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Department of Zoology, La Trobe University, Melbourne, Victoria 3086, Australia

	Abstract

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Mechanically skinned muscle fibres from the extensor digitorum longus (EDL) muscle of the rat were electrically stimulated in solutions mimicking the myoplasmic environment in the resting muscle fibre but containing different [ADP] of < 0.1 µM, 40 µM and 1.0 mM, to investigate the effects of myoplasmic ADP on the twitch response. The amplitude of the twitch response markedly and gradually decreased by 47 ± 6% (n = 9) as [ADP] was increased from < 0.1 µM to 40 µM without changing [Ca²⁺] in the myoplamsic solution (50 nM). The times for the twitch to rise from 10 to 90% (T_rise,10-90) and to decrease from 90 to 10% (T_fall,90-10) initially increased by 8 and 21% and then decreased by 16 and 30% (compared to controls), respectively, at steady state. When [ADP] was raised from < 0.1 µM to 1.0 mM and fibres were electrically stimulated, the first response was biphasic and very prolonged (by at least a factor of 10) but of an amplitude similar to that in the control solution. The following twitch response and the steady state twitch responses were much reduced in size by about a factor of 6 and more prolonged by about 40% compared to control responses. All these ADP effects were fully reversible and appear to be predominantly due to several ADP-dependent alterations in SR Ca²⁺ handling properties (ADP-dependent decrease in SR Ca²⁺ capacity together with an increase in Ca²⁺ binding to the SR pump sites facing the myoplasm). The ADP-dependent effects on the contractile apparatus and Ca²⁺ regulatory system were relatively minor. Taken together, the results demonstrate that ADP accumulation is likely to play a crucial role in metabolic fatigue of skeletal muscle and can explain the marked reduction in the amplitude and the slower time course of the twitch response during fatigue as well as the elevation of myoplasmic [Ca²⁺] in fatigued fibres at rest.

(Received 5 May 2004; accepted after revision 1 July 2004; first published online 2 July 2004)
Corresponding author D. G. Stephenson: Department of Zoology, La Trobe University, Melbourne, Victoria 3086, Australia. Email: g.stephenson{at}zoo.latrobe.edu.au

	Introduction

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Under normal resting conditions, the [ADP] within a fast-twitch skeletal muscle is maintained at approximately 10 µM (Dawson et al. 1978), primarily due to the activity of the creatine kinase reaction (ADP + CP ATP + creatine, equilibrium constant = 260, Chase & Kushmerick, 1995). During intense muscle contraction, as creatine phosphate (CP) is consumed and the creatine concentration rises, the equilibrium of the reaction is displaced towards higher [ADP] and therefore [ADP] increases. In addition, the creatine kinase reaction cannot regenerate ATP as rapidly as ADP is produced and this contributes to a large increase in [ADP] to between 200 µM and 3.0 mM during intense activity (Dawson et al. 1978; Nagesser et al. 1993; Westerblad & Lännergren, 1994). ADP concentration elevated to 200 µM–1.0 mM has been shown to have either a potentiating effect on maximum Ca²⁺-activated force and Ca²⁺ sensitivity of the contractile apparatus in both skeletal and cardiac muscle (Cooke & Pate, 1985; Kawai & Halvorson, 1986; Godt & Nosek, 1989; Karatzaferi et al. 2003), or no effect on the contractile activation characteristics of skeletal muscle (Chase & Kushmerick, 1995). However, increased [ADP] is known to have an overall depressing effect on the SR function. Thus, ADP is a weaker agonist compared to ATP for opening the ryanodine receptors (RyRs)/SR Ca²⁺-release channels (Meissner, 1984) and elevated ADP promotes an SR Ca²⁺ efflux via the SR Ca²⁺ pump (Chiesi & Wen, 1983; Inesi & de Meis, 1989; Soler et al. 1990; Duke & Steele, 2000; Macdonald & Stephenson, 2001), which reduces the ability of the SR to load Ca²⁺ (de Meis, 1988; Macdonald & Stephenson, 2001). ADP may also differently affect other steps in excitation–contraction (E–C) coupling (Stephenson et al. 1995) and therefore one cannot simply predict from the information available the overall effect of rising [ADP] on the twitch response of a skeletal muscle fibre.

In the present study we used the freshly mechanically skinned fibre preparation of rat fast-twitch muscle to investigate the effects of elevated [ADP] on action potential-induced force responses. This preparation not only permits direct access to the myoplasmic environment, thus enabling tight control of the [ADP] within the fibre (Macdonald & Stephenson, 2001), but also allows for the normal action potential-induced mechanism of force production to occur and individual steps involved in the E–C coupling to be studied separately (Posterino et al. 2000; Ørtenblad & Stephenson, 2003). Here we show that [ADP] elevation at physiologically relevant concentrations causes marked and reversible reductions to action potential-induced force responses, suggesting that ADP build-up within muscle fibres is likely to be a major contributor to muscle fatigue.

	Methods

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Dissection, preparation of fibres and apparatus

Male rats (Long-Evans, hooded, 6 months old) were killed by halothane overdose (2% v/v) in accordance with permits issued by La Trobe University Animal Ethics Committee. The extensor digitorum longus (EDL) muscles were quickly removed, well blotted on filter paper (Whatman no. 1), and then placed in a dish containing paraffin oil (Ajax Chemicals, Sydney, Australia), above a layer of Sylgard 184 (Dow Chemicals, Midland, MI, USA). Single muscle fibres were then isolated, mechanically skinned with fine forceps (jeweller's forceps no. 5) under a dissecting microscope and mounted on a force transducer (SensoNor 801, Norway) while under oil, as previously described (Fink et al. 1986; Ørtenblad & Stephenson, 2003). The length and diameter were measured using a dissecting microscope and the preparation was then stretched to 120% of its slack resting length to facilitate measurement of force production in any segment of the preparation (Lamb & Stephenson, 1990). The preparation was finally placed into a 2 ml Perspex bath containing a potassium hexamethylene diamine tetraacetate (K-HDTA) relaxing solution (Twitch₁ solution, Table 1), mimicking the myoplasmic environment. The apparatus used in these experiments and the procedure of changing solutions have been described in detail earlier (Stephenson & Williams, 1981; Ørtenblad & Stephenson, 2003).

