S-Glutathionylation of troponin I (fast) increases contractile apparatus Ca 2+ sensitivity in fast-twitch muscle fibres of rats and humans

摘要

Oxidation can decrease or increase the Ca 2+ sensitivity of the contractile apparatus in rodent fast-twitch (type II) skeletal muscle fibres, but the reactions and molecular targets involved are unknown. This study examined whether increased Ca 2+ sensitivity is due to S-glutathionylation of particular cysteine residues. Skinned muscle fibres were directly activated in heavily buffered Ca 2+ solutions to assess contractile apparatus Ca 2+ sensitivity. Rat type II fibres were subjected to S-glutathionylation by successive treatments with 2,2'-dithiodipyridine (DTDP) and glutathione (GSH), and displayed a maximal increase in pCa 50 (-log 10[Ca 2+] at half-maximal force) of ~0.24 pCa units, with little or no effect on maximum force or Hill coefficient. Partial similar effect was produced by exposure to oxidized gluthathione (GSSG, 10 mm) for 10 min at pH 7.1, and near-maximal effect by GSSG treatment at pH 8.5. None of these treatments significantly altered Ca 2+ sensitivity in rat type I fibres. Western blotting showed that both the DTDP-GSH and GSSG-pH 8.5 treatments caused marked S-glutathionylation of the fast troponin I isoform (TnI f) present in type II fibres, but not of troponin C (TnC) or myosin light chain 2. Both the increased Ca 2+ sensitivity and glutathionylation of TnI f were blocked by N-ethylmaleimide (NEM). S-Nitrosoglutathione (GSNO) also increased Ca 2+ sensitivity, but only in conditions where it caused S-glutathionylation of TnI f. In human type II fibres from vastus lateralis muscle, DTDP-GSH treatment also caused similar increased Ca 2+ sensitivity and S-glutathionylation of TnI f. When the slow isoform of TnI in type I fibres of rat was partially substituted (~30%) with TnI f, DTDP-GSH treatment caused a significant increase in Ca 2+ sensitivity (~0.08 pCa units). TnI f in type II fibres from toad and chicken muscle lack Cys133 present in mammalian TnI f, and such fibres showed no change in Ca 2+ sensitivity with DTDP-GSH nor any S-glutathionylation of TnI f (latter examined only in toad). Following 40 min of cycling exercise in human subjects (at ~60% peak oxygen consumption), TnI f in vastus lateralis muscle displayed a marked increase in S-glutathionylation (~4-fold). These findings show that S-glutathionylation of TnI f, most probably at Cys133, increases the Ca 2+ sensitivity of the contractile apparatus, and that this occurs in exercising humans, with likely beneficial effects on performance.