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Journal of Physiology (2002), 539.3, pp. 883-891
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013369

Hydrogen peroxide increases depolarization-induced contraction of mechanically skinned slow twitch fibres from rat skeletal muscles

David R. Plant, Gordon S. Lynch and David A. Williams

	ABSTRACT

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The effect of exogenous hydrogen peroxide (H₂O₂) on excitation-contraction (E-C) coupling and sarcoplasmic reticulum (SR) function was compared in mechanically skinned slow twitch fibres (prepared from the soleus muscles) and fast twitch fibres (prepared from the extensor digitorum longus; EDL muscles) of adult rats. Equilibration (5 min) with 1 mM H₂O₂ diminished the ability of the Ca²⁺-depleted SR to reload Ca²⁺ in both slow (P < 0.01) and fast twitch fibres (P < 0.05) compared to control. Under conditions when all Ca²⁺ uptake was prevented, 1 mM H₂O₂ increased SR Ca²⁺ 'leak' in fast twitch fibres by 24 ± 5 % (P < 0.05), but leak was not altered in slow twitch fibres. Treatment with 1 mM H₂O₂ also increased the peak force of low [caffeine] contracture by ~45 % in both fibre types compared to control (P < 0.01), which could be partly reversed following treatment with 10 mM dithiothreitol (DTT). The changes in SR function caused by 1 mM H₂O₂ were associated with an ~65 % increase in the peak height of depolarization-induced contractile response (DICR) in slow twitch fibres, compared to control (no H₂O₂; P < 0.05). In contrast, peak contractile force of fast twitch fibres was not altered by 1 mM H₂O₂ treatment. Equilibration with 5 mM H₂O₂ induced a spontaneous force response in both slow and fast twitch fibres, which could be partly reversed by 2 min treatment with 10 mM DTT. Peak DICR was also increased ~40 % by 5 mM H₂O₂ in slow twitch fibres compared to control (no H₂O₂; P < 0.05). Our results indicate that exogenous H₂O₂ increases depolarization-induced contraction of mechanically skinned slow but not fast twitch fibres. The increase in depolarization-induced contraction in slow twitch fibres might be mediated by an increased SR Ca²⁺ release during contraction and/or an increase in Ca²⁺ sensitivity.

(Resubmitted 8 October 2001; accepted after revision 19 December 2001)
Corresponding author D. A. Williams: Department of Physiology, University of Melbourne, Victoria 3010, Australia. Email: davidaw{at}unimelb.edu.au

	INTRODUCTION

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It is well established that skeletal muscles generate reactive oxygen species (ROS) as a by-product of aerobic metabolism (Chance et al. 1979; O'Neill et al. 1996; Kolbeck et al. 1997). Production of such oxidants is quenched by antioxidants whose presence within skeletal muscle cells tightly regulates cellular redox balance. Despite elaborate protective mechanisms against oxidation, oxidants also react with membranes and proteins within skeletal muscles when produced in excessive amounts, inducing damage or structural alteration, which has the potential to disrupt or modulate normal function (Abramson & Salama, 1989). One such example of oxidant-induced alteration of function is the increased activity of the sarcoplasmic reticulum (SR) Ca²⁺-release channel (CRC) and decreased activity of the SR Ca²⁺-ATPase, in isolated membrane vesicle preparations following equilibration with hydrogen peroxide (H₂O₂) (Salama et al. 1992; Boraso & Williams, 1994; Favero et al. 1995; Oba et al. 1996, 1998). The SR CRC plays a key role in a sequence of events known as excitation-contraction (E-C) coupling, defined as the events that include depolarization of the transverse-tubular (t-) system that activates specialized voltage sensors (dihydropyridine receptors, DHPRs), which in turn open ryanodine receptors/CRCs in the adjacent SR membrane, allowing Ca²⁺ to enter the cytoplasm (Melzer et al. 1995).

