title: Adverse effects of doxorubicin and its metabolic product on cardiac RyR2 and SERCA2A.
abstract: The use of anthracycline chemotherapeutic drugs is restricted owing to potentially fatal cardiotoxic side effects. It has been hypothesized that anthracycline metabolites have a primary role in this cardiac dysfunction; however, information on the molecular interactions of these compounds in the heart is scarce. Here we provide novel evidence that doxorubicin and its metabolite, doxorubicinol, bind to the cardiac ryanodine receptor (RyR2) and to the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA2A) and deleteriously alter their activity. Both drugs (0.01 μM-2.5 μM) activated single RyR2 channels, and this was reversed by drug washout. Both drugs caused a secondary inhibition of RyR2 activity that was not reversed by drug washout. Preincubation with the reducing agent dithiothreitol (DTT, 1 mM) prevented drug-induced inhibition of channel activity. Doxorubicin and doxorubicinol reduced the abundance of thiol groups on RyR2, further indicating that oxidation reactions may be involved in the actions of the compounds. Ca(2+) uptake into sarcoplasmic reticulum vesicles by SERCA2A was inhibited by doxorubicinol, but not doxorubicin. Unexpectedly, in the presence of DTT, doxorubicinol enhanced the rate of Ca(2+) uptake by SERCA2A. Together the evidence provided here shows that doxorubicin and doxorubicinol interact with RyR2 and SERCA2A in similar ways, but that the metabolite acts with greater efficacy than the parent compound. Both compounds modify RyR2 and SERCA2A activity by binding to the proteins and also act via thiol oxidation to disrupt SR Ca(2+) handling. These actions would have severe consequences on cardiomyocyte function and contribute to clinical symptoms of acute anthracycline cardiotoxicity.
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
authors.forename: Amy D
authors.initials: AD
authors.lastname: Hanna
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
authors.forename: Alex
authors.initials: A
authors.lastname: Lam
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
authors.forename: Steffi
authors.initials: S
authors.lastname: Tham
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
authors.forename: Angela F
authors.initials: AF
authors.lastname: Dulhunty
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia nicole.beard@canberra.edu.au.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Beard
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D054715:Cardiotoxins / Q000494:pharmacology; D004317:Doxorubicin / Q000009:adverse effects / Q000031:analogs & derivatives* / Q000494:pharmacology; D008051:Lipid Bilayers / Q000378:metabolism; D011485:Protein Binding; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D012756:Sheep
chemical_list: D054715:Cardiotoxins; D008051:Lipid Bilayers; D019837:Ryanodine Receptor Calcium Release Channel; D004317:Doxorubicin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C010013:adriamycinol; D002118:Calcium
references.citation: J Med Chem. 1979 Aug;22(8):912-8
references.pmid: 490536
references.citation: J Biol Chem. 1997 Feb 7;272(6):3739-48
references.pmid: 9013631
references.citation: Mol Pharmacol. 1983 Sep;24(2):336-40
references.pmid: 6888375
references.citation: Pediatr Cardiol. 2011 Mar;32(3):342-53
references.pmid: 21221562
references.citation: Proc Natl Acad Sci U S A. 2006 Mar 7;103(10):3604-9
references.pmid: 16505371
references.citation: Eur J Pharmacol. 2010 Nov 10;646(1-3):12-21
references.pmid: 20713040
references.citation: Circ Res. 1990 Nov;67(5):1167-74
references.pmid: 2171802
references.citation: Biochem Pharmacol. 2000 Nov 15;60(10):1435-44
references.pmid: 11020445
references.citation: Pharmacol Ther. 2005 Sep;107(3):377-91
references.pmid: 15951021
references.citation: Cell Calcium. 2008 Oct;44(4):363-73
references.pmid: 19230141
references.citation: Toxicol Appl Pharmacol. 1993 Feb;118(2):177-85
references.pmid: 8441996
references.citation: J Membr Biol. 1999 Feb 1;167(3):205-14
references.pmid: 9929372
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Anticancer Drug Des. 1995 Dec;10(8):641-53
references.pmid: 8595123
references.citation: Mol Pharmacol. 2011 Sep;80(3):538-49
references.pmid: 21697274
references.citation: J Biol Chem. 1997 Mar 14;272(11):7069-77
references.pmid: 9054399