The compositions of solutions used in experiments throughout this study are shown in Table 1. Solutions were prepared as described by Stephenson & Williams (1981) with an osmolality of 290 ± 10 mosmol kg^–1, as measured using a vapour pressure osmometer (5500 Wescor, Logan, UT, USA). Relaxing solutions contained either 50 mM EGTA and no added Ca²⁺ ([Ca²⁺] < 10^–9 M; see High EGTA Relaxing solutions in Table 1) or 0.05 mM EGTA ([Ca²⁺] 5.10^–8 M; Twitch solutions in Table 1) ensuring the complete relaxation of skinned fibres. Maximum Ca²⁺-activating solutions contained almost equimolar amounts of Ca²⁺ and EGTA as determined by potentiometric titration (Stephenson & Williams, 1981) and had an ionized [Ca²⁺] of 30 µM, which is sufficient for maximal activation of skinned fibres. The stock ADP solution contained 20 mM ADP, but otherwise was similar in composition to the Twitch solutions (see Table 1). This ensured that upon addition of ADP stock solution, alterations to solutions other than the [ADP] were minimized. All solutions also contained 126 mM K⁺, 36 mM Na⁺, 90 mM Hepes, 8 mM total ATP and an ionized [Mg²⁺] maintained close to 1.0 mM. Note that some solutions also contained 10 mM CP while others did not (see Table 1). Azide (N₃^–) at 1 mM was present in solutions used for direct Ca²⁺ activation of the contractile apparatus, but was removed from solutions used in action potential-induced force experiments, as NaN₃ has been shown to inhibit action potential-induced force responses through mitochondrial depolarization of the T-system membrane potential (Ørtenblad & Stephenson, 2003). All chemicals were obtained from Sigma (St Louis, MO, USA) except HDTA and 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), which were obtained from Fluka (Buchs, Switzerland). TBQ was added dissolved in DMSO. All experiments were conducted at room temperature (22 ± 2°C).

[ADP] in solutions

Freshly mechanically skinned fibres retain a high creatine kinase activity (Saks et al. 1978; Wegman et al. 1992; Ventura-Clapier et al. 1994; Walliman et al. 1977) and therefore, in the presence of CP, any exogenously added ADP would be rapidly converted to ATP within the myoplasmic space. To circumvent this, two approaches were taken to control and maintain the [ADP] in the preparation close to the desired levels. With the first approach, [ADP] was maintained close to its equilibrium according to the creatine kinase reaction (ADP + CP ATP + creatine, equilibrium constant = 260, Chase & Kushmerick, 1995), using the endogenous creatine kinase activity within the fibre, together with known concentrations of creatine, CP and ATP, an approach that has been successfully used previously by us and others (Chase & Kushmerick, 1995; Macdonald & Stephenson, 2001). Thus, in the presence of 8 mM ATP, 10 mM CP and only traces of creatine, [ADP] would be buffered to < 0.1 µM under our standard control conditions (see Macdonald & Stephenson, 2001), while raising the [creatine] to 12 mM in the presence of 8 mM ATP and 10 mM CP would buffer the [ADP] to close to 40 µM. In this solution creatine was added as a solid together with 40 µM ADP from stock solution. Control experiments, using sucrose to mimic the increased osmolality upon the addition of solid creatine, showed no alteration (P > 0.2) in either the action potential-induced force responses, Ca²⁺ sensitivity of the contractile apparatus or maximum Ca²⁺-activated force.

The second approach was used when investigating the effects of [ADP] in the millimolar range because the first approach of buffering [ADP] was not possible under these conditions. With this second approach CP was removed from solutions altogether (replaced with HDTA) and ADP was added from the stock ADP solution. In the absence of CP and creatine, the creatine kinase reaction does not operate, enabling the addition of exogenous ADP without the ADP being converted to ATP. Indeed, without the presence of ATP regenerating systems, the [ADP] within the fibre would increase to levels higher than that exogenously added. The added ADP is therefore an underestimation of the real [ADP] within the fibre, which will vary depending on the ATPase activities in the preparation during an experiment. Using the value for the maximum activated myofibrillar ATPase activity from Stephenson et al. (1989), it was estimated that the ATP pool in maximally activated fibres under conditions similar to those in this study would become depleted by approximately 0.3 mM ATP intramyofibrillarly. The increase in [ADP] would therefore be 0.3 mM, leading to a peak absolute [ADP] of 0.3 mM in 0 mM CP solutions and 1.3 mM in the 1.0 mM ADP solution. For the purpose of clarity, the [ADP] used in the text will be that added to solutions (e.g. 0 mM [CP] and 1.0 mM [ADP]). It must be noted that all solutions contained 8 mM [ATP], which is sufficient for preventing significant ATP depletion within the fibre during periods of maximum activation.

The myokinase enzyme is also expected to be present in mechanically skinned fibres (Ventura-Clapier et al. 1994), but as indicated below, the myokinase reaction should not significantly interfere with the myofibrillar [ADP] in experiments described here. Thus, considering that the equilibrium constant for the myokinase reaction (2ADP ATP + AMP) is 1.25 (Lehninger, 1970), and that the total [ATP] was around 8 mM, one can estimate that as a result of the myokinase reaction, the [AMP] in the myoplasmic environment would be at most 13 fM, 160 nM and 0.1 mM when the concentration of ADP is 0.1 µM, 40 µM and 1.0 mM, respectively. Therefore, [ADP] would not be expected to change by more than a few per cent even if one assumes a high myokinase kinase activity.

