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¹ Department of Physiology, University of Aarhus, DK-8000 Århus C, Denmark² Department of Zoology, La Trobe University, Bundoora, Victoria 3086, Australia

	Abstract

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Strenuous exercise causes an increase in extracellular [K⁺] and intracellular Na⁺ ([Na⁺]_i) of working muscles, which may reduce sarcolemma excitability. The excitability of the sarcolemma is, however, to some extent protected by a concomitant increase in the activity of muscle Na⁺–K⁺ pumps. The exercise-induced build-up of extracellular K⁺ is most likely larger in the T-tubules than in the interstitium but the significance of the cation shifts and Na⁺–K⁺ pump for the excitability of the T-tubular membrane and the voltage sensors is largely unknown. Using mechanically skinned fibres, we here study the role of the Na⁺–K⁺ pump in maintaining T-tubular function in fibres with reduced chemical K⁺ gradient. The Na⁺–K⁺ pump activity was manipulated by changing [Na⁺]_i. The responsiveness of the T-tubules was evaluated from the excitation-induced force production of the fibres. Compared to control twitch force in fibres with a close to normal intracellular [K⁺] ([K⁺]_i), a reduction in [K⁺]_i to below 60 mM significantly reduced twitch force. Between 10 and 50 mM Na⁺, the reduction in force depended on [Na⁺]_i, the twitch force at 40 mM K⁺ being 22 ± 4 and 54 ± 9% (of control force) at a [Na⁺]_i of 10 and 20 mM, respectively (n= 4). Double pulse stimulation of fibres at low [K⁺]_i showed that although elevated [Na⁺]_i increased the responsiveness to single action potentials, it reduced the capacity of the T-tubules to respond to high frequency stimulation. It is concluded that a reduction in the chemical gradient for K⁺, as takes place during intensive exercise, may depress T-tubular function, but that a concomitant exercise-induced increase in [Na⁺]_i protects T-tubular function by stimulating the Na⁺–K⁺ pump.

(Received 2 December 2003; accepted after revision 15 March 2004; first published online 19 March 2004)
Corresponding author O. B. Nielsen: Department of Physiology, University of Aarhus, DK-8000 Århus C, Denmark. Email: obn{at}fi.au.dk

	Introduction

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In skeletal muscles from vertebrates the signal for muscle contraction involves the propagation of action potentials along the length of the muscle fibres via the sarcolemma and into the transverse (T-) tubular system where it causes activation of voltage sensor molecules (Melzer et al. 1995). Because of the efflux of K⁺ associated with action potentials, intensive contractile activity causes a significant net loss of K⁺ from working muscle fibres, leading to increases in interstitial and T-tubular K⁺ concentration (Sejersted & Sjøgaard, 2000). This, in turn, leads to run-down of the chemical gradient for K⁺ with ensuing depolarization of the fibres. Since depolarization may interfere with fibre excitability and the function of the voltage sensors, it has repeatedly been proposed that exercise-induced increases in extracellular K⁺ concentration contribute to the development of muscle fatigue in some circumstances (Sejersted & Sjøgaard, 2000).

Since the ratio between the area of the excitable membrane and the extracellular volume it faces is much larger for the T-tubules than for the sarcolemma, the exercise-induced loss of K⁺ is likely to produce a much more pronounced build-up of [K⁺] in the T-tubules than in the interstitium (Sejersted & Sjøgaard, 2000). For this reason, the development of excitation failure during exercise is likely to be determined primarily by changes in the electrochemical gradients across the T-tubular membrane rather than across the sarcolemma.

Several reports, including both in vivo and in vitro studies, have demonstrated that muscle activity also causes an increase in the activity of muscle Na⁺–K⁺ pumps (Hallen et al. 1994; Nielsen & Clausen, 1997), which alleviates the depressing effect of increased extracellular [K⁺] on the contractile performance of muscles (Nielsen & Clausen, 2000). Measurements of muscle compound action potentials (M-waves) show that the effect of increased Na⁺–K⁺ pump activity on force is related to a recovery of fibre excitability (Overgaard et al. 1999) possibly caused by a partial repolarization of the fibres and an increase in electrochemical gradient for Na⁺ (Nielsen & Clausen, 2000). Because of difficulties in accessing the T-tubular system, much less is known about the significance of elevated Na⁺–K⁺ pump activity for the maintenance of excitability of the T-tubular membrane and voltage sensors when extracellular [K⁺] is increased during exercise.

Recently, Posterino et al. (2000) showed that in muscle fibres where the sarcolemma is peeled off mechanically in such a way that the T-tubular system seals off and repolarizes (Lamb et al. 1995), contractions can be triggered by setting up action potentials in the T-tubular system via electrical stimulation. Using this preparation, we here examine the hypotheses that (i) if the T-system is depolarized, a rise in intracellular [Na⁺] can produce significant recovery of excitation-induced force responses to single action potentials, and (ii) the reduction in the electrochemical gradient for Na⁺ produced by this rise in intracellular [Na⁺] has a conflicting deleterious effect on fibre excitability. Our findings indeed show that when T-tubules are depolarized sufficiently to reduce their excitability, increased intracellular [Na⁺] does produce significant recovery of excitation-induced force. This recovery most likely results from an increase in the activity of the Na⁺–K⁺ pump. Furthermore, we provide evidence that the rise in intracellular [Na⁺] has a separate deleterious effect in that it interferes with the ability of the T-system to support successive action potentials at close intervals. This latter effect probably arises because the electrochemical gradient for Na⁺ movement from the T-system to the cytoplasm is reduced, which hinders efficient action potential propagation through the T-system. Parts of the presented observations have briefly been reported in a preliminary form (Nielsen et al. 2002).

