Effect Of Rapamycin On PI3K-AKT-mTOR Pathway Of Myocardial Cells In Rats With Heart Failure

BackgroundHeart failure is a complex clinical syndrome caused by abnormalities in heart structure or function,impair the filling or ejection ability of the heart,result in the cardiac output can't meet the metabolic needs of the organism.Common clinical symptoms include dyspnea,limited physical activity,fluid retention,and so on.The pathophysiological mechanism of heart failure is complex.Cardiomyocyte apoptosis plays an important role in the occurrence and development of heart failure.Inhibition of cardiomyocyte apoptosis has positive significance for the prevention and treatment of heart failure.The phosphatidylinositol 3-phosphate kinase/AKT/mammalian target of rapamycin(PI3K-AKT-mTOR)signaling pathway involves in the regulation of apoptosis in mammal cells.Changes in the activity of AKT proteins are a complex process in heart failure.Long-term activation of AKT can lead to pathological hypertrophy of cardiac myocytes and enlargement of cardiac cavity,leading to cardiac systolic dysfunction and accelerating the deterioration of heart failure.AKT phosphorylation increased in patients with advanced heart failure.After left ventricular assist device treatment,AKT phosphorylation decreased significantly and cardiac function improved significantly.On contrary,several reports have showed that short-term activation of AKT is beneficial to patients with heart failure,mainly completed by inhibiting apoptosis factors,the process of promoting the energy metabolism of cardiac muscle cells and inhibiting the progress of inflammatory reaction,inversely preserved cardiac function.Rapamycin is a lipophilic,macrolide antibiotic.Recent studies have found that it has cardioprotective effects.Rapamycin can effectively reduce myocardial hypertrophy and cardiac enlargement caused by increased pressure load and improve cardiac function.Studies have shown that rapamycin,an inhibitor of mTOR,different doses have different effects on AKT activity.It can increase AKT activity by inhibiting mTORC1 at low concentrations,while rapamycin inhibits AKT activity at high concentrations,the mechanism may be mainly through the mTORC2 pathway.However,as far as we know,there is no study to investigate the effect of rapamycin on cardiomyocyte apoptosis and the role of protein AKT of PI3K-AKT-mTOR pathway in this process.ObjectiveThe aim of this study was to evaluate the intervention effects of different doses of rapamycin on the heart of rats with heart failure.Echocardiography,BNP,myocardial pathological changes,apoptosis index and AKT activation were used.To investigate the influences of rapamycin on the AKT protein changes of PI3K-AKT-mTOR signaling pathway and the effect of rapamycin on cardiomyocyte apoptosis in rats with heart failure.It is expected to provide a new theoretical basis for the protection of rapamycin on cardiomyocytes and the progress of drug therapy for heart failure.Methods65 healthy SD were fed adaptively for one week,10 rats were randomly selected as sham operation group(n=10,blank group),the other rats were treated with abdominal aortic coarctation to establish heart failure model.After 8 weeks,40 successful modeling rats were randomly divided into model group(n=10),low dose rapamycin+model group(n=10,1 mg/kg/d;low dose group),medium dose rapamycin+model group(n=10,2.5 mg/kg/d;Middle dose group),high dose rapamycin+model group(n=10,4.5 mg/kg/d;high dose group).Rapamycin was injected through the caudal vein for four consecutive weeks.At the end of the treatment,echocardiography was used to evaluate the cardiac function of the rats.ELISA kit was used to detect the level of brain natriuretic peptide(BNP)in rat serum,HE staining was used to observe the pathological changes of myocardial tissue,and TUNEL method was used to detect the apoptosis of myocardial cells.The expression of AKT,the key protein of PI3K-AKT-mTOR signaling pathway,was detected by Western Blot.To determine the effects of different doses of rapamycin on AKT protein phosphorylation and cardiomyocyte apoptosis in cardiomyocytes of heart failure rats.Results1.General state of rats:The rats in the blank group grew well,the general state and quality of life were acceptable.The rats in the model group gradually developed symptoms and signs of cardiac insufficiency,the general state and quality of life were poor.Compared with the model group,rats with heart failure in rapamycin+model groups,with the gradual increasing of rapamycin dose,the improvement of general state and quality of life were gradually increased.The most pronounced improvement was in the high dose group2.Echocardiographic results:Compared with the blank group,decreased wall motion in the model group,left ventricular end-diastolic diameter(LVIDd,mm)and left ventricular end-systolic diameter(LVIDs,mm)were significantly increased(P<0.01),while left ventricular ejection fraction(LVEF,%)and left ventricular fractional shortening(LVFS,%)were significantly decreased(P<0.01).Compared with the model group,in the rapamycin+model group,with the increasing dosage of rapamycin,wall motion increased gradually,LVIDd?LVIDS decreased gradually,LVEF?LVFS increased gradually(p<0.05,P<0.01).Compared with the low dose group,the LVIDd and LVIDs in the middle and high dose rapamycin group decreased,while the LVEF and LVFS increased(p<0.05,P<0.01)3.Histomorphological changes