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

Antioxid Redox Signal. 2015 Nov 1;23(13):1017-34. doi: 10.1089/ars.2015.6275.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Ca(2+) Mg(2+)-ATPase / metabolism
  • Calcium Signaling*
  • Cells, Cultured
  • Cysteine / analogs & derivatives*
  • Cysteine / metabolism
  • Mice, 129 Strain
  • Mice, Inbred C57BL
  • Muscle Proteins / metabolism*
  • Myocardial Contraction
  • Myocytes, Cardiac / metabolism*
  • Myofibrils / metabolism*
  • Nitric Oxide / metabolism
  • Oxidative Stress
  • S-Nitrosothiols / metabolism*
  • Sarcomeres


  • Muscle Proteins
  • S-Nitrosothiols
  • Nitric Oxide
  • S-nitrosocysteine
  • Ca(2+) Mg(2+)-ATPase
  • Cysteine