(Verbatim from the application): Reactive oxygen species (ROS) are formed during reoxygenation of ischemic hearts. These oxygen byproducts may initiate tissue damage. The inability of the myocardium to defend against ROS toxicity may lead to ischemia/reperfusion (I/R) injury. Hence, modulation of cellular stress defenses has a clinical significance regarding myocardial preservation during I/R. The inducible heat shock protein 70 (hsp70) is cardioprotective during I/R. Using H9c2 heart cells, we found that its constitutive isoform (hsc70) is also protective against stress. Furthermore, hsc70 has a lower threshold for redistribution and induction activation by mild priming stress and thus is a suitable candidate for pharmacological manipulation. Hsc70 overexpression stabilizes the membrane during hydrogen peroxide (H2O2) exposure, and a simultaneous increase in hsc70 and hsp70 confers a greater stress protection than individual hsp alone. We postulate that hsc70 alone can protect against I/R injury, and through its interaction with hsp70, this protein affords a marked I/R protection. A proteasome inhibitor, lactacystin, suppressed hsc7O-mediated membrane protection, suggesting that hsc7O overexpression may help membrane preservation by facilitating the turnover of certain stress-modified proteins. To test these hypotheses, both H9c2 cell and transgenic animal models will be employed. We will (1) explore the membrane-stabilization mechanism of hsc70 by demonstrating a redistribution of hsc70 to the plasma or mitochondrial membranes during stress, and examine the resultant change in lipid order; (2) use multiple inhibitors to confirm an involvement of proteolysis in hsc70-mediated protection; (3) examine whether hsc70 overexpression alone protects against I/R-induced myocardial infarction; and (4) study the potential interaction between hsc70/hsp70 to improve stress resistance by examining if these hsps bind and stabilize the same cellular target sites, such as the membrane, during stress. This study may enhance our understanding of the hsc70protective mechanism(s).
