Role And Mechanism Of GCN2 In Aerobic Exercise To Attenuate Myocardial Injury Induced By Doxorubicin In Mice

Background and purposeDoxorubicin is an effective cancer chemotherapy drug.Doxorubicin induced myocardial injury-dose-dependent,severely lead to heart failure and even death.The mechanism of myocardial injury is related to energy metabolism disorders,apoptosis,oxidative stress and fibrosis.Aerobic exercise has been shown to be effective as a non-pharmacological method for the prevention and alleviation of doxorubicin-induced myocardial injury,but its mechanism has not yet been fully elucidated.GCN2(general control non-depressible-2)is one of eIF2?(eukaryotic initiation factor 2?)kinases and is an amino acid sensing protein.Previous studies have shown that GCN2 activity is associated with the alleviation of cardiovascular disease,GCN2 and its downstream eIF2?/ATF4 pathway play an important role.However,the role of the GCN2 gene in the process of aerobic exercise to attenuate myocardial injury remains unclear.This study will use the doxorubicin-induced chronic myocardial injury model in mice,using a moderate-intensity treadmill exercise intervention,and to study the effect of treadmill exercise on myocardial injury in mice during and after doxorubicin injection,and explore the effect of GCN2and its downstream pathway(eIF2?/ATF4).GCN2 may be affected in this process,then we will use GCN2 knockout mice for the same doxorubicin injection and treadmill exercise intervention,and to investigate whether GCN2 knockout affect exercise-regulated myocardial injury induced by doxorubicin.Meanwhile,we will use GCN2 activator to observe whether GCN2 activation affect exercise-regulated myocardial injury induced by doxorubicin.This study will reveal the mechanism of aerobic exercise for alleviating myocardial injury and the role of GCN2.Methods and materialsPart ?:Effect of aerobic exercise on doxorubicin-induced myocardial injury in C57 mice and its effect on GCN21.Eight-week-old male C57 mice(n=36)were randomly assigned to the C57 control group(C57CON,n=6),the C57 DOX group(C57 DOX,n=12),the C57 exercise group(C57 E,n=6),and the C57 DOX+exercise group(C57 DE,n=12).Doxorubicin was injected for 4 weeks,once a week,5 mg/kg per injection,the total dose was 20 mg/kg,and the control mice were injected with the same amount of saline.2.The exercise training program used 8 weeks of moderate-intensity treadmill training.In the DE group,doxorubicin and exercise training were performed simultaneously in the first 4 weeks,and only exercise training was performed in the later 4 weeks.After 8 weeks of exercise training,echocardiography was performed to assess cardiac function in mice,including cardiac ejection fraction(EF),left ventricular fraction shortening(FS),left ventricular end diastolic diameter(LVEDD),and left ventricular end systolic diameter(LVESD).After 24 hours,the mice were tested for exercise capacity,including maximum exercise distance and exercise time.3.The mice were anesthetized and the heart tissue was obtained.The heart tissue was examined by histology,including WGA(wheat germ agglutinin)staining to observe the changes of myocardial cell size,and Sirius Red staining was used to observe the myocardial fibrosis.4.The heart tissue was examined by molecular biological,including GCN2 protein and its downstream pathways(eIF2?,P-eIF2?,ATF4),myocardial apoptosis related proteins(Caspase3,Bax,Bcl2),cardiac function related protein(ANP),energy metabolism related proteins(AMPK?,P-AMPK?,PGC1?,SIRT1)and fibrosis-related protein(Collagen I).Part ?:Effect of aerobic exercise on myocardial injury induced by doxorubicin in GCN2knockout mice1.Eight-week-old male GCN2 knockout mice(GCN2 KO,n=36)were randomly assigned to the GCN2 KO control group(GCN2 KO CON,n=6),the GCN2 KO DOX group(GCN2 KO