S-Nitrosylation of Sarcomeric Proteins Depresses Myofilament Ca2+)Sensitivity in Intact Cardiomyocytes.

Published

Journal Article

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.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.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.

Full Text

Duke Authors

Cited Authors

  • Figueiredo-Freitas, C; Dulce, RA; Foster, MW; Liang, J; Yamashita, AMS; Lima-Rosa, FL; Thompson, JW; Moseley, MA; Hare, JM; Nogueira, L; Sorenson, MM; Pinto, JR

Published Date

  • November 2015

Published In

Volume / Issue

  • 23 / 13

Start / End Page

  • 1017 - 1034

PubMed ID

  • 26421519

Pubmed Central ID

  • 26421519

Electronic International Standard Serial Number (EISSN)

  • 1557-7716

International Standard Serial Number (ISSN)

  • 1523-0864

Digital Object Identifier (DOI)

  • 10.1089/ars.2015.6275

Language

  • eng