STABLE OVEREXPRESSION OF THE CONSTITUTIVE FORM OF HEAT-SHOCK-PROTEIN-70 CONFERS OXIDATIVE PROTECTION

We have previously reported that thermal preconditioning confers an ox idative resistance in rat heart-derived H9c2 myocytes. The development of this resistance is associated with a co-expression of both inducib le (hsp70i) and constitutive (hsp70c) forms of the 70-kD heat shock pr oteins, suggesting an antioxidant role for these proteins. Overexpress ion of hsp70i has been shown to render cells more tolerant to oxidativ e challenge. The present study sought to determine whether increases i n hsp70c. the constitutive member of this protein family, are also pos itively correlated to oxidative protection. A rat cDNA encoding hsp70c was inserted into a mammalian expression vector, allowing transcripti on of the inserted gene to be regulated by a powerful cytomegaloviral promoter. After introduction of this construct into H9c2 myocytes, sta ble clones were obtained. Western and Northern blot analysis of the de rived clones showed a two-fold increase in hsp70c mRNA and protein con centrations. These clones were more resistant to thermal killing when compared to control cells transfected with the vector alone, implicati ng a functional role for the overexpressed hsp70c protein. hsp70c-enri ched cells also exhibited a marked resistance to oxidative challenges, including exposure to hydrogen peroxide (H2O2), hydroxyl radical, men adione, and hypoxia/reoxygenation. These findings indicate that hsp70c overexpression provides a protective effect against endogenous or exo genously generated reactive oxygen species (ROS), suggesting that hsp7 0c actively participates in the heat shock-induced oxidative protectio n. (C) 1998 Academic Press Limited.