Fibre activation

Fibre excitation was achieved by electrical field stimulation (2 ms pulses at 55 V cm^–1) in control solution (Twitch₁ solution; see Table 1) using two platinum wire electrodes running parallel to the skinned fibre length and eliciting action potentials in the sealed T-system (Posterino et al. 2000; Ørtenblad & Stephenson, 2003). The experiments were performed such that an action potential-induced force response was evoked as soon as possible following exposure to the test condition (elevated [ADP]) (within about 5 s) and subsequently every 20 s until steady-state twitch responses were obtained (usually within 2 min), after which the fibre was returned to control solution for recovery. At the end of an experiment, the fibre was maximally activated in Maximum Ca²⁺-activating₁ solution. Force responses were recorded in parallel at 1 kHz using a 400 series PowerLab with Chart v5 software (ADInstruments, Australia), and on a chart recorder (Linear, Reno, NV, USA). Data were expressed relative to the pre-test steady state control level.

Measurement of Ca²⁺-activated force

Ca²⁺-activated force can be used as an indicator of myoplasmic [Ca²⁺] if the direct effect of elevated [ADP] on maximum Ca²⁺-activated force and the force–pCa (– log₁₀[Ca²⁺]) relationship are known. For determining the effects of ADP on these contractile activation parameters we used preparations that were initially exposed to High EGTA relaxing₁ solution containing 2% v/v Triton X-100 for 5 min to destroy all membrane compartments (Stephenson et al. 1981). Preparations were then thoroughly washed in a separate High EGTA relaxing₁ solution (approximately 2 min, 2 separate washes) and the force–pCa relationship and the maximum Ca²⁺-activated force were examined by exposing the fibre to a sequence of solutions in which [Ca²⁺] was heavily buffered at progressively higher levels. These solutions were prepared by mixing specific proportions of High EGTA relaxing₁ solution and Maximum Ca²⁺-activating₁ solution to give strongly Ca²⁺-buffered solutions with [Ca²⁺] in the pCa range of > 9 to 4.5, the exact free [Ca²⁺] was determined by potentiometric titration (Stephenson & Williams, 1981). Exposure of the preparation to these solutions produced successively larger force responses until maximum force was achieved. The fibre was then relaxed by transfer to High EGTA relaxing₁ solution. The effect of ADP was determined by repeating this procedure with solutions containing elevated [ADP] before returning to control solutions. To compensate for the small deterioration in force between the first and last response in Maximum Ca²⁺-activating₁ solution, responses at each pCa were expressed as a percentage of the interpolated values for the maximum Ca²⁺-activated force (van der Poel & Stephenson, 2002). The force produced was expressed as a percentage of the corresponding maximum Ca²⁺-activated force for that condition, plotted as a function of pCa and fitted with a Hill equation (described by eqn (1)), using the analysis program GraphPad Prism (GraphPad Software Inc., CA, USA).

(1)

where Ca₅₀ represents the [Ca²⁺] at which 50% of maximum Ca²⁺-activated force is produced and is used as an indicator of the Ca²⁺ sensitivity of the contractile apparatus, and n_H, which is proportional to the maximum steepness of the force–pCa curve, and refers to the Ca²⁺ dependence of the contractile apparatus.

Data analysis

Results are expressed as means ± S.E.M. and n is the number of fibres investigated. The results were analysed and fitted to curves using the GraphPad Prism program. Student's t test and ANOVA were used to determine statistical significance (P < 0.05) where appropriate.

	Results

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The effects of myoplasmic ADP on E–C coupling were examined in mechanically skinned fibre preparations from the EDL muscle of the rat when the preparations were activated in the presence of three [ADP] (< 0.1 µM; 40 µM and 1 mM) by (1) generation of action potentials in the sealed transverse (T-) system by electrical depolarization and (2) direct activation of the contractile apparatus in strongly Ca²⁺-buffered solutions.

Action potential-induced twitches were achieved by applying brief square electrical pulses (see Methods) across mechanically skinned fibre preparations placed in a solution mimicking the intracellular environment (see Twitch solutions, Table 1) with respect to K⁺, Na⁺, pCa, Mg²⁺, pH, [ATP] and ionic strength (Posterino et al. 2000; Ørtenblad & Stephenson, 2003; Posterino & Lamb, 2003). The T-system seals off uniformly during the skinning procedure under paraffin oil (Lamb et al. 1995; Launikonis & Stephenson, 2001) and becomes normally polarized in solutions mimicking the intracellular environment (Lamb & Stephenson, 1990; Ørtenblad & Stephenson, 2003; Posterino & Lamb, 2003). Action potentials generated in the T-system activate the voltage sensors, which in turn activate Ca²⁺-release channels in the SR causing Ca²⁺ release and activation of the contractile apparatus just like in an intact muscle fibre (Posterino et al. 2000; Ørtenblad & Stephenson, 2003; Posterino & Lamb, 2003).

Direct activation of the contractile apparatus was performed by equilibrating the skinned fibre preparation in highly buffered Ca²⁺ solutions of different pCa values between 9 and 4.5, which were obtained by mixing in different proportions High EGTA relaxing and Maximum Ca²⁺-activating solutions with different [ADP] (see Methods and Table 1). The steady state force responses were then expressed relative to the corresponding value for the maximum force response for each steady-state value, which was calculated according to a simple protocol (see Methods).

In a previous study we used the same mechanically skinned rat EDL fibre preparation to examine the SR Ca²⁺-handling properties at different myoplasmic [ADP], under essentially the same experimental conditions to those used in the present study (Macdonald & Stephenson, 2001). The SR Ca²⁺-handling properties at different [ADP] were assessed from force measurements that could be unambiguously related to the SR Ca²⁺ content following direct activation of SR Ca²⁺ release by caffeine and low [Mg²⁺] (Macdonald & Stephenson, 2001). Results from this previous study are important for the interpretation of observations made in the present study.

