The induction of heat shock proteins during stress in part of the basic biological response of cells to injury. Previous studies have suggested that the 70 kDA heat shock protein (HSP 70) appears to protect cells against injury by stabilizing the structure of macromolecules, as well as by reactivating denatured proteins. Despite the recognized importance of HSP 70 in a variety of biological model systems, the role of HSP 70 in the cardiac myocyte is unknown. Studies from our laboratory have shown that brief ischemia in intact hearts resulted in an increase in HSP 70 mRNA, as well as in HSP 70 protein. These initial studies did not address the important question of whether HSP 70 plays a protective role in myocardial injury, or whether instead HSP 70 expression is simply a nonspecific host response to tissue injury. Given the complexity of study the role of HSP 70 in the intact heart, it has not been possible to address this question in a simple experimental system. With the advent of the AT-1 cardiac cell line, which can be passaged in culture, it is now possible to selectively overexpress the HSP 70 gene in a simple experimental system, and thereby isolate and examine the potential role of HSP 70 in protecting the cardiac cell against injury. The purpose of the present application is to test the hypothesis that HSP 70 protects cultured cardiac cells against hypoxic injury. This will be accomplished in three closely related specific aims.
In Specific Aim 1, we will overexpress the HSP 70 gene in cultured cardiac cells in the presence and absence of hypoxia, in order to determine whether overexpression HSP 70 is sufficient to protect cultured cardiac cells against hypoxic injury.
In Specific Aim 2, we will investigate whether the protection of protein synthesis by HSP 70 following hypoxia (observed in pilot experiments) is mediated through stabilization of ribosome formation of protection of RNA synthesis.
In Specific Aim 3, we will extend the findings obtained in Specific Aim 1, by mutating known functional domains of the HSP 70 protein, in order to determine how the HSP 70 molecule protects the cardiac myocyte from hypoxia. The long term objective of this research is to understand the function(s) of the heat shock proteins and their role in the cardiac cell under normal conditions and stress. This project will take an initial important first step toward this goal by demonstrating in an extremely simple experimental system that HSP 70 is capable and sufficient to protect cardiac cells from hypoxic injury. The significance of this research is that elucidation of the basic biological mechanisms whereby stress-response proteins are protective may eventually lead to new insight into methods for myocardial protection and preservation during ischemia, reperfusion, or following coronary bypass surgery.
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