	Methods

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Isolation and preparation of mechanically skinned fibres

All experiments were carried out on skinned muscle fibres prepared as previously described (Lamb & Stephenson, 1990, 1994). Male Long Evans hooded rats (16–18 weeks old) were deeply anaesthetized with halothane and killed by asphyxiation, in accordance with guidelines of the Animal Ethics Committee of La Trobe University. Whole extensor digitorum longus (EDL) muscles were dissected, blotted carefully and immersed in paraffin oil. To produce mechanically skinned fibre preparations, segments of fibres (approximately 2 mm long) were isolated from the intact muscle and skinned by rolling back the sarcolemma using jeweller's forceps under a dissecting microscope (Lamb & Stephenson, 1994). Following the skinning procedure, the segment was mounted on an isometric force transducer (AME801, SensoNor, Horten, Norway), stretched to 120% of slack length and initially immersed in a K-Na-HDTA solution (Table 1) broadly mimicking the cytosol, as in previous studies (e.g. Lamb & Stephenson, 1994; Posterino et al. 2000). All experiments were performed at 24–25°C.

Each skinned fibre segment was exposed to a number of intracellular solutions containing various concentrations of K⁺ and Na⁺; these solutions were made by appropriate mixture of the solutions shown in Table 1. All chemicals were obtained from Sigma, unless specified otherwise. The solutions used to examine twitch responses all contained 50 mM hexamethylene-diamine-tetraacetate (HDTA^2–, Fluka, Buchs, Switzerland) as the impermeant anion, 8 mM total ATP, 10 mM creatine phosphate, 1 mM free Mg²⁺, and were weakly Ca²⁺-buffered at a pCa (=–log₁₀[Ca²⁺]) of 7.0 with 50 µM total EGTA. Ca²⁺ concentrations were measured with a Ca²⁺-sensitive electrode (Orion Research Inc., Boston, MA, USA). The concentrations of K⁺ and Na⁺ were varied by appropriate mixture of the various HDTA and NH₄-HDTA solutions (K⁺ or Na⁺ based); in this way total cation concentration could be kept constant by making up any deficit with NH₄⁺ (and in the case of zero Na⁺ solutions, up to 20 mM Tris⁺), with the ionic strength and osmolality (286 ± 4 mosmol kg^–1) of all solutions kept the same. For the determination of control twitch force elicited by electrical stimulation, the fibre segment was bathed in a ‘control’ intracellular solution (mM: K⁺, 113; Na⁺, 30; NH₄⁺, 20; for other details, see Table 1), with subsequent measurements made in the same fibre segment in other solutions as appropriate, each being bracketed by measurements in the control solution.

In order to verify that the sarcoplasmic reticulum (SR) was adequately loaded with Ca²⁺, the force response of the fibre segment when exposed to a low-Mg²⁺ solution was determined at the end of each experiment. The low-Mg²⁺ solution (Table 1) was similar to the K-Na-HDTA solution but contained only 1.0 mM total Mg²⁺ (15 µM free Mg²⁺). Such a solution triggers the release of Ca²⁺ from the SR because the inhibitory effect of cytoplasmic Mg²⁺ on the release channels is removed (see Lamb & Stephenson, 1994). In all fibres used, the peak force elicited by release of SR Ca²⁺ at end of the experiment was close to the maximal force generating capacity of the fibre expected in such low Mg²⁺ conditions, indicating that the SR was indeed well loaded with Ca²⁺.

For determination of the maximal force generating capacity, the fibre segment was exposed to a heavily Ca²⁺-buffered solution with a free Ca²⁺ concentration of 30 µM (pCa 4.5) (Ca²⁺ activating solution, see Table 1). This solution was similar to the K-Na-HDTA solution but all HDTA^2– was replaced with EGTA^2–/CaEGTA^2–.

The Ca²⁺ dependence of force production by the contractile apparatus was examined by exposing a fibre segment to a sequence of solutions of progressively higher free [Ca²⁺] buffered with EGTA. Matched sets of such solutions at different [Na⁺], [K⁺] and [NH₄⁺] were made by mixing the appropriate HDTA-based and EGTA/Ca-EGTA solutions shown in Table 1. To ensure strong Ca²⁺ buffering the total [EGTA] was set at 5 mM in all the solutions (i.e. 9 parts of HDTA-based solutions to 1 part EGTA-based solutions). A fibre segment was first tested in a set of solutions with 127 mM K⁺ and 36 mM Na⁺ (K-Na-HDTA based solution), then in two further sets with different [K⁺], [Na⁺] and [NH₄⁺] (see Table 2) and then again in the original K-Na-HDTA based set. The maximum force (F_max) produced at the highest [Ca²⁺] (pCa 4.5) in each set of solutions was expressed as a percentage of the average maximum force of the two bracketing sequences in the K-Na-HDTA based solution measured in the same fibre. In addition, for each set of solutions the force response at each pCa was expressed relative to the corresponding maximum force. These data points were then fitted with a Hill curve (see Fig. 1).