Effects of 40 µM ADP on the action potential-induced twitch response

Mechanically skinned muscle fibre segments were incubated in control solutions with < 0.1 µM ADP (Twitch₁ solution in Table 1) and were electrically stimulated until stable steady state (pre-test) twitch responses were obtained. The preparations were then transferred to a solution with 40 µM ADP (see Methods) and again electrically stimulated until steady state responses were obtained, before returning them to the control solution. Representative traces of action potential-induced twitch responses in a fibre segment under control and 40 µM ADP conditions are shown in Fig. 1, with the data from nine fibres summarized in Table 2. The peak amplitude and the area of the first twitch response in the presence of 40 µM ADP were significantly decreased (P < 0.05) by 27 ± 6% and 18 ± 6% of steady state pre-test controls. The time to rise from 10 to 90% of the peak amplitude (T_rise,10-90) was also significantly slower (P < 0.05) by 8 ± 4%, while the time to fall from 90 to 10% of the peak amplitude (T_fall,90-10) was not significantly altered from control. After approximately 2 min, steady state twitch responses in the presence of 40 µM ADP were reached, with the peak amplitude and area of responses significantly further reduced (P < 0.05) compared to steady state pre-test controls. Steady state twitch responses in the presence of 40 µM ADP were significantly faster (P < 0.05) in both rising and falling than steady state pre-test controls. Returning the preparation to control solutions for recovery for 5 s caused a significant partial recovery (P < 0.05) of the peak amplitude and area of the responses, while the time course of the response had fully recovered to steady state pre-test control levels. Complete recovery of the entire response to pre-test levels was achieved after the fibre was returned to control solution for approximately 2–3 min.

View larger version (18K): [in this window] [in a new window]   Figure 1.  Effects of 40 µM ADP on action potential-induced twitch responses A, representative trace of effects of 40 µM [ADP] on action potential-induced force responses with pattern of stimulation shown under the force trace. Numbers represent: 1: steady state pre-test control; 2: first 40 µM ADP response; 3: steady-state 40 µM ADP response; 4: first post-test control response; 5: steady state post-test control response. Max force represents maximum Ca2+-activated force for that individual fibre. B, identical response as numbers indicated in panel A but at a higher time resolution to allow comparison of the following twitch parameters: amplitude, area, Trise,10-90 and Tfall,90-10. Standard control solutions were estimated to have < 0.1 µM [ADP] (see Methods).

When CP was removed from solution and replaced with 10 mM HDTA (see Table 1, Twitch₂ solution), there were significant changes in the action potential-induced twitch responses as shown in Fig. 2 and Table 3. Thus, after only 5 s in 0 mM [CP] solution, the twitch response was not altered in amplitude when compared to steady state pre-test controls, but was significantly slower (P < 0.05), taking 1.5 times longer to rise and 3 times longer to fall, and consequently the area under the first response was up to 3 times as large as that of steady state pre-test controls (P < 0.05). Four of the seven fibres produced an unusually shaped first twitch response in the 0 mM CP solution, with an exacerbated shoulder on the downward phase of the twitch as can be seen in Fig. 2 (Response 2). This shoulder is responsible for the larger area and slower time to relaxation of the first response in the absence of CP. After 1–2 min, steady state responses were achieved, which were significantly reduced (P < 0.05) in peak amplitude and area when compared to both the first response in the absence of CP and the steady-state pre-test control (Table 3). The steady state twitch responses in the absence of CP were also significantly faster (P < 0.05) than the first 0 mM CP response, but they were not different with respect to time to rise and time to fall compared with steady state pre-test twitch responses. Once returned to the control solution, the first post-test control response was significantly faster (P < 0.05) in relaxing and larger in amplitude when compared to steady state responses in the absence of CP, whereas the other parameters remained essentially unchanged from those associated with steady-state responses in the 0 mM CP solution. After 2–3 min, the twitch responses had returned to near steady state pre-test control levels with respect to all parameters analysed. Thus, the removal of CP from solutions initially potentiates, but then causes a severe but reversible inhibition of action potential-induced twitch force responses. Note that during contraction, the [ADP] in the muscle fibre preparation can increase by up to 0.3 mM in the absence of CP (see Methods), and as discussed later, the observed changes in the absence of CP can be fully explained by this rise in [ADP].

View larger version (18K): [in this window] [in a new window]   Figure 2.  Effects of CP removal on action potential-induced twitch responses A, representative trace of effects of 0 mM [CP] on action potential-induced force responses with pattern of stimulation shown under the force trace. Numbers represent: 1: steady state pre-test control; 2: first 0 mM [CP] response; 3: steady-state 0 mM [CP] response; 4: first post test control response; 5: steady state post-test control response. Max force represents maximum Ca2+-activated force for that individual fibre. B, identical response as numbers indicated in panel A but at a higher time resolution to allow comparison of the following twitch parameters; amplitude, area, Trise,10-90 and Tfall,90-10. The time scale for trace 1 in B also applies for traces 3, 4 and 5. The first responses in the absence of CP and the first post test control responses were recorded after about 5 s following the transfer of the preparation to the respective solutions.

The addition of 1.0 mM ADP to the 0 mM CP solution severely affected the action potential-induced force response. The first twitch response in the presence of 1.0 mM ADP solution was altered not only in size but also in shape (Response 2, Fig. 3). This twitch response was composed of two distinct phases, a first phase that was triggered by the action potential itself and a second phase that was both markedly potentiated and prolonged. The peak amplitude of the first phase, upon transfer from control solution ([ADP] < 0.1 µM) to 1.0 mM ADP solution, was significantly smaller (P < 0.05) when compared to both the first response in the 0 mM CP solution and to the steady state pre-test control responses in solution with [ADP] < 0.1 µM. The T_rise,10-90 was not different from that of the first response in 0 mM CP, but force was significantly slower (P < 0.05) than for controls. It was not possible to determine the T_fall,90-10 or the area of the first phase of the first response in 1.0 mM ADP, because the second phase began before the first phase had fully relaxed. By combining both phases, the first 1.0 mM ADP response was significantly slower (P < 0.05) and larger in area compared to the first response in the 0 mM CP and controls. After 1 min exposure to 1.0 mM ADP, steady state level was reached, with the resulting twitches significantly smaller (P < 0.05) with respect to all parameters when compared to the first response in the 1.0 mM ADP solution. Table 4 shows that the steady state responses in the 1.0 mM ADP solution were significantly smaller (P < 0.05) in amplitude and area but were not different in time course compared with both the steady state twitch responses in 0 mM CP solution (Table 3) and the steady state control responses.

