Study On Mechanism Of Hypertrophic Cardiomyocytes Apoptosis Induced By Hypoxia And Reoxygenation

Ischemia/reperfusion (I/R) injury is a major complication of myocardial infarction, cardiopulmonary bypass surgery, and heart transplantation . Previously, myocardial cell death in I/R injury has been considered to be mediated mainly by necrosis. However, it has been proposed that cardiomyocyte apoptosis can significantly contribute to cell death in myocardial I/R injury and that cardiomyocyte apoptosis maybe the main cause of change from cardiac hypertrophy to heart failure after repeating ischemia. At present, the mechanism of hypertrophic cardiomyocytes apoptosis induced by I/R injury is still remains uncertain. Mitochondrial dysfunction is closely associated with the pathogenesis of myocardial I/R injury, because mitochondria whose primary function is energy metabolism are abundantly present in the myocardium and are major sources of pro-apoptotic molecules. Mitochondria arose the caspase cascade by the release of cytochrome c and subsequent activation of caspase-3. Eventually, activated caspase-3 leads to apoptosis.This process is caspase-dependent cell death pathway. Therefore, it was investigated whether the inhibition of caspase-3 reduces the extent of myocardial reperfusion injury by attenuating cardiomyocyte apoptosis. Unexpectedly,it was found that caspase-3 inhibitors did not reduce infarct size. Therefore, it has been proposed that an apoptotic pathway other than the cytochrome c/caspase-3 pathway mediates the apoptosis of myocardial cells in I/R injury. In recent years, apoptosis-inducing factor(AIF) was found to be a pro-apoptotic protein which was confined to the intermembranous space of the mitochondrion. In apoptotic cells, mitochondrial AIF is released into the cytoplasm and translocates to the nucleus, and then cause apoptosis. None of these AIF efects can be blocked by caspase inhibitors,namely,that AIF mediates caspase-independent cell death pathway. However, the roles of AIF on cardiomyocyte apoptosis is still remains uncertain.On the other hand,as one of the mitochondrial dysfunction, changes of metabolic pathway are closely associated with myocardium injury. Dichloroacetate(DCA), Antimycin A ,Trimetazidine(TMZ), L-carnitine can directly or indirectly influence glucose oxidation and fatty acid oxidation, and have anti-apoptosis or pro-apoptosis effects . Therefore, changes of metabolic pathway maybe play a important role in cardiomyocyte apoptosis.At present, it is still remains uncertain whether the changes of metabolic pathway are associated with cytochrome c,caspase and AIF. Objectives 1. To investigate the relation between hypertrophic cardiomyocytes apoptosis and hypoxia/reoxygenation. 2. To discuss the roles of caspase-dependent pathway on hypertrophic cardiomyocytes apoptosis induced by hypoxia and reoxygenation. 3. To explore the roles of AIF on hypertrophic cardiomyocytes apoptosis induced by hypoxia and reoxygenation. 4. To study the effect and its mechanisms of changes of metabolic pathway on hypertrophic cardiomyocytes apoptosis induced by hypoxia and reoxygenation. Methods: 1. Myocytes from neonatal KunMing strain mice(1-2d) hearts were isolated by trypsin digesting, and purified by differential attachment technique and adding arabinosylcytosine( Ara-C).The isolated cells were cultured in DMEM/F12.The myocardial sarcomeric actin were identified by immuncytochemical staining .Myofibril and mitochondria were visible by electron microscopy. Ang Ⅱwas added in medium,after 48 h , hypertrophied cardiomyocytes was identified by measure of protein content and [3H]-Leu incorporation. Simulated ischemia/ reperfusion was achieved by hypoxia(95% N2 and 5% CO2) +low glucose DMEM and succedent reoxygenation(22%O2 and 5% CO2) in DMEM/F12. The percentage of apoptotic cells was evaluated by TUNEL assay and Hoechast33258 staining at 8h,12h after hypoxic culture condition and at 4h after reoxygenation. 2. After 8h,12h of hypoxia and hypoxia/4h of reoxygenation , cytochrome c protein expression and its translocation to cytoplasm was detected by western blot. cytochrome cmRNA expression was determined by RT-PCR. Caspase-3 activity was measured with Caspase-3/CPP32 Colorimetric Assay Kit. mitochondrial membrane potential(??m) was detected by JC-1 staining. The effect of caspase-3 inhibitor on hypertrophic ardiomyocytes apoptosis was dermined afer 12h of hypoxia and 12h of hypoxia/4h of reoxygenation (Hoechst33258 staining). 3. At the time point of 8h,12h of hypoxia and hypoxia/4h of reoxygenation, western blot was used to assay AIF protein expression and its translocation to nucleus; RT-PCR was used to detected AIF mRNA expression .The effect of AIF siRNA transfection on hypertrophic cardiomyocytes apoptosis was determined after 12h of hypoxia and 12h of hypoxia/4h of reoxygenation (Hoechst33258 staining). The efficiency of AIF gene silencing afer AIF siRNA transfection was detected by western blot and RT-PCR. 4. Cells were incubated with DCA,Antimycin A,TMZ,L-carnitine, then their effect on hypertrophic cardiomyocytes apoptosis was measured after 12h of hypoxia and 12h of hypoxia/4h of reoxygenation. Translocation of cytochrome c and AIF was detected by western blot,and their mRNA expression by RT-PCR. Results 1. The method for isolating ,purifying and culturing cardiomyocytes from neonatal mice heart was successful and reliable. 