View larger version (8K): [in this window] [in a new window]   Figure 1.  Effect of different intracellular solutions on the force–pCa relationship of a rat fibre The relationship between force and [Ca2+] in a skinned fibre from rat EDL muscle was ascertained in various intracellular solutions which differed in [Na+], [K+] and [NH4+]. In a given solution, the fibre was exposed to progressively higher free [Ca2+] (i.e. pCa from 9 to 4.5) and corresponding forces were fitted by a Hill curve. A solution set with 127 mM K+ and 36 mM Na+ was examined both before () and after () other sets containing (mM) 10 Na+, 40 K+, 113 NH4+ () and 50 Na+, 40 K+, 73 NH4+ (). The force–pCa relationships in the different conditions were virtually indistinguishable.

Fibre excitation was achieved by supramaximal electrical field stimulation (2 ms pulses at 50 V cm^–1) applied via two platinum wire electrodes positioned 4 mm apart in parallel with the skinned fibre, eliciting action potentials in the sealed T-system (Posterino et al. 2000). Force responses were amplified and then recorded at 1 kHz using a 200 series Powerlab (ADInstruments, Sydney, Australia; with Chart V4.12 software) and the results were analysed using Graphpad Prism software (San Diego, CA, USA).

In previous studies on action potential-induced responses in skinned fibres (e.g. Posterino et al. 2000), the ‘intracellular’ bathing solution contained 127 mM K⁺ and 36 mM Na⁺ (i.e. K-Na-HDTA solution in Table 1). In the present study, the [K⁺] and [Na⁺] in the ‘control’ solution were slightly lower, 113 mM and 30 mM, respectively (balanced with 20 mM NH₄⁺, see Table 1). This was because with this control solution it was possible to make a matching test solution (with unchanged osmolality and ionic strength) in which the [Na⁺] could be raised to 50 mM without altering the [K⁺], that is, without altering the electrochemical gradient for K⁺ across the T-system. It was also straightforward to make other matching solutions with lower concentrations of either or both K⁺ and Na⁺. To examine whether there was any difference between the twitch response in the K-Na-HDTA solution and in the control solution, the experiments were generally started by incubating the skinned fibre segment for 2–5 min in the K-Na-HDTA solution before moving it to the control solution. Measurements of the excitation-induced twitch response verified that the maximal twitch force was no different in the control solution from that in the K-Na-HDTA solution, the force in the control solution being 96 ± 8% of that measured in K-Na-HDTA solution in the same fibre (P= 0.58, n= 6 fibres). Subsequent determinations of the excitation-induced force response in fibre segments incubated in solutions with various concentrations of K⁺ and Na⁺ (experimental buffers) were bracketed by determinations of force in control solution. Thus, the fibre segment was placed in the control solution for 2 min and control contractions were evoked and thereafter the force response was tested after 30 s incubation in the experimental buffer. Subsequently, the preparation was again incubated for 2 min in control buffer and the force response was tested. Control experiments (not shown) demonstrated that following incubation in an experimental buffer, 2 min of incubation in the control solution was sufficient to obtain full recovery of control force.

	Results

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When the concentrations of K⁺ and Na⁺ were altered in the intracellular solutions in the examination of excitation-induced responses described below, any deficit in the total cation concentration was balanced by addition of up to 113 mM NH₄⁺ (see Methods). To test whether there was any direct effect of such manipulations on the sensitivity of the contractile apparatus to free Ca²⁺, the force–pCa relationship was examined in skinned fibre segments exposed to solutions in which the [K⁺], [Na⁺] and [NH₄⁺] were varied over the full ranges used in the twitch experiments (see Methods and Table 2). For each solution, the force of a fibre segment was determined at progressively higher free [Ca²⁺], as seen in Fig. 1. The maximum force reached at the highest [Ca²⁺] in a given fibre was not significantly different between any of the conditions examined (Table 2). Furthermore, there was no significant change in the pCa₅₀ (the pCa giving half-maximum force) or n_H (the Hill coefficient) obtained by fitting a Hill curve to the force–pCa data for each condition (Table 2). This indicates that the variations in [Na⁺], [K⁺] and [NH₄⁺] under the conditions used in this study had no detectable effect on the response of the contractile apparatus to Ca²⁺.