In addition to modulating the function of structures involved in E-C coupling, oxidants also modulate the force producing capacity of skeletal muscles. Equilibration with exogenous H₂O₂ increased submaximal force production in intact single muscle fibres (Andrade et al. 1998) and maximal force production in intact skeletal muscles (Plant et al. 2001). Subsequently, it was hypothesized that a relationship exists between muscle redox state and force production (Reid, 2001), which Andrade and colleagues (1998) suggested was mediated by an increase in sensitivity to Ca²⁺. However, further investigation demonstrated that Ca²⁺ sensitivity of membrane-permeabilized single fibres was not altered by H₂O₂, and that peak Ca²⁺-activated force was in fact decreased by elevated levels of exogenous H₂O₂ (Plant et al. 2000). We have also demonstrated recently that the effects of H₂O₂ on muscle contractility are fibre type-dependent with a greater increase in force production induced by H₂O₂ in slow than fast twitch muscles (Plant et al. 2001).

It is not known whether the effects of H₂O₂ on E-C coupling and SR membrane channel function mediate the increase in isometric contractile function in intact muscles. If H₂O₂ increased SR Ca²⁺ release and decreased Ca²⁺ reuptake in an intact muscle, cytoplasmic (intracellular) Ca²⁺ concentration ([Ca²⁺]_i) would be elevated above normal during contraction and remain elevated for an extended period following contraction. During contractions when [Ca²⁺]_i is not saturating, an increase in [Ca²⁺]_i might increase force production, due to increased Ca²⁺-troponin interaction. Whilst previous evidence demonstrates that [Ca²⁺]_i during contraction is not altered by H₂O₂ in fast twitch fibres (Andrade et al. 1998), this has not been assessed in slow twitch fibres. Exogenous application of low levels of oxidants (100 µM 2,2'-dithiodipyridine (DTDP), 100 µM 5,5'-dithionitrobenzoic acid (DTNB), 100 µM N-ethylmaleimide (NEM)) to mechanically skinned fast twitch extensor digitorum longus (EDL) fibres also did not stimulate release of Ca²⁺ from the SR (Posterino & Lamb, 1996). Similarly, exogenous application of H₂O₂ to mechanically skinned EDL fibres did not alter SR function or peak force during contraction (Brotto & Nosek, 1996). Some doubt remains about these findings, however, as the validity of the experimental techniques employed has been questioned (Lamb & Stephenson, 1997). Whilst the effects of H₂O₂ on E-C coupling and SR function are well characterized in fast twitch skeletal muscles, slow twitch muscles remain untested.

Mechanically skinned muscle fibres prepared under paraffin oil have a sealed t-system, normal voltage regulation of Ca²⁺ release and an endogenous level of SR Ca²⁺ (Lamb & Stephenson, 1990). Unlike intact single fibres and whole muscles, the effects of exogenous oxidant exposure on the SR and intracellular environment can be determined. We sought to further investigate the effects of H₂O₂ on E-C coupling and SR function of mechanically skinned fibres from slow and fast twitch skeletal muscles, utilizing the technique of Lamb & Stephenson (1990). We tested the hypothesis that H₂O₂ would increase peak depolarization-induced contractile force in mechanically skinned fibres from slow but not fast twitch muscles of the rat.

	METHODS

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All experiments were approved by the Animal Experimentation Ethics Committee of The University of Melbourne and complied with the Code of Conduct for the Care and Use of Animals as stipulated by the National Health and Medical Research Council of Australia. The soleus (predominantly slow twitch) and extensor digitorum longus (EDL, fast twitch) muscles used in these experiments were carefully excised from adult male Sprague-Dawley rats (3-4 months old) that had been killed by cervical dislocation, whilst anaesthetized deeply with sodium pentobarbital (60 mg kg^-1 I.P.). After dissection, the muscles were pinned at resting length in a Petri dish lined with Sylgard gel (Dow Corning, Midland, MI, USA) and filled with cooled liquid paraffin. Under a microscope a small bundle of fibres was dissected at one end and separated down to the midbelly of the muscle using fine jewellers' forceps. This bundle of fibres was repeatedly separated until a single muscle fibre was isolated. Each fibre was mechanically skinned by peeling the sarcolemma away from the fibre, such that 70-90 % of the fibre bulk remained (Lamb & Stephenson, 1990). One end of the peeled fibre segment was mounted to a sensitive force transducer (AE801 SensoNor, Horten, Norway) with a braided surgical silk tie (9-0, Deknatel, Falls River, MA, USA) and the other end of the fibre secured between the jaws of a pair of jewellers' forceps (no. 5, Dumont & Fils, Switzerland). Sarcomere length of the fibres was adjusted to a length slightly longer than optimal (in order to reliably measure depolarization-induced contractile responses; Owen et al. 1997). The sarcomere length was estimated from the diffraction pattern produced when the beam of a He-Ne laser (Spectra Physics, Eugene, OR, USA) was passed along the length of the fibre.

