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

Published

Journal Article

AIMS: 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. INNOVATION AND 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.

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 1, 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

Digital Object Identifier (DOI)

  • 10.1089/ars.2015.6275

Language

  • eng

Conference Location

  • United States