When the sarcolemma of a muscle fibre is skinned off by the technique used in the present study, the intracellular compartment is opened up to the bathing medium and at the same time, the T-tubular system seals off to form an isolated compartment, representing part of the former extracellular space (Lamb et al. 1995). When such fibre preparations are incubated in a solution that mimics the normal intracellular environment (high K⁺ concentration), the T-system becomes polarized and contractions can be triggered by electrical stimulation eliciting action potentials in the T-tubular system (Posterino et al. 2000). To evaluate the importance of the electrochemical gradient for K⁺ across the T-tubules for the excitability of this membrane system, the twitch response to electrical stimulation was examined in skinned fibre segments bathed in intracellular solutions having a reduced K⁺ concentration. Figure 2A shows traces of force recordings from a typical experiment in which twitch force was tested in a fibre segment bathed in a solution with 40 mM K⁺ (and 20 mM Na⁺). Compared to the average force response in the bracketing examinations of contraction in the control solution (with 113 mM K⁺ and 30 mM Na⁺), twitch force in the test conditions was reduced by 38%. Figure 2A also shows that control twitch force was well below the peak force elicited by directly releasing Ca²⁺ from the SR by exposing the fibre to a low [Mg²⁺] solution (0.015 mM free Mg²⁺; see Methods). In nine fibres examined, the force elicited by twitch stimulation in control solution at the start of the experiment was 55 ± 3% of the force elicited by release of SR Ca²⁺ upon lowering [Mg²⁺]. This shows that the Ca²⁺ release during twitch contractions was submaximal, demonstrating that twitch force was a sensitive measure of the excitation-induced Ca²⁺ release. This point is further demonstrated later when applying a pair of closely spaced electrical stimuli rather than a single stimulus. As illustrated by the control twitch elicited 27 min into the experiment (Fig. 2A), contractions in the control solution were generally well maintained throughout experiments (see also Fig. 6 later).

View larger version (15K): [in this window] [in a new window]   Figure 2.  Effect of intracellular [K+] on excitation-induced contractions in skinned muscle fibres A, traces of force recordings from a fibre segment bathed in control intracellular solution (113 mM K+ and 30 mM Na+) or in a solution with 40 mM K+ and 20 mM Na+, as indicated. Times show minutes after start of the experiment. Traces a: twitch stimulation. Trace b: force elicited by release of SR Ca2+ when exposing the fibre to the low Mg2+-solution (see Methods). Trace c: maximal Ca2+-activated force induced by exposure to the Ca-EGTA solution at pCa 4.5. B, twitch force when the bathing solution contained 20–113 mM K+ and either 20 (, ) or 50 (, •) mM Na+ expressed relative to the average of the force response in the bracketing determinations of twitch force in control solution (113 mM K+ and 30 mM Na+). Open symbols show the mean (±S.E.M.) force response at the indicated combination of K+ and Na+ for cases where 3 or more fibres were examined (3 and 4 fibres at 80 and 113 mM K+, respectively, and 5–8 fibres in other cases). Filled symbols show individual values in cases where only two fibres were examined.

View larger version (20K): [in this window] [in a new window]   Figure 6.  Contractions in a skinned muscle fibre in response to single and double pulse stimulation In each intracellular solution, the force response was tested using single and double pulses with a pulse configuration of single (s) – single (s) – double (d8) – double (d20) – single (s). The interpulse interval in the two double pulses was 8 (d8) and 20 ms (d20). Traces show force responses during consecutive exposure to control and test solutions with the indicated concentrations of Na+ and K+. Other details as in Fig. 3. Note the expanded force scale for contractions in test solutions.

To determine more precisely the role of solution [Na⁺] on the twitch force at low intracellular [K⁺], at a given [K⁺] (either 30 or 40 mM) paired experiments were conducted on each fibre segment at each of two different [Na⁺] using the protocol shown in Fig. 3A. The traces in Fig. 3A show recordings of twitch force in a typical experiment in which the effect of a reduction in intracellular [K⁺] to 30 mM was examined at 20 and 50 mM Na⁺. At both Na⁺ concentrations the reduction in K⁺ concentration led to a reduced twitch contraction but the depression was much more pronounced at the lower Na⁺ concentration. Figure 3B shows the mean data of maximum twitch force from several such experiments in which fibres were bathed in either 30 or 40 mM K⁺. At both [K⁺] the force was increased more than twofold by the increase in intracellular [Na⁺]. Figure 3B also shows that the concentration of Na⁺ necessary to maintain force at a certain level (e.g. around 50% of control force), is higher in fibres at 30 mM K⁺ than in fibres at 40 mM K⁺ ([Na⁺] about 50 and 20 mM, respectively). These results indicate that the depressing effect of a reduction in the chemical gradient for K⁺ can be alleviated by increasing the activity of the Na⁺–K⁺ pump by raising the intracellular [Na⁺] 2- to 2.5-fold within the range from 10 to 50 mM. In contrast to these findings with low intracellular [K⁺], when skinned fibres were bathed in the control solution with 113 mM K⁺ (i.e. with a near normal chemical gradient for K⁺), twitch force was not significantly affected by increasing intracellular [Na⁺] 1.7-fold from 30 to 50 mM, the difference in force being 2 ± 2%(n= 3).