Solutions for depolarization-induced responses of mechanically skinned fibres

The composition of solutions and procedures used for activation of the skinned muscle fibres has been described in detail previously (Posterino & Lamb, 1996). Briefly, the t-system of the fibres was polarized by incubating the fibre in a potassium hexamethylenediamine-tetraacetic acid (K-HDTA) solution (composition (mM): Hepes 90, K⁺ 125; Na⁺ 36; HDTA 50, EGTA 0.1, Mg (total) 8.5, NaN₃ 1, creatine phosphate 10, ATP 8) for 2 min. The fibre was then depolarized by rapidly substituting the K-HDTA with Na-HDTA, an identical solution but with all of the K⁺ replaced by equimolar Na⁺. For maximum activation, fibres were exposed to an approximately equimolar Ca-EGTA solution (composition (mM): Hepes 90, K⁺ 125; Na⁺ 36; Ca-EGTA 50, Mg (total) 8.12, NaN₃ 1, creatine phosphate 10, ATP 8; pCa < 4.7). All solutions contained 1 mM free Mg²⁺ (unless otherwise stated) and had an osmolality of ~295 mosmol (kg solvent)^-1 (Lamb & Stephenson, 1990). When required, H₂O₂ (30 % solution, Sigma, Australia) or dithiothreitol (DTT) were added directly to a given solution on the day of the experiment, with addition of either chemical not affecting solution pH. Experiments with soleus muscle fibres were performed at 25 ± 1 °C, rather than 22 ± 2 °C for EDL muscle fibres, since these fibres require a higher incubation temperature to produce consistent depolarization-induced force responses (Bakker et al. 1998).

Exogenous oxidants and reductants

H₂O₂ was chosen as an exogenous oxidant as it (1) is membrane-permeant; (2) is produced in vivo; (3) has a longer half-life than most other biological radicals and (4) is available as a stable reactant which can be added exogenously to experimental preparations in precise quantities. Application of exogenous H₂O₂ (between 100 µM and 5 mM) has been demonstrated to increase force production in skeletal muscles (Reid et al. 1993; Oba et al. 1996; Andrade et al. 1998; Plant et al. 2001). DTT was chosen as the exogenous reductant as it reverses the effects of H₂O₂ in experiments utilizing single muscle fibres (Wilson et al. 1991; Andrade et al. 1998).

Isolation of slow twitch fibres

Although the soleus muscle of rats is composed predominantly of slow twitch fibres, it also contains a small proportion (~10 %) of fast twitch fibres (Armstrong & Phelps, 1984). Other metal cations such as strontium (Sr²⁺), can replace Ca²⁺ in the activation process (Moisescu & Thieleczek, 1979). When bathed in solutions containing Sr²⁺, single fibres generate force in a manner similar to Ca²⁺ but slow twitch (type I) fibres demonstrate much greater sensitivity to Sr²⁺ activation than fast twitch (type II) fibres (Lynch & Williams, 1994). The Sr²⁺ sensitivity test for identification of slow (type I) fibres and fast (type II) has also been validated for myosin heavy composition (Bortolotto et al. 2000). When bathed in low [Sr²⁺] solutions, slow twitch fibres contract, whereas fast twitch fibres are unresponsive (Fink et al. 1986). Therefore, we used a pSr ~5.50 solution (similar to Ca-EGTA solution but containing 46.25 mM Sr²⁺ and 50 mM EGTA) to distinguish between type I (contraction) and type II fibres (no contraction) from soleus muscles. Only the responses of type I fibres from the soleus muscle were included in the results of these experiments.

