Nitric oxide is generated in skeletal muscle with activity and decreases Ca2+-sensitivity of the contractile apparatus, putatively by S-nitrosylation of an unidentified protein. We investigate the mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca2+-sensitivity in mammalian fast-twitch (FT) fibers mediated by S-glutathionylation of Cys134 on fast troponin I (TnIf). Force-[Ca2+] characteristics of the contractile apparatus in mechanically-skinned fibers were assessed by direct activation with heavily Ca2+-buffered solutions. Treatment with S-nitrosylating agents, S-nitrosoglutathione (GSNO) or S-nitroso-N-acetyl-penicillamine (SNAP), decreased pCa50 (= log10 [Ca2+] at half maximal activation) by ~-0.07 pCa units in rat and human FT fibers without affecting maximum force, but had no effect on rat and human slow-twitch fibers or toad or chicken FT fibers, which all lack Cys134. The Ca2+-sensitivity decrease was i) fully reversed with dithiothreitol or reduced glutathione, ii) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and iii) irreversibly blocked by low concentration of the alkylating agent, N-ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments caused S nitrosylation of TnIf. S-glutathionylation pretreatment blocked the effects of S-nitrosylation on Ca2+-sensitivity, and vice-versa. S-nitrosylation pretreatment prevented NEM from irreversibly blocking S-glutathionylation of TnIf and its effects on Ca2+-sensitivity, and likewise S-glutathionylation pretreatment prevented NEM block of S-nitrosylation. Following substitution of TnIf into rat slow-twitch fibers, S-nitrosylation treatment caused decreased Ca2+-sensitivity. These findings demonstrate that S-nitrosylation and S-glutathionylation exert opposing effects on Ca2+-sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnIf.
- skinned muscle fiber
- contractile apparatus
- muscle fatigue
- Copyright © 2016, American Journal of Physiology-Cell Physiology