in rats:The gross morphology of the heart was observed by naked eye.The geometry of the heart in the blank group was intact,the size of the heart chamber was normal,bright red,and the elasticity and toughness were good.In the model group,the heart lost its original geometric shape,and its cavity was obviously enlarged,dark red,with poor elasticity and toughness.The heart cavity of rapamycin+model group was enlarged to varying degrees,but compared with model group,with the increasing dosage of rapamycin,the enlargement of heart cavity was gradually reduced,the color was dark red,and the elasticity and toughness were still acceptable.HE staining was used to observe the pathological changes of myocardial tissue,including the size and edema of myocardial cells,the arrangement of myocardial fibers,the changes of interstitial cells and so on.In the blank group,myocardial cells were arranged neatly,myocardial fibers were parallel and compact,bundle-like,clear transverse striations,clear nuclei,normal shape and size of myocardial cells.in the model group,myocardial cells edema,hypertrophy,myocardial fiber breakage,arrangement disorder,wavy change,part of myocardial fiber disappeared,inflammatory cell infiltration.Compared with the model group,in the rapamycin+model group,with the increasing of the dose of rapamycin,the swelling of myocardial cells,myocardial fiber structure arrangement,inflammatory cell infiltration can be improved gradually,myocardial cell morphology,myocardial fiber arrangement degree of the high dose group has been close to the blank group,the degree of improvement is the best.4.The serum BNP levels in the HF rats:ELISA kit detection shows:the levels of BNP in the simple model group were higher than those in the blank group,there were significant statistical differences(p<0.01).However,compared with the simple model group,the levels of BNP in the rapamycin+model groups were gradually decreased with the increasing of the dose of rapamycin(p<0.05,P<0.01).Compared with the low dose group,the BNP level in the high dose group was significantly lower(p<0.05).5.Apoptosis of rat cardiomyocytes:TUNEL assay showed that:compared with the blank group,cardiac myocyte apoptosis index was increased in the model groups,there was significant statistical significance(p<0.01).Compared with the simple model group,in the rapamycin+model group,the apoptotic index of myocardial cells decreased gradually with the increasing dose of rapamycin.The apoptotic rate of the middle dose group and the high dose group decreased significantly(p<0.05,P<0.01),but there was no significant difference in the low dose group(p>0.05).Apoptotic rate of cardiomyocytes in rapamycin high dose group was significantly lower than that in low dose group(p<0.01).6.Phosphorylation of AKT protein in rat cardiomyocytes:Western Blot detection showed that:compared with the blank group,the expression of p-AKT protein in model group were increased,there were significant statistical differences(P<0.01).Compared with the simple model group,in the rapamycin+model group,the expression of p-AKT protein decreased gradually with the increasing dosage of rapamycin.The expression of p-AKT protein in rapamycin low,medium and high dose groups was lower than that in model group(p<0.05,P<0.01).Compared with low dose group,the expression of p-AKT protein in middle and high dose group was significantly lower(p<0.05).Conclusion:1.Rapamycin can improve the general state and the quality of life rats with heart failure in a dose-dependent manner.2.Rapamycin can improve the indexes of echocardiography in rats with heart failure in a dose-dependent manner,reduce LVIDd and LVIDs,increase LVEF and LVFS,and enhance the cardiac function of rats.Rapamycin has a significant preventive effect on the progress of heart failure in rats with heart failure,and can delay the progress of heart failure.3.Rapamycin can reverse naked eye changes such as cardiac enlargement and the pathological changes of myocardial cell edema,myocardial fibrous disorder and inflammatory cell infiltration in a dose-dependent manner in rats with heart failure,delay the remodeling of myocardial tissue and improve cardiac function.4.Rapamycin reduced the serum level of BNP,a marker of heart failure,in a dose-dependent manner,and improved the cardiac function of rats with heart failure.5.Cardiac myocytes apoptosis occurs in heart failure rats induced by pressure overload.Apoptosis plays an important role in the process of heart failure.Rapamycin can reduce the apoptotic rate of cardiac myocytes in heart failure rats in a dose-dependent manner,alleviate the apoptosis of cardiac myocytes and improve cardiac function.6.Rapamycin can reduce the over-phosphorylation expression of AKT,a key protein of PI3K-AKT-mTOR signaling pathway in myocardial tissue of rats with heart failure in a dose-dependent manner.7.Based on the above experimental results,we draw a preliminary conclusion,rapamycin may inhibit the over-phosphorylation of AKT protein through PI3K-AKT-mTOR signaling pathway,alleviate apoptosis of cardiac myocytes in rats with heart failure,so as to improve LVEF,reduce serum BNP level,reverse cardiac enlargement and pathological changes of myocardial tissue,delay the progress of heart failure,improve cardiac function in rats with heart failure.To provide animal experimental theoretical basis for the clinical application of rapamycin in early heart failure.