Experimental protocol

Protocol 1: effects of H₂O₂ on SR function. The effect of 1 mM H₂O₂ on the ability of the SR to load Ca²⁺ was determined in freshly prepared mechanically skinned slow and fast twitch fibres. The SR of fibres was first completely depleted of Ca²⁺ by incubation in a SR Ca²⁺ release solution (low [Mg²⁺] K-HDTA; 0.1 mM free [Mg²⁺], containing 30 mM caffeine and 0.5 mM EGTA) for 2 min. Each fibre was then incubated in a Ca²⁺-loading solution (K-HDTA with 10 µl of 0.1 M CaCl₂ added) for a set period (10, 20 or 30 s for EDL muscle fibres and 5, 10 or 15 s for soleus muscle fibres) in order to partially replenish SR Ca²⁺, with the SR depleted again of Ca²⁺ after each loading period. The entire load-deplete cycle was repeated after 5 min with 1 mM H₂O₂ K-HDTA. The area under the force response trace after partial replenishment of SR Ca²⁺ before and after H₂O₂ was compared to maximum (soleus = 15 s control Ca²⁺ loading; EDL = 30 s control Ca²⁺ loading) to establish a load time-SR Ca²⁺ content relationship.

Additional muscle fibres were used to examine the effects of H₂O₂ on Ca²⁺ leakage from the SR. As described previously, fibres were prepared by first depleting the SR Ca²⁺ by incubation for 2 min in the release solution. The SR was then reloaded with Ca²⁺ (soleus fibres = 15 s, EDL fibres = 30 s), equilibrated for 2 min in a solution in which Ca²⁺ uptake and leak were prevented (Equilibration solution (Eqbn): K-HDTA containing 10 mM free Mg²⁺ and 0.5 mM EGTA) and then the SR was allowed to 'leak' Ca²⁺ for 30 s (in K-HDTA with 1 mM total EGTA) and then the remaining Ca²⁺ in the SR was released. The entire procedure was then repeated, but with the Eqbn and leak solutions containing 1 mM H₂O₂. The area under the force-response trace caused by the release solution after the leak period was deemed the 'control' SR Ca²⁺ leak and was compared to the area under the force-response trace after the H₂O₂ equilibration and leak period.

The sensitivity to caffeine-induced Ca²⁺ release was determined by incubating another group of fibres in a K-HDTA solution containing a low concentration of caffeine. In order to reliably measure H₂O₂-induced changes in peak caffeine-induced contraction, caffeine-induced Ca²⁺ release was determined under conditions where peak caffeine-induced contraction was ~10 % of maximum Ca²⁺-activated force in the absence of H₂O₂, which in single fibres from EDL muscles was achieved by equilibration with 7 mM caffeine K-HDTA after 15 s SR Ca²⁺ loading (Posterino & Lamb, 1996). As the SR of single fibres from soleus muscles contains a much lower Ca²⁺ content than fibres from EDL muscles, a peak caffeine-induced Ca²⁺ release corresponding to ~10 % of maximum Ca²⁺-activated force was measured in single fibres from the soleus muscle after 5 s SR Ca²⁺ loading and equilibration with 1 mM caffeine K-HDTA. In each fibre, the SR was completely depleted of Ca²⁺ and then partially loaded with Ca²⁺ (5 s for soleus fibres; 15 s for EDL fibres) and contraction in the low [caffeine] solution was determined. This cycle was repeated following 5 min equilibration with 1 mM H₂O₂ and then 5 min equilibration with 10 mM DTT. For each fibre, the peak force response during low [caffeine] contracture was represented as a percentage of maximum Ca²⁺-activated force.

Protocol 2: Effects of H₂O₂ on depolarization-induced contractile responses. Additional fibres were used to examine the effect of H₂O₂ on the contractile properties of mechanically skinned EDL and soleus muscle fibres. The t-system of each fibre was depolarized by substitution of K-HDTA for Na-HDTA, causing Ca²⁺ release from the SR which initiated a transient depolarization-induced contractile response (DICR). Fibres then underwent repeated depolarization (Na-HDTA) and repolarization (30 s in K-HDTA) cycles in solutions that either contained H₂O₂ (1 mM or 5 mM) or did not contain H₂O₂ (control). When each fibre had run down to the point when DICR was less than 50 % of initial, the viability of SR CRC function was assessed by stimulating the SR Ca²⁺ release with low [Mg²⁺] (K-HDTA with 0.1 mM free Mg²⁺; Posterino & Lamb, 1996). In some fibres, reversibility of H₂O₂ treatment was assessed by 2 min incubation with 10 mM DTT (dissolved in K-HDTA) prior to low [Mg²⁺]. Maximum Ca²⁺-activated force was determined in Ca-EGTA solution for each fibre at the conclusion of testing.

