The Role of MRP1 in Protection of Cardiac Injury

The goal of the present application is to characterize the role of multidrug resistance protein I (MrpI) 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. Mrpl 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 I) Cardiac expression of MrpI protects the heart from oxidative stress and injury induced by ADR by mediating efflux of GS-HNE; 2) MrpI 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 Mrpl, overwhelming its protective role, and exacerbating oxidative injury. Four Specific Aims will test these hypotheses; Aim 1 will utilize Mrpl null mice to assess its role in protecting the heart from ADR-induced oxidative stress and injury, the ability of MnSOD to compensate for loss of Mrpl, and the function of TNF in regulating Mrpl expression. Aim 2 will characterize the subcellular localization and function of Mrpl following ADR and TNF treatment. Aim 3 will assess the role of oxidative stress in the regulation of Mrpl expression and localization. Finally, Aim 4 will characterize the ability of HNE and GS-HNE to inactivate Mrpl by alkylation of key cysteine, histidine or lysine residues. We will utilize mice of various genotypes (Mrpl null mice, MnSOD transgenic and heterozygous (+/-) mice) to assess the roles of these genes in protection against cardiac injury, confocal immunofluorescent immunohistochemistry and quantitative immunogold analysis for localization of Mrpl expression in the cardiomyocyte, and HEK293 cells for expression of Mrpl to characterize its function; proteomic analyses will be used to identify potential structural isoforms of Mrpl and the sites of HNE alkylation of Mrpl. Understanding the roles of Mrpl, 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.