Differential cytoprotection against heat stress or hypoxia following expression of specific stress protein genes in myogenic cells

Cells respond to sub-lethal heat stress by preferential synthesis and accumulation of several members of functionally and compartmentally distinct families of heat shock (or stress) proteins (such as hsp70, hsp90, hsp60 and hsp27). Some of these have been implicated in the development of thermotolerance and resistance to other environmental stresses. The aim of this investigation was to determine the ability of hsp70, hsp90 or hsp60 to individually protect embryonal rat heart-derived H9c2 myocytes against both (i) heat stress and (ii) substrate-free hypoxia. When H9c2 cells were subjected to a sub-lethal stress (43°C for 30 min) they were shown to have elevated levels of hsp70, hsp90 and hsp60 which was maximal between 14–24 h and was associated with increased survival at 24 h against a subsequent lethal heat stress (47°C for 2 h) (20.4±2.6% v 7.3±1.8%; P<0.002). H9c2 myocytes were transfected with a plasmid containing human hsp70i, hsp90β or hsp60 expressed under the control of the constitutively active human β-actin promoter or with control vector alone (containing no hsp gene). Stable colonies of primary transfectants selected for neomycin resistance showed different degrees of over-expression of hsp70i, hsp90β or hsp60 expression as determined by Western blotting using specific monoclonal antisera. Cells constitutively expressing high levels of hsp70i showed significantly higher survival against lethal heat stress compared to cells transfected with vector alone (39.2±6.5% v 4.5±1.0%; P<0.0001) and also against 20 h of substrate-free hypoxia (86.4±3.0% v 3.5±0.7%; P<0.0001). Cells constitutively expressing high levels of hsp90β also showed significantly higher survival against heat stress (21.5±3.0% v 9.8±3.7%; P<0.05) but were not resistant to 20 hours of substrate-free hypoxia (0.5±1.0% v 2.0±1.7%). Cells transfected with the hsp60 plasmid showed no increased survival against either heat stress or substrate-free hypoxia. These results demonstrate that transfection of H9c2 cells with either hsp70i, hsp90β or hsp60 genes confers different patterns of protection against heat stress and substrate-free hypoxia, indicating functional differences between stress proteins in their ability to protect against divergent stresses.