Statistical analysis

Values in the text are presented as means ± S.E.M. H₂O₂-treated and untreated (control) groups of fibres were compared using either analysis of variance with Newman-Keuls post hoc analysis or by Student's paired and unpaired t tests, where appropriate. Results were considered significant when P < 0.05.

	RESULTS

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Effect of H₂O₂ on SR function

When freshly skinned fibres were transferred from K-HDTA to the release solution, a force response was observed in both slow and fast twitch fibres, with the area under the force-response trace used as an approximate indication of SR Ca²⁺ content (Posterino & Lamb, 1996). After 2 min equilibration in the release solution and complete SR Ca²⁺ depletion, the SR was partially reloaded with Ca²⁺ by incubation in the load solution for defined intervals (soleus = 5, 10 or 15 s; EDL = 10, 20 or 30 s). The area under the force response trace after subsequent SR Ca²⁺ depletion at each 'load duration' was compared to the maximum response (15 s Ca²⁺ loading for soleus fibres and 30 s Ca²⁺ loading for EDL fibres) to establish the load duration-SR Ca²⁺ content relationship. Equilibration with 1 mM H₂O₂ for 5 min diminished the ability of the depleted SR to reload Ca²⁺ in both slow (P < 0.01) and fast twitch (P < 0.05) muscle fibres (See Fig. 1). The load duration-SR Ca²⁺ content relationship was not altered by 5 min incubation in solutions not containing H₂O₂, indicating that run-down was negligible in these experiments.

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Figure 1. The ability of Ca2+-depleted SR to reload Ca2+ is decreased by 1 mM H2O2 in both slow and fast twitch mechanically skinned muscle fibres Relationship between the time in loading solution and SR Ca2+ content of mechanically skinned slow (circles, n = 5) and fast twitch (squares, n = 6) fibres, before (continuous line, filled symbols) and after (dashed line, open symbols) 1 mM H2O2 treatment. ** P < 0.01, * P < 0.05; pre- vs. post-H2O2 treatment.

'Leak' of Ca²⁺ from the SR was determined after partial SR Ca²⁺ reloading to the Ca²⁺-depleted SR. As H₂O₂ depressed the Ca²⁺ reloading ability of the SR, Ca²⁺ leak from the SR was standardized by reloading SR Ca²⁺ under control conditions and then equilibration and 'leak' determined in the presence of H₂O₂. The ratio between the estimated SR Ca²⁺ content after equilibration and 'leak' with H₂O₂ was compared to that in control conditions (pre H₂O₂ treatment). The SR Ca²⁺ content of fast twitch fibres following 1 mM H₂O₂ 'leak' in fast twitch fibres was only 76 ± 5 % of that observed under control conditions (P < 0.05), indicating greater leak of Ca²⁺ from the SR of fast twitch fibres (see Fig. 2). Leak of Ca²⁺ from the SR of slow twitch fibres was not altered by incubation with H₂O₂.

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Figure 2. SR Ca2+ 'leak'is increased by 1 mM H2O2 in fast but not slow twitch muscle fibres Ratio of SR Ca2+ content following Ca2+ 'leak' in 1 mM H2O2 and control conditions (before H2O2 treatment) in slow (open bar) and fast twitch (hatched bar) mechanically skinned fibres (n = 5 fibres per group). Due to the suppressive effect of H2O2 on SR Ca2+ reloading (see Fig. 1), SR Ca2+ content was standardized (SR Ca2+ reloading under control conditions) before H2O2 'leak' was assessed. When all Ca2+ uptake was prevented, H2O2 increased SR Ca2+ leak from fast but not slow twitch muscle fibres. * P < 0.05 compared to control; pre-H2O2 treatment; dashed line.

