The role of MRP1 in Protection of Cardiac Injury

MRP1 在保护心脏损伤中的作用

• 批准号: 8300175
• 负责人: Mary Vore
• 金额: $ 26.19万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-25 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8300175
• 关键词: 4 hydroxynonenal; ABCC1 gene; ATP-Binding Cassette Transporters; Adriamycin PFS; Alkylation; Antibodies; Biochemical; Biological Assay; Cardiac; Cardiac Myocytes; Cardiotoxicity; Cell Line; Cell membrane; Cells; Collaborations; Coupled; Cysteine; Development; Dose; Esters; Fractionation; Genes; Genotype; Glutathione; Glutathione Disulfide; Goals; Heart; Histidine; Human; Immunohistochemistry; In Vitro; Injury; Knockout Mice; Lead; Lysine; Measures; Mediating; Messenger RNA; Mitochondria; Modality; Morphology; Mus; Normal tissue morphology; Oxidation-Reduction; Oxidative Stress; P-Glycoprotein; P-Glycoproteins; Production; Protein Isoforms; Proteins; Proteomics; Quantitative Microscopy; Regulation; Role; Sarcolemma; Schiff Bases; Site; Testing; Therapeutic; Time; Tissues; Toxic effect; Transgenic Mice; Transgenic Organisms; Tumor Necrosis Factor-alpha; Vesicle; Wild Type Mouse; adduct; base; buthionine; cancer therapy; chemotherapeutic agent; cytotoxic; functional group; in vivo; novel; overexpression; oxidant stress; oxidative damage; response; treatment effect; uptake

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 1will 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.

空间 提供了' 本申请的目的是表征多药耐药蛋白1（Mrp 1）在肿瘤细胞中的作用。 保护心脏免受氧化应激。我们推测，阿霉素（ADR）治疗癌症导致 氧化应激，这反过来又导致产生肿瘤坏死因子-a（TNF）， 并导致正常组织损伤。Mrp 1是一种ATP结合盒（ABC）转运蛋白， 谷胱甘肽（GSH）及其结合物的ATP依赖性外排，包括细胞毒性药物的GSH结合物 氧化应激产物4-羟基壬烯醛（HNE; GS-HNE）。我们将检验以下假设：1）心脏 Mrp 1的表达通过介导细胞外排保护心脏免受ADR诱导的氧化应激和损伤。 GS-HNE; 2）Mrp 1表达增加，并定位于质膜和线粒体中 氧化应激和/或TNF，和3）过量产生HNE和GS-HNE使Mrp 1失活， 超过其保护作用，并加剧氧化损伤。四个具体目标将测试这些 假设;目的1将利用Mrp 1敲除小鼠来评估其在保护心脏免受ADR诱导的损伤中的作用。 氧化应激和损伤，MnSOD补偿Mrp 1损失的能力，以及TNF在 调节Mrp 1的表达。目的2将描述Mrp 1的亚细胞定位和功能， ADR和TNF治疗。目的3探讨氧化应激在Mrp 1表达调控中的作用 和本地化。最后，目标4将通过以下方式表征HNE和GS-HNE抑制Mrp 1的能力： 关键的半胱氨酸、组氨酸或赖氨酸残基的烷基化。我们将利用不同基因型的小鼠（Mrp 1无效 小鼠、MnSOD转基因小鼠和杂合（+/-）小鼠）来评估这些基因在保护中的作用 共聚焦免疫荧光和定量免疫金 心肌细胞中Mrp 1表达的定位分析和HEK 293细胞中Mrp 1表达的分析 蛋白质组学分析将用于鉴定Mrp 1的潜在结构异构体 以及Mrp 1的HNE烷基化位点。了解Mrp 1、TNF和GSH在保护肝细胞中的作用 心脏从ADR引起的组织损伤将导致辅助治疗方式的发展， 保护免受这种伤害，从而允许使用更高剂量的这种高效的 化疗剂。