DOX,n=12),the GCN2 KO exercise group(GCN2 KO E,n=6),and GCN2 KO DOX+exercise group(GCN2 KO DE,n=12).2.Experimental methods and test indicators were the same as Part I.This part mainly to explore whether GCN2 knockout affect the regulation of aerobic exercise on doxorubicin-induced myocardial injury,and studies its mechanism.Part ?:Effect of GCN2 activation on the regulation of aerobic exercise on doxorubicin-induced myocardial injury1.Eight-week-old male C57 mice(n=24)were randomly assigned to the C57 DOX+exercise group(C57 DE,n=12)and the C57 DOX+exercise+GCN2 activator group(C57 DEL,n=12).8 weeks old male GCN2 KO mice(n=24)were assigned to GCN2 KO DOX+exercise group(GCN2 KO DE,n=12)and the GCN2 KO DOX+exercise+GCN2 activator group(GCN2 KO DEL,n=12).2.The GCN2 activator was treated with a leucine-deficiency(Leu~-)diet,and the control group was fed a normal diet.The Leu~-feed composition was identical to the normal feed except for the leucine content.One-week leucine-deficiency diet can effectively activate GCN2.3.The doxorubicin injection and the aerobic exercise regimen were the same as those of Part I,the Leu-diet intervention was performed in the last week of the 8-week aerobic exercise intervention,and the echocardiographic test and the exercise capacity test were performed after the intervention.Results Part I:1.Compared with the C57 CON group,the EF and FS of the C57 DOX group were significantly lower(P<0.01).The exercise time and distance were also significantly decreased(P<0.01).These results suggested that doxorubicin reduced heart function and exercise capacity in mice.The body weight of C57 DOX group was significantly lower from 4 weeks to the 8 weeks(P<0.01).The myocardial tissue in the C57 DOX group showed significant fibrosis(P<0.01).The expression of P-eIF2?and ATF4 in the C57 DOX group was significantly increased(P<0.01),the expression of Bax,Caspase3 and ANP was significantly increased(P<0.01),the expression of Bcl2 protein expression and the ratio of Bcl2/Bax were significantly decreased(P<0.05),the expression of energy metabolism related proteins(P-AMPK?,PGC1?,SIRT1)was significantly decreased(P<0.05),and the expression of fibrosis-related protein(Collagen I)was significantly increased(P<0.01).That is,doxorubicin induced myocardial apoptosis,energy metabolism disorders,and myocardial fibrosis in C57 mice.2.Compared with the C57 DOX group,C57 DE group attenuated the appeal symptoms,including improved EF and FS(P<0.05),improved exercise capacity(P<0.05),improved myocardial fibrosis(P<0.05),and inhibited the expression of apoptosis and fibrosis related proteins(Bax,Caspase3,Collagen I)(P<0.05),and increased the expression of Bcl2 and the ratio of Bcl2/Bax(P<0.05),and also increased the expression of energy metabolism related proteins(P-AMPK?,PGC1?,SIRT1),the expression of ANP protein tended to increase,but no significant difference.That is,aerobic exercise improved myocardial apoptosis,energy metabolism disorders,and myocardial fibrosis induced by doxorubicin in C57 mice.3.Compared with C57 DOX group,C57 DE group also inhibited the expression of GCN2 and downstream pathway(P-eIF2?/ATF4)(P<0.05).The decrease in GCN2 protein expression may be related to cardioprotection of aerobic exercise.Part?:1.Compared with the GCN2 KO group,the EF and FS of the GCN2 KO DOX group were significantly lower(P<0.01).The exercise time and distance were also significantly decreased(P<0.01).The body weight of GCN2 KO DOX group was significantly lower from 4 weeks to the8 weeks(P<0.01).The myocardial tissue in the GCN2 KO DOX group showed significant fibrosis(P<0.01).The expression of GCN2 downstream pathway-related proteins(P-eIF2?,ATF4)in the GCN2 KO DOX group was significantly increased(P<0.05),the expression of Bax and ANP proteins was significantly increased(P<0.01),the expression of