Kinetics study of heat shock protein 70 expression
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Heat shock protein 70 (HSP70) is well known for its efficacy in cardiac ischemia protection. The purpose of this study was to determine HSP70 expression kinetics in response to mild thermal stress. Currently there are no comprehensive experimental HSP70 expression kinetics data available. An efficient engineering system was designed to achieve this goal. HSP70 kinetics data will provide a rational basis for design of preconditioning protocols in cardiac surgery. Two approaches were used in this study. The first was to discover endogenous HSP70 expression kinetics. Bovine aortic endothelial cells (BAEC) were heated in a 42oC incubator for up to 5 hours and subsequently held at 37oC for up to 48 hours. Western blot and quantitative protein analysis were performed to measure the extent of HSP70 expression kinetics as a function of heating temperature and time, and post-heating duration. With 90 minutes heating at 42oC, HSP70 expression increased continuously after initiation of heat shock to a peak at about 5 hours recovery, followed by a rapid drop and a second higher peak at about 12 to 16 hours recovery that endured past 24 hours. The maximum HSP70 concentration was about 10 times higher than control. The two peak phenomenon of HSP70 expression kinetics may be an important cause of the “second window of protection” identified by other investigators of preconditioning in ischemia-reperfusion injury. The second approach was to observe HSP70 expression continuously by green fluorescence protein (GFP) on a ∆T heating stage with fluorescence microscopy. A fusion protein gene including HSP promoter, HSP70 and GFP genes, was cloned successfully and stably transfected into BAECs. Continuous observation of HSP70 expression kinetics was obtained after quantitative fluorescence image analysis. The bimodal kinetics curve from the ∆T stage is consistent with the result of endogenous HSP70 expression kinetics study. Dynamic localization of HSP70 was acquired simultaneously. HSP70 translocation from cytoplasm back to nucleus was observed in some cells. We propose that the relocalization of the HSP70 discovered in some cells may be related to cell cycles and may act as an indicator of the fate of cells. The second approach provides an effective system to obtain protein expression kinetics for concentration and localization.