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.

S-nitrosylation of connexin43 hemichannels elicits cardiac stress–induced arrhythmias in Duchenne muscular dystrophy mice

“…Iso promotes S-nitrosylation and opening of lateralized Cx43 hemichannels in the Dmd mdx heart. Previous reports show β-adrenergic stimulation activates NO synthases (NOSs) and promotes S-nitrosylation of several Ca 2+ -handling proteins in the heart (29,30). Because opening of Cx43 hemichannels has been linked to NO production in astrocytes (31), we evaluated whether Cx43 is S-nitrosylated in the hearts of WT and Dmd mdx mice upon Iso stimulation.…”

“…In addition to the canonical pathway of NO-induced protein kinase G activation, NO also induces direct posttranslational modification of thiol groups in specific cysteine residues on various proteins via S-nitrosylation (59). S-nitrosylation of several Ca 2+ -handling proteins, including RyR, SERCA2-associated phospholamban, NCX, and troponin C (29,30,60), are promoted by β-adrenergic receptors' activation. In the DMD heart, the expression of neuronal NO synthases (nNOSs) and endothelial NO synthases (eNOSs) is reduced (61); however, there is a significant increase in the levels of inducible NO synthase (iNOS) (10,61).…”

Arrhythmogenic Mechanisms in Heart Failure: Linking β-Adrenergic Stimulation, Stretch, and Calcium

“…Downstream from nNOS signaling, CaMKII was also found to modulate afterload-induced Ca 2+ sparks. The mechanically induced SR Ca 2+ leak by nNOS is expected to deplete the SR of Ca 2+ , however, SR Ca 2+ content is maintained presumably via enhanced SERCA Ca 2+ reuptake by nNOS ( Vielma et al, 2016 ).…”

Copper in the suprachiasmatic circadian clock: A possible link between multiple circadian oscillators

“…Nitric oxide (NO) production by NOS is also involved in glutamate‐induced phase shifts, and NO has been shown to activate ryanodine receptors in addition to other signaling molecules (Kakizawa, ; Kakizawa et al., ; Mikami et al., ; Vielma et al., ; Wang, Viatchenko‐Karpinski, et al., ). Therefore, we investigated the role of NO in Cu‐ and TTM‐induced phase shifts by inhibiting NOS activity with L‐NAME (Figure A), which induces no phase shifts when applied by itself (Ding et al., ; Artinian, Ding, & Gillette, ).…”