The relative abilities of caffeine and transverse tubular (T‐) system depolarisation to induce Ca2+ release in mammalian skeletal muscle were compared in mechanically skinned fibres of the rat, in order to determine whether normal excitation‐contraction (E‐C) coupling is achieved by up‐regulating the Ca2+‐induced Ca2+ release process, as caffeine is known to do. Caffeine triggered Ca2+ release in soleus (slow‐twitch) fibres at much lower concentrations than in extensor digitorum longus (EDL) (fast‐twitch) fibres when the sarcoplasmic reticulum (SR) of each type was loaded with Ca2+ at close to endogenous levels. The difference in caffeine sensitivity resulted at least in part from the SR being loaded endogenously at near maximal capacity in soleus fibres but at less than half of maximal capacity in EDL fibres. The caffeine sensitivity could be reversed by reversing the relative level of SR loading. The ability of caffeine to induce Ca2+ release was markedly reduced by lowering the level of SR loading or by raising the free [Mg2+] from 1 to 3 mm. Caffeine, even at 30 mm, triggered little or no Ca2+ release in EDL fibres (a) at 1 mm (physiological) Mg2+ when the SR was loaded at two‐thirds or less of the endogenous level, and (b) at 3 mm Mg2+ when the SR was loaded at close to the endogenous level. In contrast, depolarisation potently elicited Ca2+ release under these conditions in the same fibres. The inability of 30 mm caffeine to induce Ca2+ release under certain conditions was not attributable to desensitisation or inactivation of the release channels, because there was no response even upon initial exposure to caffeine and depolarisation always remained able to trigger Ca2+ release. It instead appeared that caffeine was a relatively ineffectual stimulus in EDL fibres except under conditions where (a) the SR was heavily loaded, (b) the free [Mg2+] was low, or (c) a high [Cl−] was present. These results show that the normal E‐C coupling mechanism in mammalian skeletal muscle does not involve just enhancing Ca2+‐induced Ca2+ release, and evidently requires the removal or bypassing of the inhibitory effect of Mg2+ on the Ca2+ release channels.

Chemically skinned single fibers from adult rat skeletal muscles were used to test the hypothesis that, in mammalian muscle fibers, myosin heavy chain (MHC) isoform expression and Ca(2+)- or Sr(2+)-activation characteristics are only partly correlated. The fibers were first activated in Ca(2+)- or Sr(2+)-buffered solutions under near-physiological conditions, and then their MHC isoform composition was determined electrophoretically. Fibers expressing only the MHC I isoform could be appropriately identified on the basis of either the Ca(2+)- or Sr(2+)-activation characteristics or the MHC isoform composition. Fibers expressing one or a combination of fast MHC isoforms displayed no significant differences in their Ca(2+)- or Sr(2+)-activation properties; therefore, their MHC isoform composition could not be predicted from their Ca(2+)- or Sr(2+)-activation characteristics. A large proportion of fibers expressing both fast- and slow-twitch MHC isoforms displayed Ca(2+)- or Sr(2+)-activation properties that were not consistent with their MHC isoform composition; thus both fiber-typing methods were needed to fully characterize such fibers. These data show that, in rat skeletal muscles, the extent of correlation between MHC isoform expression and Ca(2+)- or Sr(2+)-activation characteristics is fiber-type dependent.

