S-glutathionylation of troponin I (fast) increases contractile apparatus Ca2+ sensitivity in fast-twitch muscle fibres of rats and humans

Abstract

Oxidation can decrease or increase the Ca2+ 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 Ca2+ sensitivity is due to S-glutathionylation of particular cysteine residues. Skinned muscle fibres were directly activated in heavily buffered Ca2+ solutions to assess contractile apparatus Ca2+ 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 pCa50 (−log10 [Ca2+] 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 Ca2+ 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 Ca2+ sensitivity and glutathionylation of TnI(f) were blocked by N-ethylmaleimide (NEM). S-nitrosoglutathione (GSNO) also increased Ca2+ 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 Ca2+ 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 Ca2+ sensitivity (∼0.08 pCa units). TnIf in type II fibres from toad and chicken muscle lack Cys133 present in mammalian TnIf, and such fibres showed no change in Ca2+ 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 Ca2+ sensitivity of the contractile apparatus, and that this occurs in exercising humans, with likely beneficial effects on performance.

Figure 1. Treatment with GSSG–pH 8.5 increases Ca²⁺ sensitivity of contractile apparatus

A, isometric force production in a skinned EDL fibre to solutions with successively higher free µCa²⁺½, starting at pCa >9 (small ticks in order: pCa 6.40, 6.22, 6.02, 5.88, 5.75, 5.48, 4.7, >9). Sequence repeated twice after each treatment, giving very similar results; only the second of each pair of force–pCa staircases is shown for each treatment. Maximum Ca²⁺-activated force reached at pCa 4.7. Horizontal arrows mark force level achieved at pCa 5.75. Treatments (all applied at pCa > 9): 10 mm DTT for 10 min; pH 8.5 for 10 min; 10 mm GSSG at pH 8.5 for 10 min; 100 μm DTDP for 5 min followed by 5 mm GSH for 2 min (i.e. ‘standard DTDP–GSH treatment’). B, Hill fits to force–pCa data following each indicated treatment; data from A, with force responses in each ‘staircase’ normalised to their own maximum. C, pCa₅₀ values after indicated treatment for Hill fits in B.

Figure 2. Constancy of net increase in Ca²⁺ sensitivity with sequential GSSG and DTDP-GSH treatments

Increase in pCa₅₀ when individual EDL fibres were first subjected to a submaximal or near-maximal GSSG treatment (pCa₅₀ shift plotted as abscissa value) and then afterwards subjected to the standard DTDP–GSH treatment (additional increase in pCa₅₀ plotted as ordinate value); the total overall increase in pCa₅₀ was similar irrespective of the initial GSSG treatment. The different first treatments (all lasting 10 min) were: 10 mm GSSG at pH 7.1 (•); 2.5 mm GSSG at pH 8.5 (▾); 5 mm GSSG at pH 8.5 (◆); and 10 mm GSSG at pH 8.5 (▴). The value on the ordinate axis (▪) (i.e. +0.238 pCa units) represents the effect of DTDP–GSH treatment without any first treatment (see Table 1). In all cases subsequent DTT treatment (10 mm, 10 min) fully reversed Ca²⁺ sensitivity to its original level (i.e. by ∼−0.24 pCa units in all cases) (not shown).

Figure 3. Pre-treatment with NEM blocks action of DTDP–GSH on Ca²⁺ sensitivity

A, Hill fits for force–pCa data for a rat EDL fibre given the standard DTDP–GSH treatment both before and after treatment with NEM (25 μm, 2 min, at pCa > 9). Two successive force–pCa staircases examined after each treatment, with very similar results; data only for second in each pair plotted here. B, pCa₅₀ values following indicated treatment for data in A. Note that there is a small progressive decline in pCa₅₀ with each force–pCa staircase, which occurs even with no treatment (∼0.012 pCa units per staircase pair) (here compare ‘CON’ (control) and subsequent post ‘DTT’ responses).

