Effect Of Apoptosis Of Rat Hypertrophic Cardiomyocytes Induced With Hypoxia/reoxygenation On Intervented Metabolic Pathway

OBJECTIVES In recent years,scientific interest in mitochondria has received a newimpulse by the discovery that these energy-providing organelles aiso play a vital role in celldeath processes. A number of changes occur in mitochondria early during apoptosis. In thispaper, we set out to investigate how glucose metabolism,fatty acid metabolism andphosphotyrosine pathway are involved in apoptosis.To change metabolic pathway ofhypertrophic cardiomyocytes,we used dichloroacetate,antimycin A,trimetazidine,L-carnitine. METHODS 1.The isolated neonatal Wistar rat ventricular cardiomyocytes werecultured and purfied with the method of differential attachment.The cardiomyocytes arepurified by differential attachment technique and adding BrdU to reduce the rate ofnon-myocytes. 2. The cultured cells were indentified by morphology,spontaneouscontraction and specific immuncytochemical stain.3.Hypertrophied cardiomyocyte modelinduced by Angiotensin II was set up.By [3H]-Leu incorporation,model of hypertrophiedcardiomyocytes was assessed.4.The cultured media were replaced by low glucose DMEMsupplemented with before hypoxia.The cardiomyocytes were incubated at 370C in anair-tighe incubator where normal air was replaced by 92%N2,5%CO2,3%O2 for 24h toproduce hypoxia,and then the air was replaced by23%O2,5%CO2 for 4h to producereoxygenation;Apoptotic cell death was evaluated using a modified TUNEL(TdT-mediatedDutp Nick-End Labeling)assay(DeadEndTM Colorimetric TUNEL). The DNA ladder wasdetectable in apoptosis cell. Using SPSS10.0 for statistical analysis,the data were expressed as mean +_S.E.M.Ifdifferences were established,the statistical significance was tested by paired Student's-testor by one-way ANOVA.The results were considered statistically significant if P value wasless than 0.05. 2第三军医大学硕士学位论文 RESULTS:1.The shapes of primary cultured cardiomyocytes are spindle,triangle andirregular.Among total cultured cells, the spindle cells are majority of primary culturedcardiomyocytes.Spontaneous contraction of the spindle cell is apparent.There haveabundant brownish red filaments in cytoplasm of primary cultured cardiomyocytes with themethod of specific immunocytochemical stain.2.Ultrastrutural changes consist ofenlargement and varying shape of nuclei,enlarged mitochondria,abundant heterochromatinand accumulation of lipid droplets and glycogen.The ultrastructural alteration is unspecifiedperformance of compensatory hypertrophy.3.[3H]-Leu incorporation in cardiomyocyte ofAngiotensin II group is significantly higher than that of the control group which indicatssynthesis of protein increased induced by Angiotensin II. 4.The apoptosis rates ofcardiomyocytes increased with time of hypoxia/reoxygenation(H/R).The modle for H/Rinduced apoptosis of culturing rat hypertrophic cardiomyocyteswas establishedsuccessfully.5.When cardiomyocytes were pretreated with dichloroacetate,trimetazidine orL-carnitine, the rates of apoptosis were decreased in a dose-depend way.6.Whencardiomyocytes were pretreated with antimycin A, the rates of apoptosis were increased in adose-depend way.7. Changing metabolic pathway has effect of apoptosis of rat hypertrophiccardiomyocytes. Conclusions:1.With the method of collagense digestion,it is convenient to obtainprimary cultured neonatal Wistar rat ventricular cardiomyocytes.This method is one of vitalresearch tool in the fields of cardiac hypertrophy.2.By 10-7mol/L concertration,AngiotensinII could accelerate cellular proliferation,structural protein biosynthesis and cardiachypertrophy.3. Dichloroacetate , Trimetazidine or L-carnitine has anti-apoptosis .4.Antimycin A has anti-apoptosis.