| Background and AimsMyocardial remodeling,including cardiomyocyte hypertrophy and extracellular matrix proliferation chiefly,is the pathological basis of the progression nearly all of cardiovascular diseases.Even though myocardial remodeling is compensatory for cardiac function initially, continuing hypertrophy of myocardium may result in myocardial ischemia,cardiac dysfunction and arrhythmias.The molecular mechanisms of myocardial hypertrophy are the nucleus gene expression alteration stimulated by hypertrophic factors,through many signal pathways in cells. Among these signals,AKT,as a serine/threonine protein kinase,has comprehensive biological effect,including anti-apoptosis and facilitating cardiomyocytes survival.Besides,the role of cytokines in the procedure of myocardial hypertrophy is indispensable.One of them is cardiotrophin-1(CT-1),which,as an important factor in the physio-regulation,not only accelerates myocardium development,facilitates myocytes survival,but also takes part in the course of ventricle thickening.It may be of great value to determine relationships of correlative signal transductions and AKT changes for the alleviation and even the reverse of cardiac remodeling.At present,the roles of several signal transductions related to the myocardial hypertrophy focus chiefly on the positive regulation of cardomyocytes growth and cardiac hypertrophy,while studies in that of the negative regulation have been barely reported.Glycogen synthase kinase-3βis a member in the serine/threonine protein kinase family. Its functions include cells metabolism,gene expression and maintenance of cellular skeleton.Recently,the role of GSK-3βin cellular apoptosis is studied extensively.There are many pathways leading to apoptosis associated to GSK-3β.As a negative regulator for myocardial hypertrophy and proliferation,and basic adjustment factor in the progress of tissue proliferation,GSK-3βmay be of great significance.In this study,we used transverse aortic constriction induced left ventricular hypertrophy in rats,and cardiomyocyte culture in vitro,to determine relationships of correlative signal transductions,and the possible effects related to GSK-3β,AKT,CT-1 in the alleviation and the reverse of cardiac remodeling.MethodsThis study was divided into 2 parts,including animal experiment and cell culture experiment.(1) Pressure over-load myocardium hypertrophy of rat was induced.The expression changes of GSK-3β,AKT and CT-1 of myocardium in the process of myocardium hypertrophy were detected.The relationship of them between histocyte apoptosis and proliferation was investigated.54 adult Wistar rat were used in this experiment.They were divided into normal group(6 rats),pseudo-operation group(24 rats) and experiment group(24 rats) at random.The observing times were 6h,1d,1w and 4w post-operation.Coarctation of aorta was used in the experiment of pressure overload myocardium hypertrophy creation.The main detecting indexes were as follows:①SBP,DBP and MABP were recorded by the biological signal recorder.②Left ventricular mass index was detected too.③Hydroxyproline content of myocardium was detected.④The content of CT-1,AKT,p-AKT, GSK-3β,p-GSK-3βand caspase3 of myocardium were detected by WB.⑤The expression changes of CT-1,AKT,p-AKT,GSK-3β,p-GSK-3βand caspase3 were detected by immunohistochemistry staining.⑥Cell apoptosis was detected by TUNEL staining.⑦Histocyte cell proliferation was detected by flow cytometry.(2) Myocardium cell hypertrophy model was created in vitro.The influence of GSK-3βactivity inhibitor and reinforcement factor on the hypertrophic myocardium cell was investigated.Myocardium cell hypertrophy was induced by drug.0.1μmol/L AngⅡ+1μmol/L NE were added to the culture in the same time.In the AKT inhibitor group(Al group),AKT inhibitor SH-6 was added in the culture. The end concentration was 10-3mg/ml.In the GSK-3βinhibitor group(GI group),GSK-3β inhibitor(3-(N-[Dimethylamino]propyl-3- indolyl)-4-(3-indolyl) maleimide) was added in the culture after SH-6 was used.The end concentration was 10-3mg/ml.The observing times were 6h,24h and 48h.The main detecting indexes were as follows.①Cell area was detected under invert microscope.②Protein content of myocardium was detected by Lowry method.③Protein synthesis ration of myocardium was detected by[3H]-Leu radiometry.④Cell apoptosis of myocardium was detected by TUNEL staining.Results(1) In the over loading myocardium hypertrophy:①In the experiment group,SBP,DBP and MABP increased significantly 6h post-operation.They all had rise tendency.4 weeks after operation,SBP became as high as 201.5±22.1mmHg,DBP became 170.7±12.4 mmHg and MABP became 185.5±15.0 mmHg. In the pseudo-operation group,these indexes did not have any change.②In the normal control group,the LVMI was 2.01±0.16mg/g.In the experiment group,LVMI increased significantly in 1w and 4w observing time.In the seudo-operation group,LVMI did not have any change.③In the normal control group,the content of hydroxyproline of myocardium was 3.81±0.14 mg/g.At 1w and 4w of experiment group,hydroxyproline content increased significantly.④In the experiment group,the content of CT-1,p-AKT and p-GSK-3βincreased significantly.The content of AKT and GSK-3βdid not have any change.The content of caspase-3 decreased significantly.⑤In the experiment group,the cell apoptosis decreased significantly compared to the pseudo-operation group.⑥In the experiment group,histocyte proliferation index increased significantly.(2) In the myocardium cell hypertrophy:①Myocardium cell was cultured successfully.Positive striated muscles actin staining rate was 95%.②The area of myocardium cell in the GI group increased significantly compared to the AI group.But it was lower than that in the normal control group.③The protein content of myocardium cell in the GI group increased significantly compared to the AI group.But it was lower than that in the normal control group.④The protein synthesis ration of myocardium cell in the GI group increased significantly compared to the AI group. But it was lower than that in the normal control group.⑤The apoptosis cell in the GI group decreased significantly compared to the AI group.But it was higher than that in the normal control group.ConclusionsThe main conclusions were summarized as follows:(1) The coarctation of abdominal aorta may set up the model of pressure over-load myocardial hypertrophy in rats.(2) The content of CT-1 and p-AKT increased significantly,and GSK-3βactivity and caspase-3 decreased significantly during the occurrence of myocardial hypertrophy.(3) Cardiomyocyte apoptosis ratio decreased significantly when myocardial hypertrophy occurred by pressure over-load.(4) The changes of the content of CT-1 and activity of AKT and GSK-3βtook part in the process of myocardial hypertrophy.(5) When AKT activity was inhibited,cellular area,protein content and protein synthesis rate decreased significantly.(6) GSK-3βinhibitor cannot reverse completely the hypertrophic effect derived from the inhibition of AKT activity,i.e.the effects of inhibitor of AKT on myocardial hypertrophy are not limited to the passway of GSK-3β.(7) GSK-3βinhibitor can decrease the myocardiun apoptosis significantly. |
PDF Full Text Request