View larger version (20K): [in this window] [in a new window]   Figure 3.  Significance of the intracellular [Na+] for twitch force in skinned fibres exposed to low intracellular [K+] For determinations of control force, the fibre segment was bathed for 2 min in control solution (113 mM K+, 30 mM Na+). Force responses in solutions with low [K+] (30 or 40 mM K+) were determined after 30 s exposure to the solution. Determinations of force in test conditions were bracketed by determinations of control force. A, recordings of twitch force from an experiment in which the fibre was exposed to intracellular solutions with 30 mM K+ combined with 20 or 50 mM Na+, as indicated. B, effect of varying the Na+ concentration on the maximal twitch force in fibres exposed to 30 mM or 40 mM K+. Each fibre was only exposed to one of the two test K+ concentrations, and the tests of force at the two Na+ concentrations were done in a semi-random order. For each test condition, force is expressed relative to the average of the two bracketing control contractions. Columns show mean +S.E.M. from experiments on 4 and 5 fibres.

View larger version (13K): [in this window] [in a new window]   Figure 4.  Effect of removal of intracellular Na+ on the excitation-induced twitch force Skinned fibre segments were incubated in control solution (113 mM K+, 30 mM Na+) and control twitch was determined. At time zero, the fibres were exposed to a solution having the same constituents as the control solution but with all Na+ replaced by NH4+. The fibres were stimulated at the indicated time points using single pulses. Force responses are expressed relative to the initial control force. Curves show individual data from 3 fibres.

View larger version (13K): [in this window] [in a new window]   Figure 5.  Effect of interpulse interval on the force of fused contractions elicited by electrical stimulation with double pulses in a skinned muscle fibre The fibre segment was bathed in control solution (113 mM K+ and 30 mM Na+) and contractions were stimulated with single pulses or with double pulses with interpulse intervals from 4 to 20 ms. Contractions were evoked every 10 s and the interpulse interval was progressively increased from 4 to 20 ms and then decreased again, with the force response at a given interpulse interval being similar on both occasions. Maximal force elicited is expressed relative to the twitch force elicited by single pulse stimulation. The figure shows data from one fibre; very similar results were obtained in two other fibres.

Figure 6 shows that in general terms the force elicited by double pulse stimulation (8 or 20 ms interpulse intervals) exhibited a similar dependency on the intracellular concentrations of Na⁺ and K⁺ as did the twitch response to a single stimulus, with all responses being smaller in 40 mM K⁺ with 20 mM Na⁺ than in the control solution, and even smaller still in 40 mM K⁺ with 10 mM Na⁺. This dependency is further illustrated in Fig. 7, which shows the mean data from a number of fibre segments when lowering [K⁺] to either 30 mM (Fig. 7A) or 40 mM (Fig. 7B), at each of two Na⁺ concentrations. As with single pulse stimulation, a lowering of intracellular [K⁺] led to a reduction in the force response to double pulse stimulation, and for a given [K⁺] the reduction in force strongly depended on the prevailing [Na⁺], with the reduction in force invariably being smaller at higher [Na⁺].

View larger version (19K): [in this window] [in a new window]   Figure 7.  Effect of changes in intracellular [K+] and [Na+] on the force response to double pulse stimulation Experimental conditions as in Fig. 6. For each condition the force responses were tested using both single and double pulses with a pulse configuration of one or two single pulses followed by two double pulses and a single pulse. The interpulse interval in the two double pulses was 8 and 20 ms. The figure shows means +S.E.M. for the two double pulses and the last single pulse in the pulse configuration. The forces are expressed relative to the force elicited by the first single pulse in the bracketing determinations of control contractions. A, intracellular solutions with 30 mM K+ and 20 or 50 mM Na+, n= 5. B, solutions with 40 mM K+ and 10 or 20 mM Na+, n= 4.

View larger version (22K): [in this window] [in a new window]   Figure 8.  Effect of interpulse interval on force production at different combinations of intracellular [K+] and [Na+] Data from Fig. 7. For each condition the force responses were tested using both single and double pulses with a set pulse configuration of one or two single pulses followed by two double pulses and a single pulse. The interpulse interval in the two double pulses was 8 and 20 ms. The figure shows means +S.E.M. for the two double pulses and the last single pulse in the pulse configuration. The forces are expressed relative to the force elicited by the last single pulse in the set pulse configuration. A, intracellular solutions with 30 mM K+ and 20 or 50 mM Na+, n= 5. B, solutions with 40 mM K+ and 10 or 20 mM Na+, n= 4.

	Discussion

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Effect of reduced intracellular [K⁺] on force and T-tubular function

In the present study, the effects of reducing the chemical gradient for K⁺ on T-tubular function were examined by measurements of the force response to electrical excitation. The contractile response of the skinned fibres to excitation depends on several steps starting with the initiation of action potentials in the tubules and ending with the development of force by the contractile apparatus. Theoretically, the effect of changes in solution composition on the force response could be due to effects on any of these steps. Several findings argue, however, that the effect of low intracellular [K⁺] was caused by a reduced function of the T-tubular system, i.e. by loss of T-tubular excitability and inactivation of the voltage sensor molecules.