references.citation: Pharmacol Rep. 2009 Jan-Feb;61(1):154-71
references.pmid: 19307704
references.citation: Antioxid Redox Signal. 2011 Nov 1;15(9):2543-63
references.pmid: 21457105
references.citation: Circ Res. 2012 Sep 28;111(8):1091-106
references.pmid: 23023511
references.citation: Biophys J. 1999 Jul;77(1):189-203
references.pmid: 10388749
references.citation: Nat Med. 2004 Nov;10(11):1200-7
references.pmid: 15489859
references.citation: Front Physiol. 2012 Sep 14;3:369
references.pmid: 23049513
references.citation: Expert Opin Drug Saf. 2012 May;11 Suppl 1:S21-36
references.pmid: 21635149
references.citation: J Biol Chem. 1988 Dec 15;263(35):18750-8
references.pmid: 3198599
references.citation: J Biol Chem. 1985 Jun 25;260(12):7349-55
references.pmid: 2581966
references.citation: Mol Pharmacol. 2005 May;67(5):1505-12
references.pmid: 15705743
references.citation: J Mol Cell Cardiol. 1999 Aug;31(8):1435-46
references.pmid: 10423342
references.citation: Cancer Res. 2000 Sep 15;60(18):5158-64
references.pmid: 11016643
references.citation: Toxicol Appl Pharmacol. 2000 Dec 1;169(2):168-76
references.pmid: 11097869
references.citation: J Biol Chem. 2004 Aug 13;279(33):34514-20
references.pmid: 15197184
references.citation: Science. 1998 Jan 9;279(5348):234-7
references.pmid: 9422697
references.citation: Cochrane Database Syst Rev. 2006 Oct 18;(4):CD005008
references.pmid: 17054232
references.citation: Cardiovasc Res. 2006 Jul 15;71(2):310-21
references.pmid: 16581043
references.citation: Circ Res. 2002 Jan 11;90(1):14-7
references.pmid: 11786512
references.citation: J Mol Cell Cardiol. 1997 Oct;29(10):2631-40
references.pmid: 9344758
references.citation: J Mol Cell Cardiol. 2011 Nov;51(5):749-59
references.pmid: 21819992
references.citation: J Biol Chem. 2001 Oct 19;276(42):38814-9
references.pmid: 11477077
references.citation: Cancer Res. 2003 Oct 15;63(20):6602-6
references.pmid: 14583452
references.citation: Proc Natl Acad Sci U S A. 1988 May;85(10):3585-9
references.pmid: 2897122
references.citation: Biochem Biophys Res Commun. 1977 Jul 25;77(2):797-803
references.pmid: 20093
references.citation: Mol Pharmacol. 1999 May;55(5):821-31
references.pmid: 10220560
references.citation: Biophys J. 2002 Jan;82(1 Pt 1):310-20
references.pmid: 11751318
references.citation: Br J Cancer. 2009 Jan 27;100(2):311-4
references.pmid: 19165199
references.citation: Antioxid Redox Signal. 2007 May;9(5):609-21
references.pmid: 17465884
references.citation: Adv Pharmacol. 2010;59:1-30
references.pmid: 20933197
references.citation: Biochem Biophys Res Commun. 2002 Mar 1;291(3):433-8
references.pmid: 11855807
references.citation: Front Biosci (Landmark Ed). 2011 Jan 01;16(2):553-67
references.pmid: 21196188
references.citation: Br J Pharmacol. 1993 Nov;110(3):975-82
references.pmid: 8298821
references.citation: Chem Biol Interact. 1988;65(2):157-73
references.pmid: 2835188
references.citation: Br J Pharmacol. 2003 Jun;139(3):641-51
references.pmid: 12788824
references.citation: Am J Physiol. 1997 Jun;272(6 Pt 1):C1908-18
references.pmid: 9227420
references.citation: Mol Pharmacol. 2005 Jan;67(1):97-104
references.pmid: 15492117
references.citation: Biochem J. 2005 Aug 15;390(Pt 1):333-43
references.pmid: 15916532
references.citation: Mol Pharmacol. 1990 Apr;37(4):503-14
references.pmid: 2157959
references.citation: J Biol Chem. 1992 May 15;267(14):9474-7
references.pmid: 1374404
references.citation: Biochim Biophys Acta. 1993 Jan 15;1161(1):73-8
references.pmid: 8380722
references.citation: J Membr Biol. 1995 Sep;147(1):7-22
references.pmid: 8531200
references.citation: Eur Heart J. 2007 May;28(9):1135-42
references.pmid: 17347175
references.citation: Cancer Chemother Pharmacol. 1995;35(3):213-8
references.pmid: 7805179
references.citation: J Mol Cell Cardiol. 1998 Feb;30(2):243-54
references.pmid: 9515001
references.citation: Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4
references.pmid: 388439
references.citation: Circ Res. 2003 Sep 19;93(6):487-90
references.pmid: 14500331
references.citation: Antioxid Redox Signal. 2008 Oct;10(10):1675-86
references.pmid: 18522493
references.citation: Eur J Pharmacol. 2001 Jul 6;423(2-3):99-107
references.pmid: 11448472
references.citation: J Free Radic Biol Med. 1985;1(1):43-9
references.pmid: 3939136
references.citation: Cardiovasc Res. 2009 Dec 1;84(3):387-95
references.pmid: 19617226