View larger version (15K): [in this window] [in a new window]   Figure 3.  Effects of 1.0 mM ADP on action potential-induced twitch responses A, representative trace of effects of 1.0 mM ADP on action potential-induced force responses with pattern of stimulation shown under the force trace. Numbers represent: 1: steady state pre-test control; 2: first 1.0 mM [ADP] response; 3: steady-state 1.0 mM ADP response; 4: first post test control response; 5: steady state post-test control response. Max force represents maximum Ca2+-activated force for that individual fibre. B, identical response as numbers indicated in panel A but at a higher time resolution to allow comparison of the following twitch parameters; amplitude, area, Trise,10-90 and Tfall,90-10. The time calibration bar for trace 1 in B also applies for traces labelled 3–5 in B.

The very slow second phase of the first response in the presence of 1.0 mM ADP is most likely not triggered by electrical stimulation itself but by another mechanism that is able to activate SR Ca²⁺ release. When ADP rises, it can facilitate Ca²⁺ loss from the SR either by the slippage of the SR Ca²⁺ pump or by SR Ca²⁺ pump reversal with ATP synthesis. In a previous paper (Macdonald & Stephenson, 2001) we have argued that under our experimental conditions it was not thermodynamically possible for SR Ca²⁺ pump reversal with ATP synthesis to occur. Further support for the slippage mechanism of the SR Ca²⁺ pump, rather than SR Ca²⁺ pump reversal, accounting for the second phase of the first response in the presence of ADP is shown by the experiment illustrated in Fig. 4. This experiment was performed in the presence of Ruthenium Red (RR) to block the RyRs and at elevated [Ca²⁺] (0.2 µM) to approach the conditions prevalent at the time of initiation of the second phase of the action potential-induced response in the presence of 1.0 mM ADP. This elevated [Ca²⁺] would worsen the conditions for SR Ca²⁺ pump reversal because it would decrease the [Ca²⁺] gradient across the SR membrane, but would improve the conditions for SR Ca²⁺ pump slippage as it would increase the concentration of the SR Ca²⁺ pump intermediate, ADP · E'P · Ca₂ (Inesi & de Meis, 1989), which increases with elevating [ADP] and [Ca²⁺]. In the representative trace shown in Fig. 4 from one of the six fibres investigated, the preparation was initially electrically stimulated prior to exposure to 50 µM RR and soon after exposure to RR the force responses induced by electrical stimulation were completely abolished due to an inability of the stimulus to open the RyR to allow SR Ca²⁺ release. The preparation was then transferred to a RR solution where the [Ca²⁺] was increased from 0.05 µM to 0.2 µM (pCa 7.3 to pCa 6.7) in the presence of 1.0 mM ADP to mimic the conditions prevalent at the time of initiation of the second phase of the action potential-induced. Under these conditions, a relatively slow force transient was evoked in the presence of 50 µM RR and as shown in Fig. 4B, the response was abolished in the presence of the SR Ca²⁺-pump inhibitor TBQ, which binds tightly to the pump protein and prevents it from binding Ca²⁺, ATP (Inesi & Sagara, 1994) and consequently ADP. This experiment clearly shows that in the presence of 1.0 mM ADP and subthreshold [Ca²⁺] (pCa 6.7) for force production (see Fig. 5), there is a significant Ca²⁺ efflux from the SR through the slippage mode of the SR Ca²⁺ pump (and not via the RyR), which elicits the force transient. This rise in the Ca²⁺ efflux via the SR Ca²⁺ pump, combined with a slowing in the SR Ca²⁺ pump rate in the presence of 1.0 mM ADP (Macdonald & Stephenson, 2001), could fully account for the second phase of the large force response in the presence of 1.0 mM ADP, although it is quite possible that it would also facilitate further Ca²⁺ release from the SR via the RyRs by the mechanism of Ca²⁺-induced Ca²⁺ release.

View larger version (9K): [in this window] [in a new window]   Figure 4.  ADP-induced force response in an individual mechanically skinned EDL fast-twitch muscle fibre, in the presence of the RyR blocker, Ruthenium Red, alone or with the SR Ca2+ pump blocker, TBQ A, RR (50 µM) abolishes action potential-induced force responses but cannot eliminate the force transient in the presence of 1.0 mM ADP at pCa 6.7. B, TBQ (20 µM) blocks the force transient in the presence of 50 µM RR, 1.0 mM ADP at pCa 6.7. Panels A and B are from different individual fibres.

View larger version (14K): [in this window] [in a new window]   Figure 5.  Effects of ADP and CP on contractile apparatus of Triton X-100 treated fibres A, effect of ADP and CP on maximum Ca2+-activated force; B, representative force–pCa curves for an individual mechanically skinned EDL fast-twitch muscle fibre when activated in the absence (control, ; 0 mM [CP], •) and in the presence of 40 µM ADP () and 1.0 mM ADP (). Force responses have been normalized to maximum Ca2+-activated force under control conditions. Data points in A represent means ± S.E.M. from 9 individual fibres. (* represents significant difference from control response, P < 0.05, Student's t test.)