Firstly, since changes in solution composition were without effect on the pCa–force relationship, the effects on force were not due to changes in the responsiveness of the contractile apparatus but instead to changes in excitation-induced Ca²⁺ release. It was further apparent that the major facet of the intracellular solution influencing the response to excitation was the concentration of K⁺. As seen in Fig. 2B, a threefold decrease in the intracellular [K⁺] (from 113 mM to between 30 and 40 mM), with the [Na⁺] unchanged, caused 50% reduction in the size of the twitch response. This effect is not the result of using NH₄⁺ as the replacement cation for K⁺ or Na⁺ (see Methods), because (a) a comparable effect is seen when the [K⁺] is lowered by raising [Na⁺] rather than adding NH₄⁺ (Ørtenblad & Stephenson, 2003), (b) at low [K⁺], the size of the twitch response is greatly affected by small changes (e.g. 10 mM) in the [K⁺] and/or [Na⁺] (see Fig. 3B), with the [NH₄⁺] being varied by only 10% in such cases (e.g. from 113 to 103 mM), and (c) addition of 20 or 30 mM NH₄⁺ had no detectable effect on the twitch response when the [K⁺] was relatively high (e.g. 80 mM, see Fig. 2B and Results).

Secondly, it has previously been shown that the excitation-induced contractions in the skinned fibre preparation used here depend on the initiation and propagation of action potentials by TTX-sensitive Na⁺ channels in the T-tubular system (Posterino et al. 2000). Furthermore, Ørtenblad & Stephenson (2003) showed that in skinned fibres, where the excitation-induced twitch force was reduced by 50% by lowering intracellular K⁺, it was still possible to obtain normal force production by direct activation of the voltage sensors. This demonstrates that at this level of depolarization the reduction in force was specifically caused by loss of the excitability of the T-tubular system with no effects on the steps in the contraction mechanisms distal to voltage sensors.

Thirdly, reductions in the excitation-induced force are also seen in intact muscle or single fibres when the chemical gradient for K⁺ is reduced. In these preparations the reduction in force has been related to the ensuing depolarization of the muscle fibres, leading to slow inactivation of the TTX-sensitive Na⁺ channels and loss of excitability (Ruff, 1999). Measurements of single fibre action potentials and muscle compound action potentials show that depending on the level of depolarization increased extracellular K⁺ increases the pulse strength necessary to initiate an action potential, reduces the action potential amplitude, causes propagation block or even renders the fibres completely inexcitable (Renaud & Light, 1992; Cairns et al. 1997; Overgaard & Nielsen, 2001; Rich & Pinter, 2001, 2003; Yensen et al. 2002). In all cases the depolarization and loss of excitability results in a reduction in the excitation-induced release of Ca²⁺ from the SR and thereby in force production (Caputo et al. 1984). Since the Na⁺ channels responsible for the excitability of the sarcolemma are similar to the Na⁺ channels of the T-tubular membranes, it is likely that similar loss of T-tubular excitability takes place when this membrane system is depolarized by a reduction in intracellular [K⁺]. This notion is supported by the similarity in the sensitivity of the excitability of skinned fibres and intact muscles to depolarizations. Thus, the twitch force in the skinned fibres here was well preserved when the intracellular [K⁺] was decreased from 113 mM to 80 mM (Fig. 2), only decreasing to 50% of the control level when the [K⁺] was lowered to around 30–40 mM. This indicates that a considerable depolarization safety margin exists in the skinned fibres, which is similar to findings in intact muscle (Cairns et al. 1995, 1997; Yensen et al. 2002).

The net effects on the twitch response observed here with a threefold decrease in intracellular [K⁺] is similar to that occurring in an intact muscle fibre when raising the extracellular [K⁺] from 4 to 12 mM, which corresponds to a depolarization to around –65 mV (Cairns et al. 1995; Clausen & Overgaard, 2000). The effect of such a depolarization on the excitation-induced activation of the voltage sensors and, thus, the force production depends not only on the size and duration of the action potential but also on its ability to propagate throughout the whole T-system. In addition, if EDL fibres are depolarized to –60 mV, there is a substantial degree of inactivation of the voltage sensors (Chua & Dulhunty, 1988), which reduces the pool of voltage sensors available for activation. This effect would be expected to further reduce the Ca²⁺ release and force response to any action potentials that are propagated. It seems likely therefore that inactivation of voltage sensors added to the reduction in force induced here in the skinned fibres by depolarization.

Although the potential of the T-system cannot be readily measured in skinned fibres, the resting potential in the standard conditions with 113–126 mM intracellular K⁺ present must be approximately –80 to –90 mV in the rat EDL fibres used here. This is evident from a comparison of the activation and steady-state inactivation properties of depolarization-induced force responses in skinned EDL fibres (Posterino & Lamb, 1998) with those in intact EDL fibres (Chua & Dulhunty, 1988), and by the consistency of findings when adding agents affected by the T-system potential, such as nifedipine (Posterino & Lamb, 1998), or altering other conditions, such as the intracellular [Cl^–] (Coonan & Lamb, 1998). Furthermore, to explain such a resting potential, it can also be concluded that the [K⁺] in the T-system in the skinned fibres under the standard conditions must be approximately 4 mM, which is similar to that in an intact fibre in situ under rested conditions.