In this study the effects of oxidation and reduction on various steps in the excitation-contraction (E-C) coupling sequence was examined in mammalian skeletal muscle. In mechanically skinned fast-twitch fibres, electric field stimulation was used to generate action potentials in the sealed transverse-tubular (T-) system, thereby eliciting twitch responses, which are a sensitive measure of Ca2+ release. Treatment of fibres with the oxidant H2O2 (200 microM and 10 mM) for 2-5 min markedly potentiated caffeine-induced Ca2+ release and the force response to partial depolarisation of the T-system (by solution substitution). Importantly, such H2O2 treatment had no effect at all on any aspect of the twitch response (peak amplitude, rate of rise, decay rate constant and half-width), except in cases where it interfered with the T-system potential or voltage-sensor activation, resulting in a reduction or abolition of the twitch response. Exposure to strong thiol reductants, dithiothreitol (DTT, 10 mM) and reduced glutathione (GSH, 5 mM), did not affect the twitch response over 5 min, nor did varying the glutathione ratio (reduced to oxidised glutathione) from the level present endogenously in the cytosol of a rested fibre (30:1) to the comparatively oxidised level of 3:1. In fibres that had been oxidised by H2O2 (10 mM) (or by 2,2'-dithiodipyridine, 100 microM), exposure to GSH (5 mM) caused potentiation of twitch force (by approximately 20 % for H2O2); this effect was due to the increase in the Ca2+ sensitivity of the contractile apparatus that occurs under such circumstances and was fully reversed by subsequent exposure to 10 mM DTT. We conclude that: (a) the redox potential across the sarcomplamsic reticulum has no noticeable direct effect on normal E-C coupling in mammalian skeletal muscle, (b) oxidising the Ca2+-release channels and greatly increasing their sensitivity to Ca2+-induced Ca2+ release does not alter the amount of Ca2+ released by an action potential and (c) oxidation potentiates twitches by a GSH-mediated increase in the Ca2+ sensitivity of the contractile apparatus.

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1. The effect on sarcoplasmic reticulum (SR) function of exposure to high intracellular [Ca¥] was studied in mechanically skinned fibres from the extensor digitorum longus muscle of the rat, using caffeine to assay the SR Ca¥ content. 2. A 15 s exposure to 50 ìÒ Ca¥ irreversibly reduced the ability of the SR to loadÏretain Ca¥ and completely abolished depolarization-induced Ca¥ release, whereas a 90 s exposure to 10 ìÒ Ca¥ had no detectable effect on either function. The reduction in net SR Ca¥ uptake: (a) was near-maximal with treatment at 50 ìÒ Ca¥, (b) was unrelated to voltage-sensor function, and (c) persisted unchanged for > 20 min. The reduction was primarily due to a threefold increase in leakage of Ca¥ out of the SR. This increased leakage was not substantially blocked by the presence of 10 mÒ Mg¥ or 2 ìÒ Ruthenium Red. 3. The adverse effect on SR function of exposure to high [Ca¥] could also be observed by the reduction in the ability of the SR to maintain a low [Ca¥] within the skinned fibre in the face of elevated [Ca¥] METHODS Isolation of skinned fibresLong-Evans hooded rats (Rattus norvegicus) aged 14 to 20 weeks were anaesthetized by halothane (2 % vÏv) and killed by asphyxiation in accordance with guidelines of the La Trobe University Animal Ethics Committee. As described previously (Lamb & Stephenson, 1994), both extensor digitorum longus muscles were then removed and placed in paraffin oil and kept cool on ice. Single muscle fibres were dissected free at one end from the muscle and mechanically skinned. The skinned fibre segment was mounted onto a force transducer (AME875, SensoNor, Horten, Norway), and stretched to 120% of its resting length. The fibre was then placed into a 2 ml Perspex bath containing a potassium or sodium 1,6-diaminohexane-N,N,N' ,N'-tetraacetic acid (HDTA) solution to equilibrate before being stimulated by rapid substitution of an appropriate solution. All experiments were performed at room temperature (23 ± 2°C). SolutionsAll chemicals were obtained from Sigma unless stated otherwise. The make-up of the solutions used in the different parts of the experiments is set out below. All solutions had a pH of 7·10 ± 0·01 and an osmolality of 295 ± 5 mosmol kg¢, and unless stated otherwise had 1 mÒ free Mg¥ and 8 mÒ total ATP. Free [Ca¥] at ü 0·1 ìÒ was verified with a Ca¥-sensitive electrode (Orion, Cambridge, MA, USA) . Leak solution. K-HDTA solution with 2 mÒ total EGTA (pCa > 8), but otherwise similar to pre-equilibration solution. Used to allow Ca¥ leakage from the SR in the absence of Ca¥ uptake. Additional matching solutions were also made with 10 mÒ free Mg¥ (22·7 mÒ total magnesium) or with all K¤ replaced with Na¤ (for chronic T-system depolarization).Contractile apparatus experiments Relaxing solution. Potassium solution with 50 mÒ EGTA (pCa > 9), which is similar to the polarizing solution with all HDTA replaced with EGTA (see Stephenson & Williams, 1981).Maximum-activation solution. Same as Relaxing solution but with 50 mÒ CaEGTA (pCa 4·5). Referred to as 'M...