S-nitrosylation and S-glutathionylation of Cys134 on troponin I have opposing competitive actions on Ca2+ sensitivity in rat fast-twitch muscle fibers

Abstract

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 investigated 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 (TnI_f). 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 1) fully reversed with dithiothreitol or reduced glutathione, 2) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and 3) 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 TnI_fS-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 TnI_f and its effects on Ca²⁺ sensitivity, and likewise S-glutathionylation pretreatment prevented NEM block of S-nitrosylation. Following substitution of TnI_f 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 TnI_f.

Keywords: contractile apparatus; muscle fatigue; oxidation; skinned muscle fiber.