Figure 4. DTDP–GSH treatment causes S-glutathionylation of troponin I fast isoform (TnI_f)

A, upper panel: Western blot with anti-GSH antibody in EDL fibre samples (5 fibre segments per sample) subjected to indicated treatment (all at pCa > 9) (15% SDS-PAGE; non-reducing). In all cases, fibres washed initially for at least 5 min in pCa > 9. Lane 1, control treatment (Con), 10 min washing at pCa > 9; Lane 2, 5 mm SNAP for 5 min; Lane 3, 100 μm DTDP for 5 min, 5 mm GSH for 2 min (i.e. ‘standard DTDP–GSH treatment’); Lane 4, 10 mm DTT for 10 min. All samples blocked with 5 mm NEM for 5 min before adding SDS (see Methods). Arrows indicate bands corresponding to TnI. The bands at ∼43 kDa correspond to actin. Lower panel: subsequent reprobe of same membrane for troponin I (TnI); EDL fibres contain only the fast isoform, TnI_f. Positions of molecular weight markers shown on left (see Methods). B, middle panel: anti-GSH blot of EDL fibres given indicated treatments as in A (12.5% SDS-PAGE). Lane 3: fibres treated with NEM (200 μm, 2 min), followed by DTT (10 mm, 10 min), then standard DTDP–GSH treatment. Membrane reprobed for TnI (bottom) and then actin (top). C, mean +SEM ratio of band density for anti-GSH signal relative to corresponding TnI_f signal for indicated treatments, expressed relative to DTDP–GSH case on same gel. Number of independent gels shown in each bar. *Significantly different (P < 0.001) from DTDP–GSH case.

Figure 5. Biotin labelling of reactive cysteines

Western blot of single skinned EDL fibre segments treated in relaxing solution with a biotin-tagged thiopyridine reagent (Biotin–HPDP) for either 1 or 5 min, and probed for biotin with strepatividin (see Methods). The 1 min exposure to the reagent produced clear labelling of the TnI isoform present (TnI_f), but an even more prolonged (5 min) exposure produced no detectable labelling of either TnC or MLC2 (both run at ∼18 kDa). 12.5% SDS-PAGE Tropomyosin (Tm). Bottom panel: reprobe for TnI.

Figure 6. Freshly prepared GSNO produces S-glutathionylation of TnI_f

EDL type II fibres (4 fibre segments per sample) were subjected to control treatment (‘Con’, 10 min wash only) or to a 2 min treatment with GSNO (2 mm) either immediately after it was prepared (‘Imm’) or 10 min after it was prepared (delayed, ‘Del’). Fibres first washed for 5 min before either GSNO treatment. All solutions were at pCa > 9. Middle panel: Western blot with anti-GSH antibody. Lower panel: reprobe of membrane for TnI. Top panel: MHC band in coomassie-stained gel post-transfer. 15% SDS-PAGE.

Figure 7. Effect of S-glutathionylation following partial TnI_f exchange into a type I fibre

A, Ca²⁺-activated force responses in skinned segment of a rat soleus type I fibre following exchange of porcine fast-twitch troponin (see Methods). µCa²⁺½ initially pCa > 9 and raised progressively (small ticks) in order: pCa 6.70, 6.40, 6.22, 6.02, 5.88, 5.75, 5.48, 4.7, >9). Fibre treated successively with DTDP (100 μm, 5 min), GSH (5 mm, 2 min) and DTT (10 mm, 10 min). µCa²⁺½ sequence repeated twice after each treatment, giving very similar force responses; only second of each pair shown. Horizontal arrows indicate force level at pCa 6.40 in each case. B, Hill fits to force–pCa data in A. C, Western blotting for TnI, TnC and actin in soleus single fibre segments with (Exch) and without (Con) troponin exchange. The segment producing the force responses in A and B was run in Lane 2; virtually all of the endogenous TnC, and ∼35% of the TnI, was replaced with the respective fast isoform (see text). Lane 4: another fibre following troponin exchange. Lanes 1 and 3: untreated control fibres. Lane 5: 0.5 pmol of the exogenous porcine troponin complex (TnX). 8–16% Criterion Stain Free gel.

Figure 8. Glutathionylation treatment causes increased Ca²⁺ sensitivity in human type II fibres but not type I fibres

Segments of individual fibres from vastus lateralis muscle biopsy from a rested human subject were mounted on the force transducer and force–pCa staircases recorded. A, Hill curves for human type II fibre subjected to standard DTDP–GSH treatment and then DTT treatment. B, Western blots of human single fibres, probed first for TnI, then TnC, MHCII and finally MHCI. Fibre in A run in Lane 1. Force response of fibres to pSr 5.2 in accord with TnC isoform in every case (see text). 4–15% Criterion TGX Stain Free gel. C, Hill curves for human type I fibre subjected to successive treatments with DTDP (100 μm, 5 min), GSH (5 mm, 2 min), and finally DTT (10 mm, 10 min).