Role of the Na⁺–K⁺ pump in maintaining T-tubular function

Measurements on frog muscle have shown that the T-tubular membranes contain a substantial amount of Na⁺–K⁺ pumps (Clausen, 2003). In the present study, the activity of the T-tubular Na⁺–K⁺ pumps was manipulated by varying the intracellular [Na⁺] from 0 to 50 mM. Studies on intact cells or vesicles from heart and skeletal muscle have given values for the intracellular Na⁺ affinity of the Na⁺–K⁺ pump from 9 to 43 mM (Semb & Sejersted, 1996), indicating that the range of intracellular [Na⁺] used in the present study was within the dynamic range for the Na⁺–K⁺ pump. Thus, although the activity of the Na⁺–K⁺ pump was not measured in the present study, an increase in intracellular [Na⁺] was most likely associated with a higher activity of the Na⁺–K⁺ pump. Several studies have shown that in intact muscle incubated at high extracellular [K⁺], stimulation of the Na⁺–K⁺ pump produces recovery of force and sarcolemma excitability (Clausen & Everts, 1991; Overgaard & Nielsen, 2001). It is most likely therefore that the recovery of force obtained by increased intracellular [Na⁺] here (Figs 2B and 3) was caused by improved T-tubular polarization associated with an increase in the activity of the T-tubular Na⁺–K⁺ pump.

The present experiments were done at temperatures between 24 and 25°C. The reason for this was that the skinned fibre preparation, like isolated intact muscles (Segal & Faulkner, 1985; Lännergren & Westerblad, 1987), was unable to maintain viability for prolonged periods at physiological temperatures (see Lamb & Stephenson, 1994). A recent study on isolated whole muscle demonstrated, however, that qualitatively the loss of force induced by depolarization and the subsequent recovery of force by stimulation of the Na⁺–K⁺ pump was the same at 20, 30 and 35°C (Pedersen et al. 2003). The same study showed that the loss of force and excitability induced by a reduction in the chemical gradient for K⁺ was less at the highest temperatures. It was further suggested that this effect was the result of a temperature-induced increase in the activity of the Na⁺–K⁺ pump and a lesser tendency for the Na⁺ channels to enter a state of slow inactivation at high temperatures. These findings indicate that stimulation of the Na⁺–K⁺ pump also improves the excitability of K⁺-depressed fibres at physiological temperatures, but that more severe reductions in the chemical gradient for K⁺ has to take place before excitability is lost.

In the preparation here the intracellular K⁺ concentration is determined by the K⁺ concentration of the bathing solution and is not changed by stimulation of the Na⁺–K⁺ pump. However, because of the small volume of the T-tubules (Clausen, 2003), any increase in Na⁺–K⁺ pump activity in response to increased intracellular [Na⁺] will lead to a decrease in the T-tubular [K⁺] in skinned fibres, just as it does in intact fibres. In depolarized fibres this will tend to restore the membrane potential and improve excitability, voltage sensor availability and force responses. In addition to its action in lowering tubular [K⁺], an increase in the Na⁺–K⁺ pump activity will also help polarize the T-system by its direct electrogenic effect.

The importance of the balance between active and passive Na⁺ and K⁺ fluxes for the maintenance of T-tubular excitability in skinned fibres is, perhaps, best illustrated by the dramatic loss of force that took place when the T-tubular Na⁺–K⁺ pump was inhibited by incubation of the fibres at 0 Na⁺. In these experiments, the intracellular concentration of Na⁺ and K⁺ was initially optimal for action potentials. The loss of the ability of the fibres to respond to excitation was therefore most likely related to a net flux of Na⁺ and K⁺ between the T-tubules and the intracellular compartment with ensuing decrease of the chemical gradients for the two ions. A similar loss of force has been reported in skinned muscle fibres where ouabain was used to inhibit the Na⁺–K⁺ pump (Costantin & Podolsky, 1967; Lamb & Stephenson, 1990). In intact fibres, the maintenance of T-tubular Na⁺–K⁺ homeostasis may be assisted by diffusion of ions between the T-tubules and the interstitium. However, based on computer simulations that took into account the dimensions of the T-tubular system, Wallinga et al. (1999) concluded that because of diffusion limitations in the T- tubules, the maintenance of Na⁺–K⁺ homeostasis in the inner T-tubular compartment depends mainly on ion fluxes across the T-tubular membrane with little contribution from diffusion between the T-tubules and the interstitium. Thus, also in intact muscle, T-tubular Na⁺–K⁺ homeostasis may be critically dependent on the activity of the Na⁺–K⁺ pump.

Dual mechanisms by which the Na⁺–K⁺ pump helps prevent muscle fatigue

The ways in which problems in excitability can contribute to muscle fatigue can be viewed as follows. If a muscle fibre is repeatedly stimulated at a comparatively high frequency, the muscle fibre will experience a progressive net loss of intracellular K⁺ and a net gain of intracellular Na⁺. Of these, the initial major problem is that efflux of K⁺ from the fibre causes an increase in the [K⁺] in the interstitium and in the T-system, with the latter most likely reaching a critical level earlier owing to the relatively small volume of the T-system. The present results show that a threefold decrease in the chemical gradient for K⁺ across the T-system causes a large depolarization of the T-system (probably by 25 mV to –60 mV) and a reduced twitch response to single action potential stimuli. This situation should mimic what happens in an intact fibre if the T-system [K⁺] rises from 4 mM to 12 mM with unchanged intracellular [Na⁺]. The reduced size of the twitch is due both to slow inactivation of Na⁺ channels and inactivation of voltage sensors, with it being evident that a sufficient proportion of the Na⁺ channels must still remain activatable to support propagation of an action potential and hence give any twitch response at all. During this action potential, as with any action potential, the activated Na⁺ channels will undergo fast inactivation (Rich & Pinter, 2003), but it seems that enough of these channels recover from this fast inactivation to propagate another action potential 8–20 ms later in at least some of the T-system. This is indicated by the fact that with 40 mM K⁺ and 10 mM Na⁺ the d₈ and d₂₀ responses were 50% or more bigger than the response to a single action potential (Fig. 8A).

