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 (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 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 TnIfS-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.
Highlights
NITRIC OXIDE (NO) is generated in skeletal muscle at rest, primarily by neuronal nitric oxide synthase, and production increases markedly with contractile activity [5, 20, 30]
We tested this by examining 1) whether the Ca2ϩ sensitivity changes in various mammalian and nonmammalian muscle fibers are in accord with the presence of Cys134, 2) whether S-nitrosylation and S-glutathionylation treatments show competitive effects, similar sensitivity to irreversible block by N-ethylmaleamide (NEM), and a reciprocal ability to protect Cys134 and the Ca2ϩ sensitivity changes of the opposing treatment from block by NEM, and 3) whether NO treatment results in reduced Ca2ϩ sensitivity in slowtwitch mammalian fibers following exchange of fast-twitch troponin
To demonstrate that this signal reflected labeling of TnIf and not myosin light chain 1 (MLC1), which runs at a similar apparent molecular weight, these proteins were physically separated by dissociating myosin and the associated light chains with a high [KCl] solution, and the dissociated proteins and those remaining in the fiber were run in adjacent lanes on SDS-PAGE for Western blotting
Summary
NITRIC OXIDE (NO) is generated in skeletal muscle at rest, primarily by neuronal nitric oxide synthase (nNOS), and production increases markedly with contractile activity [5, 20, 30]. We tested this by examining 1) whether the Ca2ϩ sensitivity changes in various mammalian and nonmammalian muscle fibers are in accord with the presence of Cys134, 2) whether S-nitrosylation and S-glutathionylation treatments show competitive effects, similar sensitivity to irreversible block by N-ethylmaleamide (NEM), and a reciprocal ability to protect Cys134 and the Ca2ϩ sensitivity changes of the opposing treatment from block by NEM, and 3) whether NO treatment results in reduced Ca2ϩ sensitivity in slowtwitch mammalian fibers following exchange of fast-twitch troponin. The findings provide strong evidence that the NO decreases Ca2ϩ sensitivity in skeletal muscle by inducing S-nitrosylation of Cys134 on TnIf, and that this effect competitively antagonizes the ability of oxidant-induced S-glutathionylation to increase Ca2ϩ sensitivity, which would adversely affect skeletal muscle performance in various circumstances.
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