STABLE HIGH-LEVEL EXPRESSION OF A TRANSFECTED HUMAN HSP70 GENE PROTECTS A HEART-DERIVED MUSCLE-CELL LINE AGAINST THERMAL-STRESS

Heat shock protein 70 (HSP70) has been shown to play a fundamental role in the induction of thermotolerance in many biological systems. Elevated synthesis of HSP70 in response to diverse stresses such as heat, anoxia, ischaemia, ethanol and heavy metals has been correlated with protection against subsequent more severe stress and cross-tolerance to differing stresses. In this respect, exposure of the mammalian heart to sublethal heat treatment or ischaemia has been shown to protect against subsequent myocardial ischaemia with concomitant elevation of HSP70. However, direct demonstration that HSP70 can alone confer thermotolerance has until recently been restricted to transfection of fibroblasts with an HSP70 gene, although preliminary data from others suggests that transfection of H9c2 myocytes with an HSP70 gene can confer tolerance to substrate-free hypoxia. The purpose of this study was, therefore, to test directly whether a myocyte cell line which retains certain features of cardiac cells (as opposed to non-myocyte cells) can be protected against lethal thermal stress by transfection with a single HSP70 gene. Rat heart-derived H9c2 cells were transfected either with a plasmid from which high level expression of a human HSP70 gene is driven by a strong, heterologous (human),β-actin promoter (APr-HS70), or with an analogous control plasmid containing no HSP70 gene (pAPr-1 neo). Following selection with the neomycin analogue G418, several clones were isolated which either expressed no HSP70 (control pAPr-1 neo-derived) or stably expressed high constitutive levels of an inducible isoform of HSP70 (HSP70i) (APr-HS70-derived) as determined by Western blotting with a specific monoclonal antibody to HSP70i. It was found that cells constitutively expressing HSP70i were significantly protected (P≤0.01) against lethal heat stress (47°C for 2 h) compared to cells transfected with the control plasmid. The degree of protection was slightly greater than that obtained by ‘preconditioning’ control (untransfected) H9c2 cells with a mild pre-heat stress (43°C for 30 min followed by 24 h recovery) which was associated with elevation of endogenous HSP70i. These results demonstrate that stable, constitutive over-expression of an inducible isoform of HSP70 can confer a significant degree of thermotolerance to heart-derived muscle cells. © 1994 Academic Press Limited.