The Protective Effect Of Heat Shock Proteins On Cardiomyocytes Against Apoptosis Induced By Hydrogen Peroxide

There is a growing body of evidence that cardiac dysfunction induced bymyocardial ischemia-reperfusion injury, cardiomyopathy, miocardialinfarction, heart failure and so on involves not only necrosis but alsoapoptosis, which is due in part to the generation of reactive oxygenspecies(ROS). Clarifying the mechanisms contributing to cardiomyocyteapoptosis may provide valuable information for the treatment and preventionof many cardiovascular diseases. Apoptosis plays important roles in many pathophysiological processesas well as physiological states. The mechanisms of apoptosis involve mainlytwo signal transduction pathways including mitochondrial pathway and celldeath receptor pathway. The key element of mitochondrial pathway is theefflux of cytochrome c from mitochondria to cytosol, where it subsequentlyforms a complex(apoptosome) with Apaf-1 and caspase-9, leading toactivation of the latter. The cell death receptor pathway is charactered by thebinding between cell death ligands(FasL, TNF) and cell death receptors (Fas,TNFR), and subsequent the activation of caspase-8. In some kinds of cells,the activation of caspase-8 can promote cleavage of Bid, a member of Bcl-2family, to tBid, which then activate mitochondrial pathway. However, whether this two pathways play roles in H2O2-inducedapoptosis of neonatal rat cardiomyocytes and C2C12 myogenic cells is stilluncertain. A number of studies have recently demonstrated the effect of cellularendogenous protective mechanism in the treatment and prevention ofcardiovascular diseases, in which attention has primarily been focused on therole of heat shock proteins(HSPs). HSPs is a family of molecular chaperons,which are indispensable in physiological states and exserts protective role inpathological processes. Some evidences showed that HSPs could protectmany kinds of cells from apoptosis induced by a variety of stressors. But themechanism by which HSPs protect cells from apoptosis is still unclear.Whether HSPs inhibit apoptosis through mitochondrial pathway or cell deathreceptor pathway or both needs to be confirmed.In our studies, apoptosis of neonatal rat cardiomyocytes and mouseembryonic myogenic cell line C2C12 cells was induced by exposure the cellsto 500 μM hydrogen peroxide (H2O2) for different durations, and themechanisms of apoptosis induced by H2O2 and the mechanisms by whichheat shock proteins protected cardiomyocytes against apoptosis wereinvestigated. The main results and findings are as follows:1,Mitochondria and death receptor signal pathways were both involvedin apoptosis of cardiomyocytes and C2C12 cells induced by H2O2. 1) Exposureof the cells to 500 μM H2O2 for 24 hours resulted in apoptosis in neonatal ratcardiomyocytes and C2C12 cells as shown by TUNEL and Hoechst 33258staining;2) The activities of caspase -3, 8, 9 were significantly increased after4 hours of H2O2 treatment, and reached peak at 8-12 hour;3) The release ofcytochrome C from mitochondria to cytoplasm was observed at 1-2 hoursafter treatment of H2O2;4) The cleavage of Bid to tBid was obvious after 1-2hours treatment of H2O2.2,Heat shock response protected cardiomyocytes and C2C12 cells againstapoptosis by inhibiting the activation of both mitochondrial and deathreceptor pathways. The expression of HSP70 and αB-crystallin significantlyincreased at 3h and reached peak at 6-12 hour after heat shock response.H2O2 -induced release of cytochrome c from mitochondria to cytoplasm,activation of caspase -3, 8, 9, cleavage of Bid to tBid and subsequentapoptosis were significantly inhibited by Heat shock response .3,HSP70 protected C2C12 cells against apoptosis by inhibiting theactivation of both mitochondrial and death receptor pathways.pcDNA3.1-HSP70 recombinant plasmid was constructed and transfected intoC2C12 cells. The expression of HSP70 was increased at 36h after transfectionand H2O2-induced activation of caspase -3, 8, 9, cleavage of Bid to tBid andapoptosis were significantly inhibited.In conclusion, hydrogen peroxide could induce the release ofcytochrome C from mitochondria to cytoplasm, the activation of caspase -3,8, 9, cleavage of Bid to tBid and subsequent apoptosis in cardiomyocytesand C2C12 cells, which indicated that mitochondria and death receptor signalpathways were both involved in apoptosis induced by H2O2;Heat shockresponse and HSP70 over-expression protected cardiomyocytes fromapoptosis by inhibiting the activation of both mitochondrial and deathreceptor pathways.