SPACE PROVIDED. ' The goal of the present application is to characterize the role of multidrug resistance protein 1 (Mrp1) in protecting the heart from oxidative stress. We postulate that cancer treatment with Adriamycin (ADR)leads to oxidative stress, which in turn leads to production of tumor necrosis factor-a (TNF) that amplifies oxidative stress and causes normal tissue injury. Mrp1 is an ATP-binding cassette (ABC) transporter that mediates the ATP-dependent efflux of glutathione (GSH) and its conjugates, including the GSH conjugate of the cytotoxic product of oxidative stress, 4-hydroxynonenal (HNE; GS-HNE). We will test the hypotheses that 1) Cardiac expression of Mrp1 protects the heart from oxidative stress and injury induced by ADR by mediating efflux of GS-HNE; 2) Mrp1 expression increases and is localized in plasma membrane and mitochondria in response to oxidative stress and/or TNF, and 3) excessive production of HNE and GS-HNE inactivates Mrp1, overwhelming its protective role, and exacerbating oxidative injury.
Four Specific Aims will test these hypotheses;
Aim 1 will utilize Mrp1 null mice to assess its role in protecting the heart from ADR-induced oxidative stress and injury, the ability of MnSODto compensate for loss of Mrp1, and the function of TNF in regulating Mrp1 expression.
Aim 2 will characterize the subcellular localization and function of Mrp1 following ADR and TNF treatment.
Aim 3 will assessthe role of oxidative stress in the regulation of Mrp1 expression and localization. Finally, Aim 4 will characterize the ability of HNEand GS-HNE to inactivate Mrp1 by alkylation of key cysteine, histidine or lysine residues. We will utilize mice of various genotypes (Mrp1 null mice, MnSOD transgenic and heterozygous (+/-) mice) to assessthe roles of these genes in protection against cardiac injury, confocal immunofluorescent immunohistochemistry and quantitative immunogold analysis for localization of Mrp1 expression in the cardiomyocyte, and HEK293 cells for expression of Mrp1 to characterize its function; proteomic analyses will be used to identify potential structural isoforms of Mrp1 and the sites of HNE alkylation of Mrp1. Understanding the roles of Mrp1, TNF and GSH in protecting the heart from ADR-induced tissue injury will lead to the development of ancillary therapeutic modalities to protect against such injury, and thus permit utilization of higher doses of this highly effective chemotherapeutic agent.
