<i>S</i> -nitrosylation and <i>S</i> -glutathionylation of Cys134 on troponin I have opposing competitive actions on Ca ²⁺ 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 <i>S-</i>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 <i>S-</i>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 <i>S-</i>nitrosylating agents, <i>S-</i>nitrosoglutathione (GSNO) or <i>S-</i>nitroso-<i>N</i>-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>1</i>) fully reversed with dithiothreitol or reduced glutathione, <i>2</i>) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and <i>3</i>) irreversibly blocked by low concentration of the alkylating agent, <i>N</i>-ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments caused <i>S-</i>nitrosylation of TnI_f<i>S-</i>glutathionylation pretreatment blocked the effects of <i>S-</i>nitrosylation on Ca²⁺ sensitivity, and vice-versa. <i>S-</i>nitrosylation pretreatment prevented NEM from irreversibly blocking <i>S-</i>glutathionylation of TnI_f and its effects on Ca²⁺ sensitivity, and likewise <i>S-</i>glutathionylation pretreatment prevented NEM block of <i>S-</i>nitrosylation. Following substitution of TnI_f into rat slow-twitch fibers, <i>S-</i>nitrosylation treatment caused decreased Ca²⁺ sensitivity. These findings demonstrate that <i>S-</i>nitrosylation and <i>S-</i>glutathionylation exert opposing effects on Ca²⁺ sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnI_f.