Figure 9. TnI_f runs more slowly when S-glutathionylated

A, Western blots of tissue sections from rat EDL and human vastus lateralis muscles subjected either to a wash only (lanes 1 and 3, ‘–’) or to wash with DTDP (100 μm) and then GSH (5 mm) (lanes 2 and 4, ‘+’). (Adjacent transverse sections of frozen muscles used; treated at room temperature, all in K-HDTA ‘intracellular’ solution at pCa > 9.) Middle panel: anti-GSH antibody shows DTDP–GSH treatment increased glutathionylation of TnI_f in rat EDL muscle and of both TnI_f and TnI_s in human muscle (lower and upper bands, respectively). Bottom panel: reprobe with TnI antibody: only fast isoform (TnI_f) is present in rat EDL muscle, whereas both TnI_f and TnI_s are present in the human muscle. Note that TnI_f runs at a slightly higher molecular weight following treatment with DTDP–GSH in both the rat and human muscle. 8–16% Criterion Stain Free gel. B, diagrammatic representation of exposed and hidden cysteine residues on the troponin complex in mammalian fast-twitch (type II) muscle fibres (modified from Chong & Hodges, 1982; © 1982 The American Society for Biochemistry and Molecular Biology). Letters I, C and T indicate TnI_f, TnC_f and TnT_f subunits, and numbers indicate cysteine residues on TnI_f and TnC_f (there are none on TnT_f). Only Cys133 on TnI_f is readily accessible to cysteine reagents. TnI_s (not shown) lacks an equivalent of Cys133, having Cys38, Cys65 and Cys85, with Cys65 being homologous to Cys 64 on TnI_f.

Figure 10. No S-glutathionylation of TnI_f in toad fibres

Western blot with anti-GSH (upper panel) and subsequent reprobe for TnI (lower panel). Rat EDL fibres in lanes 1 & 2 and 5 & 6, and toad iliofibularis fibres in lanes 3 & 4 and 7 & 8 (4 fibre segments in each sample). Fibres in left lane of each pair (i.e. in lanes 1, 3, 5 and 7) given standard DTDP–GSH treatment (+), and fibres in adjacent lanes (2, 4, 6 and 8) washed in same solution without DTDP and GSH (–). In the rat EDL fibres the DTDP–GSH treatment caused a very marked increase in the anti-GSH signal corresponding to TnI_f (arrow) (compare lanes 1 & 3 with 2 & 6), but no other proteins in the rat fibres showed much increase with treatment. (Note that the anti-GSH signal at ∼43 kDa arising from actin is overexposed and appears white.) TnI_f in the toad fibres displayed no anti-GSH signal, either with or without the treatment; note that the anti-GSH signal seen in the toad fibres running similar to the rat TnI_f signal does not arise from the toad TnI_f, which runs at an appreciably lower molecular weight, as seen in the TnI reprobe below. M, molecular markers (see Methods). 4–15% Criterion TGX Stain Free gel.

Figure 11. Increased S-glutathionylation of TnI_f in human muscle with prolonged exercise

A, Western blot with anti-GSH, and subsequent reprobe for TnI, of transverse sections from vastus lateralis muscle biopsies taken from two human subjects both before (‘pre’) and after 40 min cycling exercise at ∼60% of (‘exerc’). The anti-GSH signal indicated by arrow overlaid with the TnI_f bands (lower of the two TnI bands). Increased glutathionylation was also evident at ∼14 kDa in these two post-exercise cases. Top panel shows corresponding MHC bands imaged in stain-free gel before protein transfer. 4–20% Criterion Stain Free gel. B, mean data (+SEM) from 5 subjects for the TnI_f S-glutathionylation signal before and after exercise (all samples run on same gel, repeated three times). The density of each TnI_f glutathionylation signal was first normalised by the corresponding TnI_f band density, and then each value was re-expressed relative to the mean of the pre-exercise cases on that gel (effectively declaring latter as ‘1’); values from 3 repetitions averaged to yield a single value for each pre- and post-exercise sample for each subject. *Significantly greater than ‘pre’ (paired t test, n= 5 subjects, P < 0.05, one-sided Wilcoxin signed rank test).