title: S-Nitrosylation of Sarcomeric Proteins Depresses Myofilament Ca2+)Sensitivity in Intact Cardiomyocytes.
abstract: OBJECTIVE
  The heart responds to physiological and pathophysiological stress factors by increasing its production of nitric oxide (NO), which reacts with intracellular glutathione to form S-nitrosoglutathione (GSNO), a protein S-nitrosylating agent. Although S-nitrosylation protects some cardiac proteins against oxidative stress, direct effects on myofilament performance are unknown. We hypothesize that S-nitrosylation of sarcomeric proteins will modulate the performance of cardiac myofilaments.
  
  
  RESULTS
  Incubation of intact mouse cardiomyocytes with S-nitrosocysteine (CysNO, a cell-permeable low-molecular-weight nitrosothiol) significantly decreased myofilament Ca(2+) sensitivity. In demembranated (skinned) fibers, S-nitrosylation with 1 μM GSNO also decreased Ca(2+) sensitivity of contraction and 10 μM reduced maximal isometric force, while inhibition of relaxation and myofibrillar ATPase required higher concentrations (≥ 100 μM). Reducing S-nitrosylation with ascorbate partially reversed the effects on Ca(2+) sensitivity and ATPase activity. In live cardiomyocytes treated with CysNO, resin-assisted capture of S-nitrosylated protein thiols was combined with label-free liquid chromatography-tandem mass spectrometry to quantify S-nitrosylation and determine the susceptible cysteine sites on myosin, actin, myosin-binding protein C, troponin C and I, tropomyosin, and titin. The ability of sarcomere proteins to form S-NO from 10-500 μM CysNO in intact cardiomyocytes was further determined by immunoblot, with actin, myosin, myosin-binding protein C, and troponin C being the more susceptible sarcomeric proteins.
  
  
  CONCLUSIONS
  Thus, specific physiological effects are associated with S-nitrosylation of a limited number of cysteine residues in sarcomeric proteins, which also offer potential targets for interventions in pathophysiological situations.
authors.affiliation: 1 Department of Biomedical Sciences, College of Medicine, Florida State University , Tallahassee, Florida.|2 Instituto de Bioquímica Médica Leopoldo de Meis (IBqM), Federal University of Rio de Janeiro , Rio de Janeiro, Brazil .|3 Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami , Miami, Florida.
authors.forename: Cícero
authors.initials: C
authors.lastname: Figueiredo-Freitas
authors.affiliation: 4 Interdisciplinary Stem Cell Institute, University of Miami , Miami, Florida.
authors.forename: Raul A
authors.initials: RA
authors.lastname: Dulce
authors.affiliation: 5 Pulmonary, Allergy and Critical Care Medicine, Duke University Medical Center , Durham, North Carolina.|6 Proteomics and Metabolomics Shared Resource, Duke University Medical Center , Durham, North Carolina.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Foster
authors.affiliation: 3 Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami , Miami, Florida.
authors.forename: Jingsheng
authors.initials: J
authors.lastname: Liang
authors.affiliation: 2 Instituto de Bioquímica Médica Leopoldo de Meis (IBqM), Federal University of Rio de Janeiro , Rio de Janeiro, Brazil .
authors.forename: Aline M S
authors.initials: AM
authors.lastname: Yamashita
authors.affiliation: 2 Instituto de Bioquímica Médica Leopoldo de Meis (IBqM), Federal University of Rio de Janeiro , Rio de Janeiro, Brazil .
authors.forename: Frederico L
authors.initials: FL
authors.lastname: Lima-Rosa
authors.affiliation: 6 Proteomics and Metabolomics Shared Resource, Duke University Medical Center , Durham, North Carolina.
authors.forename: J Will
authors.initials: JW
authors.lastname: Thompson
authors.affiliation: 6 Proteomics and Metabolomics Shared Resource, Duke University Medical Center , Durham, North Carolina.
authors.forename: M Arthur
authors.initials: MA
authors.lastname: Moseley
authors.affiliation: 4 Interdisciplinary Stem Cell Institute, University of Miami , Miami, Florida.
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Hare
authors.affiliation: 2 Instituto de Bioquímica Médica Leopoldo de Meis (IBqM), Federal University of Rio de Janeiro , Rio de Janeiro, Brazil .
authors.forename: Leonardo
authors.initials: L
authors.lastname: Nogueira
authors.affiliation: 2 Instituto de Bioquímica Médica Leopoldo de Meis (IBqM), Federal University of Rio de Janeiro , Rio de Janeiro, Brazil .
authors.forename: Martha M
authors.initials: MM
authors.lastname: Sorenson
authors.affiliation: 1 Department of Biomedical Sciences, College of Medicine, Florida State University , Tallahassee, Florida.|3 Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami , Miami, Florida.
authors.forename: José Renato
authors.initials: JR
authors.lastname: Pinto
mesh_terms: D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D020013:Calcium Signaling; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D009124:Muscle Proteins / Q000378:metabolism*; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D018384:Oxidative Stress; D026403:S-Nitrosothiols / Q000378:metabolism*; D012518:Sarcomeres
chemical_list: D009124:Muscle Proteins; D026403:S-Nitrosothiols; D009569:Nitric Oxide; C021315:S-nitrosocysteine; D017301:Ca(2+) Mg(2+)-ATPase; D003545:Cysteine