Inesi & de Meis (1989) have shown that elevation of [ADP] causes an alteration in the distribution of the SR Ca²⁺ pump intermediate states, altering the number of SR Ca²⁺ pump Ca²⁺ binding sites available for rapidly binding Ca²⁺ on the myoplasmic side. Intuitively, when Ca²⁺ rises rapidly in the myoplasmic space in response to electrical stimulation, there will be a sharp increase in the concentration of the pump intermediate ADP · E'P · Ca₂, the intermediate responsible for the pump slippage (Inesi & de Meis, 1989) in the presence of ADP, causing more Ca²⁺ from the myoplasm to bind to the SR Ca²⁺ pump sites at higher [ADP]. Using the model proposed by Inesi & de Meis (1989), one can show that the concentration of SR Ca²⁺ binding sites rapidly binding Ca²⁺, when myoplasmic [Ca²⁺] rises from 0.1 to 10 µM (as it would be expected during the twitch), increases by tens of µM as [ADP] increases from 0.1 µM to 1.0 mM. This increased ability of the SR Ca²⁺ pump sites to bind Ca²⁺ in the presence of elevated [ADP] may cause a decrease in the twitch size. The reduction in the first twitch response in the presence of 40 µM and 1.0 mM [ADP] (Figs 1 and 3) could therefore be the result of an increase in the number of myoplasmic Ca²⁺ binding sites associated with the SR Ca²⁺ pump. To determine if such a change in the concentration of the SR Ca²⁺ pump Ca²⁺ binding sites could elicit a significant drop in the twitch response, experiments were performed where an extra 10 µM EGTA was added to the standard control solution (Twitch₁ solution, Table 1) in addition to the 50 µM EGTA already present, to mimic the increase in the concentration of Ca²⁺ binding sites in the myoplasm. Since addition of 10 µM extra EGTA to the control solution would decrease the [Ca²⁺] in solution from 0.05 µM to 0.04 µM, the [Ca²⁺] of the solution was adjusted to maintain [Ca²⁺] at 0.05 µM (pCa 7.3). The results from this experiment are summarized in Table 5. The first response in the presence of the extra 10 µM EGTA in the myoplasmic environment was significantly smaller (P < 0.05) in amplitude and area, and faster in time course when compared to steady state pre-test controls responses, with no further alterations in a second subsequent twitch response. Returning to the control solution, without the extra 10 µM EGTA added, the first post-test control response showed a significant recovery (P < 0.05) with respect to all parameters except time to fall, when compared to the second response in the presence of the extra 10 µM EGTA and in less than 1 min all twitch parameters had returned to the pre-test steady state control response levels. Thus, increasing the number of Ca²⁺ binding sites in the myoplasmic environment by only 10 µM markedly reduced action potential-induced twitch responses.

Under our control conditions the tetanic force is close to the maximal Ca²⁺-activated force (Posterino et al. 2000), but the twitch force response is far from saturation (Figs 1–3). Therefore, the twitch response but not the tetanic force response can be used to assess changes in myoplasmic [Ca²⁺] in the presence of ADP, from which effects of ADP on Ca²⁺ movements can be inferred. For this it is necessary to determine whether elevated [ADP] has any direct effects on the Ca²⁺-activation properties of the contractile apparatus under our experimental conditions. Using Triton X-100 treated fibres, elevating [ADP] in solution from < 0.1 µM to 40 µM did not alter the maximum Ca²⁺-activated force (Fig. 5A). Further elevation of [ADP] to 1.0 mM in the absence of CP did not cause a significant change to the maximum Ca²⁺-activated force, but, when compared to the 10 mM CP control solution ([ADP] < 0.1 µM), it was significantly increased (P < 0.05) by 4.9 ± 1.4%. Simply reducing [CP] from 10 mM to 0 mM in the absence of any added ADP caused a small but statistically significant increase (P < 0.05) in the maximum Ca²⁺-activated force by 6.6 ± 1.5%, indicating that the small effect on the maximum force compared to control responses is caused by the removal of 10 mM CP rather than the addition of 1.0 mM ADP.

The effect of elevated [ADP] on the force–pCa relationship of an individual EDL fibre is shown in Fig. 5B. In the nine fibres that were examined, increasing the [ADP] in solutions from < 0.1 to 40 µM did not alter either the pCa₅₀ (5.98 ± 0.02 versus 5.98 ± 0.02) or the n_H (3.85 ± 0.24 versus 4.14 ± 0.26) of the force–pCa curve. There was also no change in the pCa₅₀ and and n_H values when 1.0 mM ADP was added to the 0 mM CP solution itself (6.03 ± 0.01 versus 6.03 ± 0.02 for pCa₅₀ and 4.75 ± 0.41 versus 4.62 ± 0.33 for n_H, respectively) although they were slightly, but statistically significantly, different (P < 0.05) when compared to controls. The removal of CP from solutions without any added ADP significantly (P < 0.05) changed the pCa₅₀ from 5.98 ± 0.02 to 6.03 ± 0.02 and the n_H from 3.85 ± 0.24 to 4.62 ± 0.33, indicating that the small effect on the force–pCa relationship in the presence of 1.0 mM ADP compared to that for control conditions (< 0.1 µM) is caused by the removal of 10 mM CP rather than the addition of 1.0 mM ADP.

