1 C-00334-2016 Revised 1 2 3 4 5 S-Nitrosylation and S-Glutathionylation of Cys 134 on troponin I have 6 opposing competitive actions on Ca 2 + sensitivity in rat fast-twitch muscle fibers

44 Nitric oxide is generated in skeletal muscle with activity and decreases Ca-sensitivity of the 45 contractile apparatus, putatively by S-nitrosylation of an unidentified protein. We investigate the 46 mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca47 sensitivity in mammalian fast-twitch (FT) fibers mediated by S-glutathionylation of Cys134 on 48 fast troponin I (TnIf). Force-[Ca] characteristics of the contractile apparatus in mechanically49 skinned fibers were assessed by direct activation with heavily Ca-buffered solutions. Treatment 50 with S-nitrosylating agents, S-nitrosoglutathione (GSNO) or S-nitroso-N-acetyl-penicillamine 51 (SNAP), decreased pCa50 (=-log10 [Ca] at half maximal activation) by ~-0.07 pCa units in rat 52 and human FT fibers without affecting maximum force, but had no effect on rat and human slow53 twitch fibers or toad or chicken FT fibers, which all lack Cys134. The Ca sensitivity decrease 54 was i) fully reversed with dithiothreitol or reduced glutathione, ii) at least partially reversed with 55 ascorbate, indicative of involvement of S-nitrosylation, and iii) irreversibly blocked by low 56 concentration of the alkylating agent, N-ethylmaleimide (NEM). The biotin-switch assay showed 57 that both GSNO and SNAP treatments caused S-nitrosylation of TnIf. S-glutathionylation 58 pretreatment blocked the effects of S-nitrosylation on Ca sensitivity, and vice-versa. 59 S-nitrosylation pretreatment prevented NEM from irreversibly blocking S-glutathionylation of 60 TnIf and its effects on Ca-sensitivity, and likewise S-glutathionylation pretreatment prevented 61 NEM block of S-nitrosylation. Following substitution of TnIf into rat slow-twitch fibers, 62 S-nitrosylation treatment caused decreased Ca-sensitivity. These findings demonstrate that 63 S-nitrosylation and S-glutathionylation exert opposing effects on Ca-sensitivity in mammalian 64 FT muscle fibers, mediated by competitive actions on Cys134 of TnIf. 65