%-60%VO2max)speed for 50min/d for 60min,5d/Wk×4wk,and the slope of the platform was 0°.According to the method reported in the literature,1wk after surgery,the mice of SE,ME and MEN group started adaptive incremental treadmill training.The formal training was:Warm up with 5M/min(40%-50%VO2max)for 10min/d,and then run alternately with 8M/min×3min(50%-60%VO2max)and 12M/min×7min(80%-90%VO2max)for 50min/d,60min/d,5d/wk×4wk at a gradient of 0°.2 Cell experiment:(1)Cell culture and intervention:H9c2 cells were treated with H2O2 intervention to prepare oxidative stress model,AICAR intervention to prepare exercise effect model,LPS intervention to prepare inflammation model,and exogenous rhIGF-1,LY294002,NVP-AEW541 intervention.Groups:Normal control group(C),H2O2 group(H),AICAR group(A),H2O2+AICAR group(HA),LPS group(L),LPS+AICAR group(LA),LPS+rhIGF-1 group(LR),LPS+AICAR+rhIGF-1 group(LAR),LPS+LY294002+rhIGF-1 group(LY),LPS+LY294002+rhIGF-1 group(LRY),LPS+LRY+LY294002+AICAR group(LAY),LPS+NVP-AEW541 group(LN),LPS+AICAR+NVP-AEW541 group(LAN),LPS+rhIGF-1+NVP-AEW541 group(LRN).(2)Cell culture and intervention:Exogenous rhIGF-1 and NVP-AEW541 were performed on H9c2 cells in oxidative stress and exercise effect model.Endogenous intervention H9c2 cells were transfected with Smyd1 interference and overexpressed viral vectors respectively,followed by H2O2,AICAR,rhIGF-1 or/and NVP-AEW541 intervention.Groups:Normal control group(C),H2O2 group(H),H2O2+AICAR group(HA),H2O2+NVP-AEW541 group(HN),H2O2+AICAR+ NVP-AEW541 group(HAN),H2O2+rhIGF-1+NVP-AEW541 group(HRN),H2O2+rhIGF-1+AICAR group(HRA),Scramble-shRNA group,Smyd1-shRNAgroup,Smyd1-shRNA+H2O2 group,Smyd1-shRNA+AICAR group,Smyd1-shRNA+ AICAR+H2O2 group,Smyd1-shRNA+rhIGF-1 group,Vehicle group,Smyd1 OE group,Smyd1 OE+H2O2 group,Smyd1 OE+AICAR group,Smyd1 OE+AICAR+H2O2 group,Smyd1 OE+rhIGF-1 group?Smyd1 OE+rhIGF-1+NVP-AEW541 group.3 Testing indexes of heart and cells:Common carotid artery intubation was used to detect hemodynamic indexes of rats to evaluate cardiac function.Cardiac function indexes of mice were detected by echocardiography.The percentage of myocardial collagen volume(CVF%)was observed and analyzed by Masson staining.Cross sectional area(CSA)of myocardial cells was observed and analyzed by HE staining.ROS content in cardiac cells was observed and analyzed by DCFH-DA staining.The apoptosis of myocardial cells,the positive expression of Smyd1,F-actin and ?-actinin were observed by immunofluorescence staining.The expressions of igf-1,smyd1,trx1,hsp90,murfl and mafbx were detected by RT-qPCR.IGF-1,IGF-1R,Smyd1,HSP90,MuRF1,MAFbx,p-PI3K/PI3K,p-Akt/Akt,p-NF-?B/NF-?B TNF-?,IL-1?,IL-6,IL-10,BNP,cTnI,?-actinin,Cleaved Caspase3,Bax,Bcl-2,p-AMPK/AMPK,F-actin and G-actin were detected by Western Blotting.The expression of T-SOD,MDA,GSH-Px,CAT and Chymotrypsin-like Proteasome in myocardium were detected by enzyme activity detection kit.CCK-8 was cultured in H9c2 cells to detect cell activity.Fluorescence intensity was used to verify cell transfection efficiency.Results:1 Interval exercise inhibited oxidative stress and upregulated endogenous the IGF-1/IGF-1R expression to reduce cell apoptosis(1)Interval exercise or Tempol intervention significantly upregulated the expression of IGF-1,IGF-1R and Smyd1 in the myocardium of S and MI rats(p<0.05,p<0.01)and p-AMPK/AMPK ratio(p<0.01).They can significantly increased cTnI,?-actinin expression(p<0.01)and F-actin assembly(p<0.05,p<0.01),increased the cross-sectional area of myocardial cells(p<0.01),and significantly decreased the expression of MuRF1 and HSP90(p<0.05,P<0.01).The expression of Smydl was positively correlated with AMPK phosphorylation and cross-sectional area of cardiomyocytes(p<0.01).Interval exercise was more effective than Tempol.