Abstract
Multidrug resistance-associated protein 1 (Mrp1; Abcc1) is expressed in sarcolemma of murine heart, where it probably protects the cardiomyocyte by mediating efflux of endo- and xenobiotics. We used doxorubicin (DOX), a chemotherapeutic drug known to induce oxidative stress and thereby cardiac injury, as a model cardiotoxic compound and observed changes in the Mrp1 expression pattern in cardiac tissue of DOX-versus saline-treated mice. Confocal immunofluorescent and immunogold electron microscopy, together with subcellular fractionation followed by immunoblot analyses and transport measurements, localized functional Mrp1 to mitochondria after DOX. Expressions of Mrp1 in heart homogenate, sarcolemma, and submitochondrial particles (SMP) were increased 1.6-, 2-, and 3-fold, respectively, at 24 h after DOX. Mitochondrial Mrp1 expression was markedly increased 72 h after DOX, whereas transport of Mrp1 substrates in SMP was maximal at 24 h. ATP-dependent transport in SMP occurred into an osmotically sensitive space and was inhibited by the anti-MRP1 antibody QCRL3. Adduction of a 190-kDa protein with the reactive lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was detected in SMP and was maximal at 72 h after DOX; immunoprecipitation confirmed Mrp1-HNE adduction. In vitro, HNE (10 μM) inhibited mitochondrial respiration and transport activity in SMP, suggesting that Mrp1 is adversely affected by oxidative stress. These data demonstrate that after DOX, functional Mrp1 is detected in mitochondria in addition to that in sarcolemma; however, adduction with HNE inhibits Mrp1 activity. Mrp1 may serve to protect the heart by mediating the efflux of toxic products of oxidative stress from mitochondria and cardiomyocytes.
Footnotes
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This work was supported by the National Institutes of Health National Cancer Institute [Grants CA94853, CA139844] and the National Institutes of Health National Institute of General Medical Sciences [Grant GM55343].
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ABBREVIATIONS: DOX, doxorubicin; E217G, estradiol 17β-d-glucuronide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSTα, glutathione transferase α; HEK, human embryonic kidney; HEKMrp1, pCEBV7-Mrp1-transfected human embryonic kidney 293 cells; HNE, 4-hydroxy-2-trans-nonenal; LAMP2, lysosome-associated membrane protein 2; Mrp1, murine multidrug resistant-associated protein 1; SDHB, succinate dehydrogenase subunit B; SMP, submitochondrial particles; VDAC, voltage-dependent anion channel; ABC, ATP-binding cassette; LTC4, leukotriene C4; mAb, monoclonal antibody; TBS, Tris-buffered saline; DMSO, dimethyl sulfoxide; GS-HNE, glutathione conjugate of 4-hydroxy-2-trans-nonenal; Na+/K+-ATPaseα1, anti-sodium/potassium-ATPase α1; pAb, polyclonal antibody.
- Received September 22, 2008.
- Accepted February 18, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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