title: HNO enhances SERCA2a activity and cardiomyocyte function by promoting redox-dependent phospholamban oligomerization.
abstract: OBJECTIVE
  Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca(2+) uptake and myofilament Ca(2+) sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in a redox-dependent manner, improving Ca(2+) handling in isolated myocytes/hearts.
  
  
  RESULTS
  Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln(-/-)) mice. Compared to WT, pln(-/-) myocytes displayed enhanced resting sarcomere shortening, peak Ca(2+) transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca(2+) transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln(-/-) cells/hearts. HNO enhanced SR Ca(2+) uptake in WT but not pln(-/-) SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca(2+)-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available.
  
  
  METHODS
  HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a.
  
  
  CONCLUSIONS
  PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca(2+) handling in failing hearts.
authors.affiliation: 1 Division of Cardiology, Johns Hopkins Medical Institutions , Baltimore, Maryland.
authors.forename: Vidhya
authors.initials: V
authors.lastname: Sivakumaran
authors.forename: Brian A
authors.initials: BA
authors.lastname: Stanley
authors.forename: Carlo G
authors.initials: CG
authors.lastname: Tocchetti
authors.forename: Jeff D
authors.initials: JD
authors.lastname: Ballin
authors.forename: Viviane
authors.initials: V
authors.lastname: Caceres
authors.forename: Lufang
authors.initials: L
authors.lastname: Zhou
authors.forename: Gizem
authors.initials: G
authors.lastname: Keceli
authors.forename: Peter P
authors.initials: PP
authors.lastname: Rainer
authors.forename: Dong I
authors.initials: DI
authors.lastname: Lee
authors.forename: Sabine
authors.initials: S
authors.lastname: Huke
authors.forename: Mark T
authors.initials: MT
authors.lastname: Ziolo
authors.forename: Evangelia G
authors.initials: EG
authors.lastname: Kranias
authors.forename: John P
authors.initials: JP
authors.lastname: Toscano
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Wilson
authors.forename: Brian
authors.initials: B
authors.lastname: O'Rourke
authors.forename: David A
authors.initials: DA
authors.lastname: Kass
authors.forename: James E
authors.initials: JE
authors.lastname: Mahaney
authors.forename: Nazareno
authors.initials: N
authors.lastname: Paolocci
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002316:Cardiotonic Agents / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004220:Disulfides; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D008861:Microsomes / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism*; D009589:Nitrogen Oxides / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization / Q000187:drug effects*; D055550:Protein Stability / Q000187:drug effects; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D000099211:Phospholamban
chemical_list: D000255:Adenosine Triphosphate; D000975:Antioxidants; D002118:Calcium; D002135:Calcium-Binding Proteins; D002316:Cardiotonic Agents; D017868:Cyclic AMP-Dependent Protein Kinases; D004220:Disulfides; D009589:Nitrogen Oxides; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C039900:nitroxyl
references.citation: Biochemistry. 1996 Sep 3;35(35):11247-59
references.pmid: 8784178
references.citation: Proc Natl Acad Sci U S A. 2011 Feb 15;108(7):2735-40
references.pmid: 21282613
references.citation: Biochemistry. 1991 Jul 23;30(29):7171-80
references.pmid: 1649624
references.citation: Nat Rev Mol Cell Biol. 2003 Jul;4(7):566-77
references.pmid: 12838339
references.citation: Biophys J. 1986 Apr;49(4):937-42
references.pmid: 3013331
references.citation: Antioxid Redox Signal. 2011 Mar 15;14(6):1023-37
