title: S-nitrosylation andS-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 byS-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 byS-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 withS-nitrosylating agents,S-nitrosoglutathione (GSNO) orS-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 was1) fully reversed with dithiothreitol or reduced glutathione,2) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and3) irreversibly blocked by low concentration of the alkylating agent,N-ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments causedS-nitrosylation of TnIfS-glutathionylation pretreatment blocked the effects ofS-nitrosylation on Ca2+sensitivity, and vice-versa.S-nitrosylation pretreatment prevented NEM from irreversibly blockingS-glutathionylation of TnIfand its effects on Ca2+sensitivity, and likewiseS-glutathionylation pretreatment prevented NEM block ofS-nitrosylation. Following substitution of TnIfinto rat slow-twitch fibers,S-nitrosylation treatment caused decreased Ca2+sensitivity. These findings demonstrate thatS-nitrosylation andS-glutathionylation exert opposing effects on Ca2+sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnIf.
authors.affiliation: School of Life Sciences, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: T L
authors.initials: TL
authors.lastname: Dutka
authors.affiliation: School of Life Sciences, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: J P
authors.initials: JP
authors.lastname: Mollica
authors.affiliation: School of Life Sciences, La Trobe University, Melbourne, Victoria, Australia.|Institute of Sport, Exercise and Active Living (ISEAL), Victoria University, Melbourne, Victoria, Australia; and.
authors.forename: C R
authors.initials: CR
authors.lastname: Lamboley
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: V C
authors.initials: VC
authors.lastname: Weerakkody
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: D W
authors.initials: DW
authors.lastname: Greening
authors.affiliation: School of Life Sciences, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: G S
authors.initials: GS
authors.lastname: Posterino
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: R M
authors.identifier: http://orcid.org/0000-0003-3697-589X
authors.initials: RM
authors.lastname: Murphy
authors.affiliation: School of Life Sciences, La Trobe University, Melbourne, Victoria, Australia; g.lamb@latrobe.edu.au.
authors.forename: G D
authors.initials: GD
authors.lastname: Lamb
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002478:Cells, Cultured; D002645:Chickens; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007537:Isometric Contraction / Q000502:physiology*; D008297:Male; D018656:Muscle Fibers, Fast-Twitch / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D020318:Rats, Long-Evans; D017207:Rats, Sprague-Dawley; D013045:Species Specificity; D019210:Troponin I / Q000737:chemistry / Q000378:metabolism*; D055815:Young Adult
chemical_list: D019210:Troponin I; D009569:Nitric Oxide; D005978:Glutathione; D003545:Cysteine; D002118:Calcium
keywords: contractile apparatus; muscle fatigue; oxidation; skinned muscle fiber