Peak caffeine-induced Ca²⁺ release was determined after partial SR Ca²⁺ reloading in fibres with a Ca²⁺-depleted SR. Before each treatment, the SR of single fibres from EDL and soleus muscles was depleted and then partially reloaded with Ca²⁺ (5 s for soleus fibres and 15 s for EDL fibres). Peak caffeine-induced contraction was increased markedly in both slow and fast twitch fibres following equilibration with 1 mM H₂O₂ (P < 0.01; see Fig. 3). Subsequent treatment with 10 mM DTT fully reversed the effects of H₂O₂ in slow twitch fibres, but only partially reversed the effects in fast twitch fibres, with the caffeine-induced response remaining above pre-treatment levels (P < 0.05).

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Figure 3. H2O2 increases the sensitivity of both slow and fast twitch mechanically skinned muscle fibres to caffeine-induced Ca2+ release Peak force responses during low [caffeine] contracture under control conditions, 1 mM H2O2 equilibration (5 min), and 10 mM DTT equilibration (5 min) in slow (open bars) and fast twitch (hatched bars) mechanically skinned fibres (n = 5 fibres). The sensitivity to caffeine-induced Ca2+ release was increased after equilibration with 1 mM H2O2, indicating increased activity of the SR CRC. The effect of H2O2 was fully reversed in slow twitch fibres but only partly reversed in fast twitch fibres exposed to 10 mM DTT. * P < 0.05, ** P < 0.01 pre- vs. post-H2O2 treatment for each fibre.

Effect of H₂O₂ on contractile function

Rapid substitution of K-HDTA for Na-HDTA solution resulted in depolarization of the t-system, a rapid release of Ca²⁺ and a rapid transient depolarization-induced contractile response (DICR) that was 47 ± 5 % (n = 5, soleus fibres) and 72 ± 5 % (n = 7, EDL fibres) of maximum Ca²⁺-activated force (see Fig. 4A and B). The t-system of the muscle fibres could be repolarized by 30 s incubation in K-HDTA with 7 ± 1 (n = 5, soleus fibres) and as many as 20 ± 1 (n = 7, EDL fibres) depolarizations elicited before rundown (DICR < 50 % of initial).

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Figure 4. Depolarization-induced force responses of mechanically skinned slow and fast twitch muscle fibres Original recordings from (A) a slow twitch fibre from the soleus muscle and (B) a fast twitch fibre from the EDL muscle repeatedly depolarized and repolarized (30s in K-HDTA) between successive depolarizations. Many fewer depolarizations could be elicited from the slow than fast twitch fibres before run-down. Incubation with a low [Mg2+] solution (0.1 mM free Mg2+) elicited a transient force response indicative of functional CRC, even at the point of fibre rundown. Soleus fibres were also incubated with an Sr-EGTA solution (pSr ~5.50) to distinguish slow (type I) from fast (type II) fibres (see text). At the conclusion of all experiments, each fibre was bathed in a Ca-EGTA solution (pCa < 4.7) to determine maximum Ca2+-activated force. Here and in subsequent figures: Depol, depolarization with Na-HDTA solution; Low [Mg2+], 0.1 mM free Mg2+ in K-HDTA; Max, Ca-EGTA (pCa < 4.7); Sr2+, Sr-EGTA (pSr ~5.50).

The effect of H₂O₂ on depolarization-induced force was fibre type dependent. On average, treatment of slow twitch fibres with 1 mM H₂O₂ increased DICR by ~65 % (4th depolarization in sequence: 1 mM H₂O₂ treated DICR = 170 ± 15 % of initial DICR (n = 6 fibres) vs. control DICR = 106 ± 2 % of initial (n = 5 fibres), P < 0.05; see Fig. 5A). Spontaneous force production was also caused by 1 mM H₂O₂ in most of the slow twitch fibres tested. In contrast, 1 mM H₂O₂ did not alter the peak force or number of DICRs obtainable from fast twitch fibres (see Fig. 5B).