references.pmid: 20649472
references.citation: J Mol Cell Cardiol. 2001 Jul;33(7):1345-53
references.pmid: 11437540
references.citation: Cardiovasc Res. 1987 Aug;21(8):601-5
references.pmid: 3328648
references.citation: Circulation. 2006 Feb 21;113(7):995-1004
references.pmid: 16476846
references.citation: Biophys J. 1986 Apr;49(4):921-35
references.pmid: 3013330
references.citation: Biochemistry. 1986 Jan 14;25(1):194-202
references.pmid: 3006752
references.citation: J Biol Chem. 1988 Jul 5;263(19):9178-86
references.pmid: 2837480
references.citation: Pflugers Arch. 2009 Jan;457(3):687-700
references.pmid: 18415121
references.citation: Protein Expr Purif. 2004 Mar;34(1):56-67
references.pmid: 14766300
references.citation: Circulation. 1999 Dec 7;100(23):2308-11
references.pmid: 10587333
references.citation: Circ Res. 2012 Sep 28;111(8):1091-106
references.pmid: 23023511
references.citation: Front Biosci (Elite Ed). 2010 Jan 01;2:614-26
references.pmid: 20036906
references.citation: Eur Heart J. 2011 Aug;32(15):1838-45
references.pmid: 21388993
references.citation: Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5537-42
references.pmid: 12704230
references.citation: Circulation. 2002 Feb 26;105(8):904-7
references.pmid: 11864915
references.citation: Nat Clin Pract Cardiovasc Med. 2008 Sep;5(9):554-65
references.pmid: 18665137
references.citation: Circ Res. 2009 Mar 27;104(6):720-3
references.pmid: 19265039
references.citation: Circ Res. 2012 Sep 28;111(8):1002-11
references.pmid: 22851540
references.citation: Biochemistry. 2003 Sep 16;42(36):10674-82
references.pmid: 12962492
references.citation: Circ Res. 2007 Jan 5;100(1):96-104
references.pmid: 17138943
references.citation: Ann N Y Acad Sci. 2003 Apr;986:338-40
references.pmid: 12763844
references.citation: J Card Fail. 2009 Apr;15(3):171-81
references.pmid: 19327618
references.citation: J Physiol. 2007 May 1;580(Pt.3):951-60
references.pmid: 17331988
references.citation: Circ Res. 2012 Jun 8;110(12):1646-60
references.pmid: 22679139
references.citation: Biochemistry. 2005 May 31;44(21):7713-24
references.pmid: 15909986
references.citation: Circ Res. 2003 Mar 7;92(4):350-8
references.pmid: 12623873
references.citation: Circ Res. 1999 Jul 9;85(1):38-46
references.pmid: 10400909
references.citation: Circ Res. 1993 Feb;72(2):463-9
references.pmid: 8418995
references.citation: J Clin Invest. 2001 Apr;107(8):967-74
references.pmid: 11306600
references.citation: Biochemistry. 2007 Feb 20;46(7):1999-2009
references.pmid: 17261028
references.citation: J Biol Chem. 1997 Jun 13;272(24):15061-4
references.pmid: 9182523
references.citation: Biochemistry. 2008 Dec 16;47(50):13150-2
references.pmid: 19053265
references.citation: Biochemistry. 2009 Mar 24;48(11):2411-21
references.pmid: 19191503
references.citation: Circulation. 2001 Sep 18;104(12):1424-9
references.pmid: 11560860
references.citation: Circ Res. 2001 Jun 8;88(11):E66-7
references.pmid: 11397790
references.citation: Antioxid Redox Signal. 2011 May 1;14(9):1687-98
references.pmid: 21235349
references.citation: J Biol Chem. 1997 Jun 20;272(25):15872-80
references.pmid: 9188486
references.citation: Circulation. 2005 May 31;111(21):2837-49
references.pmid: 15927992
references.citation: J Biol Chem. 1949 Feb;177(2):751-66
references.pmid: 18110453
references.citation: Circ J. 2009 Sep;73(9):1561-7
references.pmid: 19667488
references.citation: J Biol Chem. 2001 Jun 29;276(26):24145-52
references.pmid: 11328820
references.citation: Mol Ther. 2008 Jun;16(6):1002-4
references.pmid: 18500238
references.citation: Biochemistry. 1997 Oct 21;36(42):12903-10
references.pmid: 9335549
references.citation: Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10463-8
references.pmid: 11517312