title: Nitric Oxide Synthase 1 Modulates Basal and β-Adrenergic-Stimulated Contractility by Rapid and Reversible Redox-Dependent S-Nitrosylation of the Heart.
abstract: S-nitrosylation of several Ca2+ regulating proteins in response to β-adrenergic stimulation was recently described in the heart; however the specific nitric oxide synthase (NOS) isoform and signaling pathways responsible for this modification have not been elucidated. NOS-1 activity increases inotropism, therefore, we tested whether β-adrenergic stimulation induces NOS-1-dependent S-nitrosylation of total proteins, the ryanodine receptor (RyR2), SERCA2 and the L-Type Ca2+ channel (LTCC). In the isolated rat heart, isoproterenol (10 nM, 3-min) increased S-nitrosylation of total cardiac proteins (+46±14%) and RyR2 (+146±77%), without affecting S-nitrosylation of SERCA2 and LTCC. Selective NOS-1 blockade with S-methyl-L-thiocitrulline (SMTC) and Nω-propyl-l-arginine decreased basal contractility and relaxation (-25-30%) and basal S-nitrosylation of total proteins (-25-60%), RyR2, SERCA2 and LTCC (-60-75%). NOS-1 inhibition reduced (-25-40%) the inotropic response and protein S-nitrosylation induced by isoproterenol, particularly that of RyR2 (-85±7%). Tempol, a superoxide scavenger, mimicked the effects of NOS-1 inhibition on inotropism and protein S-nitrosylation; whereas selective NOS-3 inhibitor L-N5-(1-Iminoethyl)ornithine had no effect. Inhibition of NOS-1 did not affect phospholamban phosphorylation, but reduced its oligomerization. Attenuation of contractility was abolished by PKA blockade and unaffected by guanylate cyclase inhibition. Additionally, in isolated mouse cardiomyocytes, NOS-1 inhibition or removal reduced the Ca2+-transient amplitude and sarcomere shortening induced by isoproterenol or by direct PKA activation. We conclude that 1) normal cardiac performance requires basal NOS-1 activity and S-nitrosylation of the calcium-cycling machinery; 2) β-adrenergic stimulation induces rapid and reversible NOS-1 dependent, PKA and ROS-dependent, S-nitrosylation of RyR2 and other proteins, accounting for about one third of its inotropic effect.
authors.affiliation: Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, PO Box 114-D, Santiago, Chile.
authors.forename: Alejandra Z
authors.initials: AZ
authors.lastname: Vielma
authors.affiliation: Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, PO Box 114-D, Santiago, Chile.
authors.forename: Luisa
authors.initials: L
authors.lastname: León
authors.affiliation: Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, PO Box 114-D, Santiago, Chile.
authors.forename: Ignacio C
authors.initials: IC
authors.lastname: Fernández
authors.affiliation: Departamento de Ciencias Básicas Biomédicas, Facultad de Ciencias de la Salud, Universidad de Talca, Av. Lircay S.N., Talca, Chile.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: González
authors.affiliation: Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, PO Box 114-D, Santiago, Chile.
authors.forename: Mauricio P
authors.identifier: http://orcid.org/0000-0001-6735-6675
authors.initials: MP
authors.lastname: Boric
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020836:Protein Structure, Quaternary; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D026403:S-Nitrosothiols / Q000378:metabolism*; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D007545:Isoproterenol; D052248:Nitric Oxide Synthase Type I; D017382:Reactive Oxygen Species; D011943:Receptors, Adrenergic, beta; D026403:S-Nitrosothiols; D000099211:Phospholamban