Stable Overexpression of the Constitutive Form of Heat Shock Protein 70 Confers Oxidative Protection

Abstract

We have previously reported that thermal preconditioning confers an oxidative resistance in rat heart-derived H9c2 myocytes. The development of this resistance is associated with a co-expression of both inducible (hsp70i) and constitutive (hsp70c) forms of the 70-kD heat shock proteins, suggesting an antioxidant role for these proteins. Overexpression of hsp70i has been shown to render cells more tolerant to oxidative challenge. The present study sought to determine whether increases in hsp70c, the constitutive member of this protein family, are also positively correlated to oxidative protection. A rat cDNA encoding hsp70c was inserted into a mammalian expression vector, allowing transcription of the inserted gene to be regulated by a powerful cytomegaloviral promoter. After introduction of this construct into H9c2 myocytes, stable clones were obtained. Western and Northern blot analysis of the derived clones showed a two-fold increase in hsp70c mRNA and protein concentrations. These clones were more resistant to thermal killing when compared to control cells transfected with the vector alone, implicating a functional role for the overexpressed hsp70c protein. hsp70c-enriched cells also exhibited a marked resistance to oxidative challenges, including exposure to hydrogen peroxide (H2O2), hydroxyl radical, menadione, and hypoxia/reoxygenation. These findings indicate that hsp70c overexpression provides a protective effect against endogenous or exogenously generated reactive oxygen species (ROS), suggesting that hsp70c actively participates in the heat shock-induced oxidative protection.