Apart from the increase in extracellular [K⁺] excitation of muscles may also produce an increase in intracellular [Na⁺] (Clausen, 2003). The results here (Figs 6–8) show that when the number of activatable Na⁺ channels is reduced by low intracellular [K⁺] an increase in intracellular [Na⁺] from 10 to 20 mM reduces the capacity of the T-system to generate a propagating action potential shortly after another (i.e. with 8–20 ms intervals). Fully comparable results have also been very recently obtained in intact muscle when reducing the electrochemical gradient for Na⁺ by lowering the extracellular [Na⁺] (Cairns et al. 2003). Thus, even though the build-up of T-system [K⁺] by itself may cause only a moderate degree of depolarization and inactivation, an accompanying rise in intracellular [Na⁺] can result in the T-system being unable to support trains of action potentials. Therefore, the rise in intracellular [Na⁺] can lead to greatly reduced tetanic force responses, substantially contributing to muscle fatigue. At the same time, however, the rise in intracellular [Na⁺] will potently stimulate the Na⁺–K⁺ pump, which during muscle contractions will protect excitability by limiting the increase in T-system [K⁺].

The conclusions above are quite consistent quantitatively with the expected levels of depolarization and polarization produced by the changes in intracellular [K⁺] and [Na⁺]. The mean data for twitch responses to single action potential stimulation in Fig. 2B shows that the force response at 40 mM K⁺ and 10 mM Na⁺ was very similar to that at 30 mM K⁺ and 20 mM Na⁺. Thus, the depolarizing effect of lowering the intracellular [K⁺] from 40 to 30 mM was evidently fully countered by raising the intracellular [Na⁺] from 10 to 20 mM. This indicates that the rise in intracellular [Na⁺] stimulated the Na⁺–K⁺ pump sufficiently to allow the reduction in T-system [K⁺] to keep the electrochemical gradient for K⁺ approximately unchanged (e.g. reducing T-system [K⁺] from 4 to 3 mM). Consequently, the considerable improvement in the twitch response seen when intracellular [Na⁺] was raised from 10 to 20 mM (Fig. 2B) was apparently produced by a repolarization of the T-system by 7 mV [i.e. 58 log₁₀(4/3) mV]. Such improvement is consistent with observations in intact EDL fibres of the rat showing complete failure of action potentials at –61 mV and generation of action potentials of 50% peak amplitude at –65 mV (Fig. 1 in Rich & Pinter, 2003), the difference being due to the extent of slow Na⁺ channel inactivation. In the skinned fibre experiments here the twofold rise in intracellular [Na⁺] would have also caused a decrease in the electrochemical gradient for Na⁺ by 10 mV [i.e. 58 log₁₀(20/10) – 58 log₁₀(4/3) mV] to 115 mV (assuming 140 mM Na⁺ in the T-system). Though reducing the gradient to this level is not sufficient to prevent propagation of single action potentials with the level of slow Na⁺ channel inactivation prevailing, it evidently is enough to interfere with propagation of closely spaced action potentials (Figs 6 and 7), that is when compounded with the total level of slow and fast inactivation of Na⁺ channels. Significantly, this very closely matches recent findings in intact muscle (Cairns et al. 2003) where action potential failure occurred when extracellular [Na⁺] was decreased to 40 mM and stimuli were applied 8 ms apart (i.e. with 125 Hz stimulation). Assuming the intracellular [Na⁺] was 10 mM in those fibres, the electrochemical gradient for Na⁺ at the failure point was 113 mV, which is similar to that found here in the skinned fibres.

In conclusion, this study provides evidence that the responsiveness of a muscle fibre can be depressed after repeated stimulation both because the ensuing increase in T-tubular [K⁺] causes chronic T-system depolarization, and because the accompanying rise in intracellular [Na⁺] interferes with the ability of the T-system to support successive action potentials at close intervals. These depressive effects, however, are normally resisted in the muscle fibre to some extent because the rise in intracellular [Na⁺] stimulates the Na⁺–K⁺ pump, which reduces both T-system [K⁺] and intracellular [Na⁺] thereby helping maintain excitability and force production during intensive activation of the fibre

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	Acknowledgements

 
The study was supported by grants from the Danish Biomembrane Research Centre, the Australian National Health & Medical Research Council and the Institute of Advanced Science, La Trobe University.

Author's present address
N. Ørtenblad: Institute of Sports Science and Clinical Biomechanics, University of Southern Denmark, DK-5230 Odense, Denmark.

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