96% of total cells was cardiomyocytes verified by actin ICC and electron microscopy. Compared with the control group, protein content and [3H]-Leu incorporation of cardiomyocytes increased significantly at 48h after AngⅡstimulation.; The rates of cardiomyocytes apoptosis at 8h and 12h of hypoxia groups were significantly higher than that of control group, reached at 10.7% and 13.4% respectively. The rates of apoptosis of cardiomyocytes at 8h and 12h of hypoxia /4h of reoxygenation groups were significantly higher than hypoxia group, reached at 17.8% and 24.9% respectively. 2. The expression of cytochrome c protein and mRNA was increased significantly after 8h, 12h of hypoxia, and was further increased after hypoxia/4h of reoxygenation. Furthermore, when cells were exposed to hypoxia and hypoxia/ reoxygenation, cytochrome c was release from Mitochondria to cytoplasm; The caspase-3 activity of hypertrophic cardiomyocytes of hypoxia (8h,12h) groups was significantly higher than that of control group, and was further increased after hypoxia/4h of reoxygenation. After incubated withthe caspase-3 inhibitor, Ac-DEVD-cmk, the activity of caspase-3 of hypoxia groups and reoxygenation groups has no difference compared with control group; ? ? m did not decrease when cells were exposed to hypoxia alone, however, it decreased significantly when cells were exposed to hypoxia/ reoxygenation; After inhibition of caspase-3 activity , the rates of apoptosis of cardiomyocytes at 12h of hypoxia and 12h of hypoxia /4h of reoxygenation groups decreased significantly compared with control group, from 13.4% and 24.9% to 6.7% and 17.9% respectively. 3. The expression of AIF protein and mRNA was increased significantly after 8h, 12h of hypoxia, and was further increased after hypoxia/4h of reoxygenation; AIF was translocated to nucleus after 8h and 12h of hypoxia followed by 4h of reoxygenation, but not after 8h and 12h of hypoxia alone. inhibition of caspase-3 activity did not effect significantly AIF translocation to nucleus, After 30h,54h of AIF siRNA transfection into hypertrophic cardiomyocytes, the level of AIF protein decreased by 88.4%和93.7% respectively, at the same time,AIF mRNA decreased by 91.6% and 94.1%,which indicate satisfying effect of gene silencing; AIF siRNA transfection into hypertrophic cardiomyocytes had no significant effect on the rates of apoptosis of hypoxia groups, but decreased significantly the rates of apoptosis of hypoxia /reoxygenation groups.By combination AIF siRNA transfection and inhibition of caspase-3 activity, the rates of apoptosis of hypoxia /reoxygenation groups further decreased signicantly ,however was still higher than that of normal control group. 4. At 12h of hypoxia and 12h hypoxia/4h of reoxygenation, Dichloroacetate, Trimetazidine and L-carnitine decreased significantly the rates of hypertrophic cardiomyocytes apoptosis, but Antimycin A increased significantly it; When cells were exposed to hypoxia followed by reoxygenation, Dichloroacetate, Trimetazidine and L-carnitine decreased significantly level of AIF translocating to nucleus,but Antimycin A increased significantly it; When cells were exposed to hypoxia alone and hypoxia followed by reoxygenation, Dichloroacetate, Trimetazidine and L-carnitine decreased significantly level of cytochrome c translocating to cytoplasm,but Antimycin A increased significantly it. at the same time, Dichloroacetate, Trimetazidine and L-carnitine decreased significantly the expression of protein and mRNA of cytochrome c and AIF and the activity of caspase-3,but Antimycin A increased significantly them.Conclusion 1. Hypertrophic cardiomyocytes from neonatal mice(1-2d) hearts were cultured successfully by trypsin digesting, differential attachment technique, adding arabinosylcytosine and AngⅡstimulation. Exposed to condition of Hypoxia or hypoxia/ reoxygenation, hypertrophic cardiomyocytes apoptosis could be induced,which was the ideal model for study on cardiomyocytes apoptosis. 2. The caspase-dependent cell death pathway was one of important mechanism by which hypertrophic cardiomyocytes apoptosis was induced by hypoxia alone and hypoxia/ reoxygenation. 3. AIF mediated a caspase-independent cell death pathway,and maybe played a important role in apoptosis of hypertrophic cardiomyocytes induced by hypoxia /reoxygenation. 4. Dichloroacetate, Trimetazidine and L-carnitine has function of anti-apoptosis, however Antimycin A has function of pro-apoptosis. The altered energy metabolic pathways maybe the important cause by which hypoxia alone ane hypoxia /reoxygenation induced apoptosis of hypertrophic cardiomyocytes and its concrete mechanism may be related to caspase-dependent cell death pathway and caspase-independent pathway meciated by AIF.