We have evidence that the amount of creatine kinase in the skinned fibre preparation decreases after Triton X-100 treatment but this decrease does not alter either the maximum Ca²⁺-activated force or the force–pCa relationship under our experimental conditions. Our laboratory has repeatedly shown this by comparing submaximal and maximal isometric force responses in heavily Ca²⁺-buffered solutions of the same type as those used in this study before and after Triton X-100 treatment. The reduced creatine kinase activity would nevertheless reduce the ability of the fibre to recycle ADP to ATP in the presence of CP. Therefore, one could expect a larger ADP build up in the Triton X-100 treated fibres than in non-Triton treated fibres and the apparent complete lack of effect of ADP on the Ca²⁺-activation characteristics in the Triton X-100 treated fibres further emphasizes the point that under our conditions, [ADP] up to 1.0–1.3 mM does not alter either the maximum Ca²⁺-activated force or the force–pCa relationship. It is important to point out that what was shown here is a lack of ADP effect on the steady state Ca²⁺-activated isometric force. This does not mean that the [ADP] used does not have effects on the kinetic parameters of Ca²⁺-activated force. Earlier studies from this laboratory have shown that not only the steady-state isometric force but also the rate of force rise depends on [Ca²⁺] (see, e.g. Stephenson & Williams, 1981) and it is quite conceivable that under our conditions ADP may affect the kinetics of isometric force development at a given [Ca²⁺] by altering the cross-bridge kinetics without altering the overall force–pCa relationship. The isometric force response belongs to the class of Ca²⁺ indicators that respond relatively slowly to changes in myoplasmic [Ca²⁺]. Therefore, if the changes in myoplasmic [Ca²⁺] were relatively slow compared with the kinetics of force changes, then the time course of the force response would closely resemble the time course of [Ca²⁺] changes. However, if the [Ca²⁺] changes were very fast compared with the changes in force, as is the case with the twitch responses (Hollingworth et al. 1996), then the force response would follow more closely the time integral of the [Ca²⁺] changes (Ashley & Moisescu, 1972). Under our conditions, we have previously estimated that the force response would follow closely [Ca²⁺] changes that occur at rates much slower than 10 s^–1 (Stephenson & Williams, 1981).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Effect of elevated [ADP] on action potential-induced force responses

The present results indicate that the elevation of [ADP] to levels expected to occur during skeletal muscle fatigue alters all aspects of action potential-induced force responses, demonstrating that ADP has significant and reversible effects on E–C coupling in fast-twitch muscle.

Mechanism of reduced action potential-induced force response at elevated [ADP]

Effects of elevated [ADP] on contractile apparatus.  We report that buffering the [ADP] to 40 µM had no effect on steady-state contractile activation characteristics, which is in agreement with Chase & Kushmerick (1995), who, using similar conditions, showed no effect of elevated [ADP] up to 50 µM. In a separate study, Karatzaferi et al. (2003) found that 50 µM ADP increased maximal Ca²⁺-activated force substantially by 21% at 50 µM ATP, but had a much smaller inhibitory effect of only 6% when the [ATP] was increased to 0.5 mM. Our results here show that increasing the [ATP] levels to 8 mM could potentially abolish this effect of elevated [ADP] altogether. Indeed, in vivo, an increase in [ADP] in the micromolar range would be accompanied by only a very small reduction in [ATP].

To facilitate the addition of 1.0 mM ADP, CP was removed from solution, which itself caused a small increase in both maximum Ca²⁺-activated force and the Ca²⁺ sensitivity of the contractile apparatus. Considering the smaller concentration difference used in our experiments (10 mM to 0 mM CP), the data agree with previously published work by Godt & Nosek (1989) and Fryer et al. (1995), who also showed slight potentiation with the removal of CP.

The lack of ADP effects on the maximum Ca²⁺-activated force and Ca²⁺ sensitivity of the contractile apparatus at 1.0 mM ADP and 8 mM ATP is consistent with previous investigations where [ATP] was lower than the 8 mM ATP used throughout this study. Cooke & Pate (1985) added 1.0 mM ADP to skinned rabbit psoas muscles in the presence of 4 mM MgATP and 0 mM CP and found an increase in maximum Ca²⁺-activated force by only 10%. In a similar study, Godt & Nosek (1989) found that addition of 0.7 mM ADP to skinned rabbit psoas muscles increased maximum force by 6% in the presence of 4.7 mM ATP and 0 mM CP, while Karatzaferi et al. (2003) showed that 1.0 mM of the ADP analogue, SL-ADP, in the presence of 3 mM ATP increased force by 10%. Using caged ADP, Lu et al. (1993) showed that 0.5 mM ADP increased maximum force by 5% in the presence of 4 mM ATP; however, this increase was significantly larger (14%) when only 1 mM ATP was present in the activating solution. In a study using similar concentrations of adenine nucleotides, Kawai & Halvorson (1986) demonstrated that increasing the [ADP] from 0 mM to 8 mM in the presence of 6.1 mM ATP, caused no significant increase in force production.

The small increase in maximal Ca²⁺-activated force by ADP is thought to arise from ADP competing with ATP for the myosin binding site, resulting in inhibition of the dissociation of ADP from the acto-myosin–ADP complex to form acto-myosin (Cooke & Pate, 1985; Dantzig et al. 1991). This would lead to crossbridges being attached for a longer period of time, thereby causing an increase in the average force produced per crossbridge. If crossbridges were unable to dissociate as rapidly, the time course of contraction would also become more prolonged, a characteristic observed at high [ADP] in this study. It may be argued that this mechanism may help explain the slower time course of action potential-induced force responses at 0 mM CP and 1.0 mM ADP (Figs 2 and 3, Tables 2 and 4) although the lack of ADP effects on both maximum Ca²⁺-activated force and force–pCa curve under our experimental conditions would make this explanation less likely.

The kinetics of the twitch force changed in a gradual and biphasic way with time upon the rise in [ADP] from < 0.1 µM to 40 µM or 1.0 mM (see Tables 2 and 4), which is not consistent with an expected relatively fast and monotonic change in the twitch characteristics if the effects were mediated simply by changes in the Ca²⁺-activation kinetics of the contractile apparatus.

Thus, the direct effects of elevated [ADP] on the contractile apparatus cannot be responsible for the marked reduction in the size of the action potential-induced force responses, indicating that some other steps in the E–C coupling are affected by ADP.

ADP effects on the SR.  Possible SR-dependent mechanisms for reduced action potential-induced force responses in the presence of ADP include either the reduction in SR Ca²⁺ release or the increase in the rate of Ca²⁺ removal from the myoplasmic space, or a combination of both. In turn, a reduction in the SR Ca²⁺ release could be due either to ADP-induced inhibitory effects on the RyR/SR Ca²⁺ release channel or to a decrease in the Ca²⁺ electrochemical gradient across the SR membrane. Conversely, the increase in the rate of Ca²⁺ removal from the myoplasmic space could be due to increased SR Ca²⁺-pump activity or to increased number and/or affinity of myoplasmic Ca²⁺-binding sites.