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Figure 5. Peak depolarization force is increased by 1 mM H2O2 in mechanically skinned slow but not fast twitch muscle fibres Original experimental trace recorded from (A) a slow and (B) a fast twitch fibre repeatedly depolarized (Na-HDTA) and repolarized (30 s in K-HDTA) during 1 mM H2O2 exposure. H2O2 treatment increased the peak height of DICR in slow but not fast twitch fibres.

All fibres transferred to 5 mM H₂O₂ (K-HDTA) exhibited spontaneous force production in both slow and fast twitch fibres, indicative of SR Ca²⁺ release (see Fig. 6A and B). Peak DICR was also increased ~40 % in slow twitch fibres by 5 mM H₂O₂ (4th depolarization in sequence: 5 mM H₂O₂ treated DICR = 145 ± 17 % of initial DICR (n = 5 fibres) vs. control DICR = 106 ± 2 % of initial (n = 5 fibres), P < 0.05; see Fig. 6A). While in some fast twitch fibres 5 mM H₂O₂ increased peak DICR, on average, DICR was not different from control (2nd depolarization in sequence: control DICR = 104 ± 5 % of initial (n = 7 fibres) vs. 5 mM H₂O₂ treated DICR = 116 ± 7 % of initial DICR (n = 5 fibres), P > 0.05; see Fig. 6B). In some instances 5 mM H₂O₂ caused peak DICR to exceed maximum Ca²⁺-activated force in both slow and fast twitch fibres. In fibres treated with 5 mM H₂O₂ spontaneous force production was elicited prior to depolarization of the t-system, but force returned to baseline levels after DICR, indicating a net active SR Ca²⁺ reuptake. Spontaneous force production soon returned when fibres were transferred back into 5 mM H₂O₂ K-HDTA. A 2 min treatment with 10 mM DTT reduced the spontaneous force production and restored DICR, but to a lower peak DICR value than observed initially (see Fig. 6A and B).

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Figure 6. Spontaneous force production is caused by 5 mM H2O2 in both slow and fast twitch mechanically skinned muscle fibres Original experimental recording of a mechanically skinned (A) slow and (B) fast twitch muscle fibre during exposure to 5 mM H2O2. Spontaneous force production was observed soon after exposure to 5 mM H2O2. On average the peak height of DICR was increased following H2O2 treatment in slow but not fast twitch fibres. The spontaneous force production induced by H2O2 was reduced by treatment with 10 mM DTT.

	DISCUSSION

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This study compared the effects of exogenous H₂O₂ application on SR function and the resultant contractile activation characteristics of slow and fast mechanically skinned skeletal muscle fibres. The experiments indicate that H₂O₂ has fibre type specific effects on mechanically skinned fibres. The increase in peak depolarization-induced force production observed in slow but not fast twitch fibres correlates with observations of a much greater H₂O₂-induced increase in maximum isometric force producing capacity of intact slow than fast twitch skeletal muscles (Plant et al. 2001). The effects of H₂O₂ were also concentration dependent. A greater increase in peak DICR was evident in slow twitch fibres during 1 mM than 5 mM H₂O₂ treatment, and spontaneous force production was induced by 5 mM H₂O₂ but not by 1 mM H₂O₂.

The increase in the peak DICR in slow twitch fibres is likely to be mediated by an increase in force per active crossbridge and/or an increase in the rate of crossbridge attachment and detachment following depolarization and SR Ca²⁺ release. If Ca²⁺-troponin-C binding sites were not all occupied following SR Ca²⁺ release, then an increase in [Ca²⁺]_i during each discrete contractile event would have the potential to increase total Ca²⁺-troponin interaction, thereby increasing the rate of active crossbridge cycling, and thus increase force production. An increase in [Ca²⁺]_i following depolarization might be caused by an increase in the rate of Ca²⁺ release of the SR CRC. Stimulation of the SR CRC was evidenced by spontaneous force production upon exposure to 5 mM H₂O₂, in addition to increased peak force during low [caffeine] contracture in 1 mM H₂O₂-treated fibres. The spontaneous force evident during 5 mM H₂O₂ equilibration was absent when fibres were incubated in a solution in which SR Ca²⁺ release and re-uptake was prevented (Eqbn: 10 mM free Mg²⁺ and 0.5 mM EGTA), and when fibres were incubated in an EGTA-based solution that contained 5 mM H₂O₂. The observations provide direct evidence for the activation of the SR CRC. Similar observations of a sustained activation of the CRC were observed in membrane vesicle preparations from cardiac myocytes incubated with 5 mM H₂O₂ (Boraso & Williams, 1994).

