| Cytoprotection derived from preconditioning such as heat, ischemia, hypoxia and medication, has been well established in various cell types including cardiac cells, endothelial cells and neurons. Induction of endogenous protective proteins including heat shock proteins (HSPs) has been demonstrated to be a key mechanism through which preconditioning induces cytoprotection. In previous studies, the 78-kD glucose-regulated protein (GRP78), an endoplasmic reticulum-resident protein belonging to the HSP 70 family, was shown to be cytoprotective against a variety of stresses when overexpressed. Expression of GRP78 was shown to be induced by hypoxia, oxidative stress and glucose-depletion. However, the involvement of GRP78 in preconditioning-induced cytoprotection had not been described until recently in a neuron study. Based on the inducibility and cytoprotective property of GRP78, we hypothesized that GRP78 induction serves as a common mechanism in preconditioning-induced cytoprotection. To test the hypothesis, the present study was designed to determine GRP78 protein level and function in cultured hypoxic cardiomyocytes receiving different preconditionings. Specifically, hypoxia and endothelin-1 (ET-1) were utilized as preconditioning to produce protection in cultured hypoxic cardiomyocytes, and GRP78 protein levels were measured to estimate its induction. Furthermore, GRP78 expression was down-regulated by antisense oligodeoxynucleotides (AS-ODN) to suppress preconditioning-induced cytoprotection, and was over-expressed by gene transfer via recombinant adenoviral vector encoding GRP78 cDNA to produce cytoprotection. In consideration that HSP70 has been established as anendogenous protective protein that contributes to cytoprotection induced by various preconditionings, HSP70 was employed as positive control and investigated in parallel with GRP78.Materials and Methods1. Estimation of hypoxia-induced injury in cultured cardiomyocytes: All experiments were performed in primary cultured neonatal rat cardiomyocytes. Hypoxia was prepared by incubating cardiomyocytes in 3%O2-5%CO2 atmosphere at 37'C for 12h. Cell injury was assessed by supernatant lactate dehydrogenase (LDH) activity, and cell viability was assessed by MTT assay. Supernatant superoxide dismutase( SOD) activity and malondialdehyde (MDA) content were measured as index of lipid peroxidation. In addition, [Ca2+] was measured with fluorescent probe Fluo-3/AM under a laser confocal microscope. The hypoxia model used in [Ca2] measurement was produced by superfusing cardiomyocytes with 95%N2-5%CO2 bubbled DMEM solution containing 1mM N32S2O4.2. Protocol of hypoxia preconditioning: Hypoxia preconditioning was produced by incubating cardiomyocytes in a hypoxic atmosphere of 95%N2-5%CO2 at 37 for 30 min, followed by a normoxic atmosphere of 95%air-5%CO2. Twenty-four hours after the hypoxic preconditioning, the cells were subjected to hypoxia (3%O2-5%C02)for12h.3. Protocol of ET-1 pretreatment: Cardiomyocytes were incubated in DMEM containing ET-1 0.01 , 0.1 , 1nmol/Lfor 10 min, and then in ET-1 -free DMEM for 10 min. Subsequently, cells were subjected to hypoxia (3%O2-5%CO2) for 12h. In experiments observing the effects of ET-1 preconditioning on [Ca2] , the ET-1 preconditioning protocol was three cycles of 5-min ET-1 perfusion following with 10-min ET-1-free DMEM.4. Determination of HSP70 and GRP78 protein levels: In cardiomyocytesexposed to hypoxic and ET-1 preconditioning and hypoxia, the profile of HSP70 and GRP78 protein levels was determined by Western blotting.5. Administration of GRP78 AS-ODN: Prior to ET-1 pretreatment, phosphorothioate AS-ODN against rat GRP78 mRNA was applied with calcium phosphate precipitation in the cardiomyocytes to the final concentration of 1 mol/L for 24h, and phosphorothioate sense-ODN and scrambled-ODN were also administered as control. The localization of GRP78 AS-ODN in cardiomyocytes was monitored using FAM-conjugated GRP78 AS-ODN unde...
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