(2)Interval exercise or Tempol intervention significantly increased the expression of T-SOD,CAT and GSH-Px in myocardium of MI rats(p<0.05,p<0.01),decreased the expression of MDA and Trx1(p<0.05,p<0.01),improved the level of oxidative stress in MI,and effectively activated PI3K/Akt pathway in MI(p<0.01).Smyd1 expression was positively correlated with T-SOD expression(p<0.01),and negatively correlated with MDA expression(p<0.01).Interval exercise was more effective than Tempol.(3)Interval exercise or Tempol intervention significantly decreased CVF%,LVDEP,apoptosis and BNP expression in MI rats(p<0.05,p<0.01),increased LVSP,ądp/dtmax and Bcl-2/Bax ratios(p<0.01).The expression of IGF-1 was positively correlated with the expression of Smyd1 and the cross-sectional area of cardiomyocytes(p<0.01).Smyd1 was positively correlated with LVSP and ądp/dtmax(p<0.01),was significantly negatively correlated with LVDEP(p<0.01).Interval exercise was more effective than Tempol.(4)H9c2 cells treated with H2O2 or/and AICAR significantly up-regulated the expression of IGF-1,IGF-1R and Smyd1(p<0.01),and increased the expression of cTnI,?-actinin and F-actin in cell volume and skeleton((p<0.05,p<0.01).AICAR significantly activated the PI3K/Akt pathway(p<0.05,p<0.01),decreased the expression of HSP90 and MuRF1 in H2O2-induced cells(p<0.05,p<0.01),decreased cell apoptosis,increased Bcl-2/Bax ratio and cell survival rate(p<0.05,p<0.01).2 Interval exercise inhibited inflammation and upregulated endogenous IGF-1/IGF-1R expression to improve cardiac function(1)Interval exercise or/and Etanercept effectively activated PI3K/Akt pathway in myocardium of MI rats(p<0.01),significantly upregulated the expression of IGF-1 and IGF-1R(p<0.01),and decreased the expression of TNF-?,IL-6,MuRF1,MAFbx and NF-?B(PP65)(p<0.05,p<0.01).The efficacy of interval exercise intervention was better than that of Etanercept intervention,and the efficacy of combined exercise and Etanercept intervention was better than that of single intervention.(2)Interval exercise or/and Etanercept significantly decreased CVF%,LVDEP,apoptosis,and Cleaved caspase 3 expression in MI rats(p<0.05,p<0.01),increased LVSP,ądp/dtmax and Bcl-2/Bax ratio(p<0.05,p<0.01).The efficacy of interval exercise intervention was better than that of Etanercept intervention,and the efficacy of combined exercise and Etanercept intervention was better than that of single intervention.(3)Interval exercise or/and Etanercept intervention significantly up-regulated the expression of Smyd1 and cytoskeleton,such as cTnI and ?-actinin in MI rats(p<0.01).Meanwhile,they can also decreased the expression of HSP90(p<0.05,p<0.01),increased the cross-sectional area of myocardial cells(p<0.01).The expression of MuRF1 was negatively correlated with Smydl,LVSP,ądp/dtmax(p<0.01),and it was significantly positively correlated with LVEDP(p<0.01),Smyd1 expression was positively correlated with LVSP and ądp/dtmax(p<0.01),was significantly negatively correlated with LVEDP(p<0.01).The efficacy of interval exercise intervention was better than that of Etanercept intervention,and the efficacy of combined exercise and Etanercept intervention was better than that of single intervention.(4)AICAR or/and rhiGF-1 could significantly increase the expression of IGF-1,IGF-1R and skeleton protein in LPS-induced H9c2 cells(p<0.01),effectively activated the PI3K/Akt signaling pathway(p<0.01),inhibited apoptosis and protein ubiquitination degradation(p<0.01),improved cell inflammatory state and cell survival rate(p<0.01).LY294002 intervention significantly inhibited PI3K/Akt pathway activation(p<0.01),inhibited the positive regulation of AICAR or rhIGF-1 on LPS-induced H9c2 cell state.LY294002 can also increased the expression of TNF-?and Cleaved caspase 3(p<0.05,p<0.01),decreased IL-10 expression and Bcl-2/Bax ratio(p<0.05,p<0.01)and promote cell apoptosis.3 Interval exercise regulates Smyd1 levels through IGF-1/IGF-1R to regulate sarcomere assembly(1)Interval exercise significantly activated PI3K/Akt pathway in MI mice(p<0.01),and increased EF%and FS%(p<0.01),and up-regulated the expression of IGF-1,IGF-1R,Smyd1,?