In some instances, H₂O₂ treatment increased peak DICR above maximum Ca²⁺-activated force, indicating a possible increase in force per active crossbridge during contraction. We have previously demonstrated that H₂O₂ does not affect either Ca²⁺ sensitivity, or the total number of crossbridges active during contraction, leaving the possibility of an increase in force per active crossbridge (Plant et al. 2000). However, H₂O₂ decreased maximum Ca²⁺-activated force production in a time-dependent manner (Plant et al. 2000). Therefore, it is most likely that the ratio of peak DICR to maximum Ca²⁺-activated force production during H₂O₂ treatment was overestimated in these experiments due to the confounding time dependent effects of H₂O₂ on maximum Ca²⁺-activated force production. The conclusions drawn from our results regarding the effect of H₂O₂ on depolarization-induced contraction are based on a comparison of initial DICR for each fibre, therefore the time dependent influence of H₂O₂ is not problematic.

In addition to greater release of Ca²⁺ from the SR, the results indicate that H₂O₂ reduced the rate of SR Ca²⁺ reuptake, which would further potentiate the increase in [Ca²⁺]_i after depolarization. This finding confirms previous observations of the depressive effect of H₂O₂ on the activity of the SR Ca²⁺-ATPase in membrane vesicle preparations (Scherer & Deamer, 1986; Viner et al. 1997; Xu et al. 1997). Ca²⁺ permeability of the SR membrane of slow twitch fibres was not altered by H₂O₂, and therefore would not contribute to any alteration in [Ca²⁺]_i. The small increase in the 'rate' of Ca²⁺ leak observed in fast twitch fibres would not be likely to be a major contributor to changes [Ca²⁺]_i (see Discussion in Posterino & Lamb, 1996).

No evidence of spontaneous force production was seen in mechanically skinned fast twitch muscle fibres after treatment with other oxidants (100 µM 2,2'-dithiodipyridine (DTDP), 100 µM 5,5'-dithionitrobenzoic acid (DTNB), 100 µM N-ethylmaleimide (NEM)) (Posterino & Lamb, 1996). Instead, low concentrations of various oxidants were reported to have no discernible effects on SR Ca²⁺ release, but they did suppress voltage sensor activation and subsequent contractile function (Posterino & Lamb, 1996). Our results indicate that oxidant concentrations of 5 mM are necessary to stimulate SR Ca²⁺ release in fast twitch fibres but whether this is a physiological concentration in vivo has yet to be determined. In addition, we also demonstrated that mechanically skinned slow twitch muscle fibres have greater potential to increase peak contractile force than fast twitch fibres and therefore, low levels of exogenous DTDP, DTNB and NEM may have the potential to affect the contractility of slow twitch fibres.

Reversal of the effects of H₂O₂ was only partially achieved by incubation with DTT. The absence of complete reversal of the effects of H₂O₂ is similar to our previous investigations of intact skeletal muscles (Plant et al. 2001), suggestive of some form of long lasting covalent modification to key regulatory sites, which cannot be reversed by the presence of a sulfhydryl donor (Castilho et al. 1996).

Our findings indicate that exogenous H₂O₂, in a defined concentration range, stimulated caffeine-induced Ca²⁺ release in both slow and fast twitch mechanically fibres from mammalian skeletal muscles. H₂O₂ treatment also increased peak depolarization-induced force production in slow but not fast twitch fibres and diminished SR Ca²⁺ reuptake in fast but not slow twitch mechanically skinned fibres from the rat. From these experiments we conclude that the H₂O₂ increases depolarization-induced contraction in mechanically skinned slow twitch muscle fibres, which is likely mediated by an increase in SR Ca²⁺ release and/or an increase in Ca²⁺ sensitivity.

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Acknowledgements

We thank Associate Professor Graham Lamb for helpful discussion regarding depolarization-induced responses in mechanically skinned muscle fibres. This work was supported by the National Health and Medical Research Council of Australia (NHMRC).

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