Bcl2 protein expression and the ratio of Bcl2/Bax were significantly decreased(P<0.05),the expression of energy metabolism related proteins(P-AMPK?,PGC1?,SIRT1)was significantly decreased(P<0.05).That is,doxorubicin induced myocardial apoptosis,energy metabolism disorders and myocardial fibers in GCN2 KO mice.2.Compared with GCN2 KO DOX group,GCN2 KO DE group relieved the symptoms of appeal,including improved EF and FS(P<0.05),improved exercise capacity(P<0.05),improved myocardial fibrosis(P<0.05),and inhibited the expression of apoptosis and cardiac function related proteins(Bax,ANP)(P<0.05),and increased the expression of Bcl2 and the ratio of Bcl2/Bax(P<0.05),and increased the expression of energy metabolism-related proteins(P-AMPK?,PGC1?,SIRT1),and inhibited the expression of GCN2 downstream protein(P-eIF2?,ATF4)(P<0.05).That is,aerobic exercise improved myocardial apoptosis,energy metabolism disorders,and myocardial fibrosis induced by doxorubicin in GCN2 KO mice.3.The study also compared the myocardial injury of GCN2 KO mice and C57 mice.Compared with C57 DOX group,the body weight,exercise capacity,EF and FS in the GCN2 DOX group were higher(P<0.05),the level of myocardial fibrosis in the GCN2 DOX group was significantly lower(P<0.05).Compared with C57 DE group,EF and FS in the GCN2 DE group were significantly higher(P<0.05),the other indicators were higher,but no significant difference.GCN2 knockout attenuated myocardial injury induced by doxorubicin in mice,and GCN2 knockout can also promote exercise-improved myocardial injury in mice,which related to the inhibition of GCN2 activity,and then inhibit eIF2?/ATF4 pathway,which in turn inhibit myocardial apoptosis,fibrosis,and energy metabolism disorders.Part ?:1.Compared with C57 DE group,the expression of GCN2 downstream pathway protein(P-eIF2?/ATF4)in C57 DEL group was significantly increased(P<0.05).2.Compared with C57 DE group,exercise capacity was significantly decreased(P<0.05),EF and FS were significantly decreased(P<0.05),the level of myocardial fibrosis tended to increase,but no significant difference.GCN2 activation caused a decrease in body weight,heart function and exercise capacity in the C57 DE group.Compared with GCN2 KO DE group,the body weight in the GCN2 KO DEL group was significantly decreased(P<0.05),EF,FS and exercise ability tended to decrease,but no significant difference,the level of myocardial fibrosis tended to increase,but no significant difference.3.Compared with C57 DEL group,the body weight in the GCN2 KO DEL group was no significant difference.EF and FS were higher,but no significant difference.Exercise capacity was significantly higher(P<0.05).The level of myocardial fibrosis was significantly lower(P<0.05).The expression of downstream protein of GCN2(P-eIF2?/ATF4)was also significantly lower(P<0.05).Conclusion1.8 weeks of aerobic exercise attenuated myocardial injury induced by doxorubicin in C57 mice,the mechanism was related to the regulation of myocardial apoptosis,energy metabolism disorders and myocardial fibrosis-related protein.Aerobic exercise also inhibited the expression of GCN2protein and its downstream P-eIF2?/ATF4.The decreased expression of GCN2 protein may be related to aerobic exercise to attenuate myocardial injury.2.GCN2 knockout attenuated myocardial injury induced by doxorubicin,and can also promote the improved effect of exercise on myocardial injury,its mechanism may be related to inhibit the expression of GCN2 protein,regulation of eIF2?/ATF4 pathway,and thus inhibited myocardial apoptosis,energy metabolism disorders and fibrosis.3.GCN2 activation inhibited the improved effect of exercise on myocardial injury in C57 mice.GCN2 gene is an important gene for exercise-relieving myocardial injury,and its downstream pathway(eIF2?/ATF4)play an important role in this process.