-Actinin and Bcl-2/Bax ratio(p<0.05,p<0.01),decreased the expression of CVF%,LVIDd,LVIDs,MuRF1,Cleaved caspase 3(p<0.01),improve cardiac function and reduce cell apoptosis.(2)NVP-AEW541 significantly inhibited PI3K/Akt pathway activation in MI mice(p<0.01),decreased EF%,FS%,decreased the expression of myocardial IGF-1,IGF-1R,Smyd1,?-actinin and decreased the ratio of Bcl-2/Bax(p<0.01),and increased the expression of CVF%,LVIDd,LVIDs,MuRF1,Cleaved caspase 3(p<0.01),reduce cardiac function and promote cell apoptosis.(3)NVP-AEW541 intervention significantly increased AICAR or/and rhIGF-1 induced H2O2-induced H9c2 cell apoptosis(p<0.01),up-regulated Cleaved caspase 3 expression(p<0.01)and inhibited the expression of IGF-1,IGF-1R,Smyd1 and?-actinin(p<0.01),decreased the Bcl-2/Bax ratio and the rate of cell survival(p<0.01).(4)Smyd1-shRNA interference significantly reduced the expression of H9c2 cytoskeleton proteins and IGF-1/IGF-1R.H2O2 up-regulated the expression of H9c2 cytoskeleton protein and the expression of IGF-1 and IGF-1R transfected with Smyd1-shRNA.AICAR can further up-regulate the expression of H9c2 cytoskeleton protein and the expression of IGF-1 and IGF-1R transfected by H2O2-induced Smyd1-shRNA.(5)Smyd1-OE significantly increased the expression of H9c2 cytoskeleton related proteins and the expression of IGF-1 and IGF-1 R;H2O2 can further up-regulate the expression of H9c2 cytoskeleton related proteins and the expression of IGF-1 and IGF-1R in Smyd1-OE.AICAR can up-regulate the expression of Smyd1-OE H9c2 cytoskeleton related proteins and the expression of IGF-1 and IGF-1R induced by H2O2.Conclusion:(1)Interval exercise or Tempol intervention significantly increased IGF-1,IGF-1R,PI3K/Akt phosphorylation and Smyd1 expression in normal and MI myocardium,promoted F-actin assembly in muscle segment,decreased Trx1,MuRF1 and HSP90 expression and oxidative stress level,inhibited protein ubiquitination degradation,promoted physiological hypertrophy of cardiomyocytes and improved cardiac systolic function.After the intervention of H9c2 cells treated with H2O2 by AICAR,the PI3K/Akt pathway was significantly activated,the expression of Smyd1 was increased and the oxidative stress level was reduced.These results indicated that interval exercise could inhibit oxidative stress,up-regulate endogenous IGF-1/IGF-1R expression,and reduce cell apoptosis.(2)Interval exercise and Etanercept intervention both up-regulated the expression of IGF-1 and IGF-R in MI,decreased the level of inflammation,activated the PI3K/Akt signaling pathway,and inhibited the TNF-?/NF-?B/MuRF1 pathway.In H9c2 cells,AICAR and rhiGF-1 significantly activated PI3K/Akt pathway and inhibited TNF-?/NF-?B/MuRF1 pathway.These results indicated that interval exercise inhibited TNF-?/NF-?B/MuRF1 pathway,up-regulated endogenous IGF-1/IGF-1R expression,promoted sarcomere assembly,reduced myocardial fibrosis,and improved cardiac function.(3)Interventional exercise promoted the expression of IGF-1 and IGF-1R,activated PI3K-Akt pathway,up-regulated the expression of Smyd1,and was involved in regulating the expression of cTnI and ?-actinin,regulating the sarcomere assembly and cardiomyocyte hypertrophy,reducing cell apoptosis,and improving MI function.The effect could be inhibited by NVP-AEW541.These results suggested that interval exercise regulates Smyd1 level and sarcomere assembly through IGF-1/IGF-1R.In conclusion,interval exercise can improve myocardial function by inhibiting oxidative stress and inflammation,up-regulating endogenous IGF-1/IGF-1R expression,reducing cell apoptosis,and promoting sarcomere assembly by regulating Smyd1 expression. |
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