996-15 Adenovirus Mediated Inducible Heat Shock Protein 70 Gene Transfer Protects Against Simulated Ischemia in a Muscle Derived Cell Line

Abstract

The inducible heat shock protein 70 (HSP70i) is a stress related factor that has been shown to have a protective effect against ischemia. In order to study further this protective effect we have constructed a recombinant replication deficient adenovirus that constitutively expresses HSP70i at high levels under the direction of the viral CMV promoter. Expression was verified in neonatal cardiomyocytes in cell culture infected at a multiplicity of infection of 2:1. The majority of cells stained strongly positive after 24 hours with immunohistochemistry using a monoclonal antibody against HSP70i. Neonatal cardiomyocytes infected similarly with a recombinant adenovirus encoding beta-galactosidase did not stain with the HSP70i antibody. C2C12 cells, a muscle cell line, were infected with either the HSP70i adenovirus or betagalactosidase encoding adenovirus as above. After 24 hours the cells were incubated with rhodamine, afluorescent marker retained by viable cells, and were then subjected to simulated ischemia (slightly hypotonic Hanks buffer in a GasPak hypoxic jar) for 6 hours. After a 1 hour non-hypoxic recovery period in isosmotic media the cells were examined with a fluorescent microscope. There was a readily apparent increase in survival of the HSP70i adenovirus infected cells relative to the beta-galactosidase infected cells based on rhodamine mediated fluorescence. These studies demonstrate the feasibilityof using adenoviral vectors to effect expression of HSP70i in cardiac myocytes and also demonstrate the efficacy of this approach for providing protection against simulated ischemia.