Meissner (1984) showed that ADP acts as an agonist for the RyR but is less powerful than ATP. It may therefore, compete with ATP for the binding site on the RyR, impairing opening of the RyR, thus reducing Ca²⁺ release from the SR. Assuming that both ATP and ADP compete for the same binding site, one can calculate from the apparent affinities of ATP and ADP to the RyR (K_d of the ATP analogue AMP-PCP = 0.11 mM and K_d ADP = 0.17 mM, Meissner, 1984), that at 40 µM ADP, in the presence of 8 mM ATP, only 0.3% of the RyRs would have ADP instead of ATP bound. This clearly shows that the 27% reduction in first action potential-induced force response in the presence of elevated [ADP] at 40 µM (Fig. 1) cannot be primarily due to elevated [ADP] inhibiting the SR Ca²⁺ release. Therefore the reduction in action potential-induced force response observed with elevated [ADP] is unlikely to be due to any direct effects of ADP altering the properties of the RyR and thus reducing SR Ca²⁺ release.

In fibres that had an SR Ca²⁺ content significantly lower than endogenous SR Ca²⁺, Posterino & Lamb (2003) found a marked reduction in action potential-induced force responses, while Pape et al. (1998) showed similar results in amphibian muscle fibres. A reduction in the SR Ca²⁺ content in a fibre means that when stimulated, less Ca²⁺ can be released from the SR, resulting in a reduction in force. Importantly, we have recently shown using the same preparation and ADP-containing solutions (Macdonald & Stephenson, 2001) that the SR Ca²⁺ content is considerably reduced in the presence of 40 µM and 1.0 mM ADP at resting physiological [Ca²⁺] levels. Therefore the gradual reduction of action potential-induced force responses during exposure to elevated [ADP] (Figs 1–3) can be explained to a large extent by a gradual reduction of ADP-dependent SR Ca²⁺ content. The gradual recovery of action potential-induced force response upon return to control solutions further supports this explanation since once ADP has been removed the SR can re-load back to endogenous Ca²⁺ capacity.

The reduced SR Ca²⁺ capacity at elevated [ADP] (Macdonald & Stephenson, 2001) was the result of a small decrease in the SR Ca²⁺ pump rate and a marked increase in the SR Ca²⁺ leak rate. A reduction in SR Ca²⁺ pump rate would contribute to the larger area of the action potential-induced force responses in the presence of elevated [ADP]. A slower SR Ca²⁺ pump rate would impair the sequestration of Ca²⁺ back into the SR, prolonging the myoplasmic Ca²⁺ transient, and leading to a prolonged force response. Using TBQ to inhibit the SR Ca²⁺ pump (Posterino & Lamb, 2003) showed significantly larger action potential-induced responses in both amplitude and area, as well as a markedly longer response, which required many seconds to fully relax. Thus, the SR Ca²⁺ pump is of major importance in determining the time course of the twitch response. Duke & Steele (1999) observed a similar prolongation of the force response upon CP removal and suggested that inhibition of ATP regeneration was the underlying cause. In the absence of CP there would be a concomitant increase in [ADP], due to the lack of ATP regenerating systems, which in light of the results presented here could explain the prolonged force response observed by Duke & Steele (1999).

Interestingly, returning the preparation to control solutions for recovery increased the time course of the entire twitch response, before restoration back to steady state control levels. Under normal conditions, SR Ca²⁺ pump rate would be influenced by feedback inhibition, where increasing SR Ca²⁺ content slows the SR Ca²⁺ pump rate, preventing further accumulation of SR Ca²⁺ (Inesi & de Meis, 1989). Considering that at 1.0 mM ADP there is less Ca²⁺ in the SR than at < 0.1 µM ADP (Macdonald & Stephenson, 2001), the level of feedback inhibition will be reduced immediately upon transfer back into control solutions compared with the situation at steady-state under control conditions, resulting in a transient increase in the SR Ca²⁺ pump rate. The transiently elevated SR Ca²⁺ pump rate under these conditions would lead to a transiently faster removal of Ca²⁺ from the myoplasm, and therefore, a transiently faster fall in twitch response as can be seen in Tables 2 and 4.

Along with a reduced SR Ca²⁺ pump rate, elevated [ADP] also causes a marked increase in the SR Ca²⁺ leak rate by a factor of 3 and 4 times, respectively, at 40 µM and 1.0 mM [ADP] (Macdonald & Stephenson, 2001). The increased SR Ca²⁺ leak rate would hamper the ability of the SR Ca²⁺ pump to re-sequester Ca²⁺ into the SR, resulting in an increase in the myoplasmic Ca²⁺ transient and contributing to a prolongation of the force response, as observed with action potential-induced force responses (Figs 1–3).

The mechanism for the ADP-induced leak through the SR Ca²⁺ pump is via SR Ca²⁺ pump slippage, as first proposed by Inesi & de Meis (1989), which we showed also exists in mechanically skinned fibres (Macdonald & Stephenson, 2001), rather than by SR Ca²⁺ pump reversal (see above and Macdonald & Stephenson, 2001 for a detailed explanation). In short, the Ca²⁺ binding sites of the ADP-sensitive state of the SR Ca²⁺ pump are able to face either the luminal or myoplasmic side of the SR membrane, and can therefore transport Ca²⁺ in either direction by slipping or flicking from one side of the membrane to the other. In the presence of elevated [ADP], the proportion of SR Ca²⁺ pumps in the ADP-sensitive state will be higher, thus increasing the amount of Ca²⁺ that can leak from the SR via this slippage mechanism. Using the model of the SR Ca²⁺ pump cycle proposed by Inesi & de Meis (1989), one can see that the SR Ca²⁺ pump intermediate state (ADP · E'P · Ca₂) that is directly responsible for the loss of SR Ca²⁺ from the SR to the myoplasm would increase significantly with elevated [ADP] and [Ca²⁺], facilitating the leak of Ca²⁺ via this mechanism. The markedly slower rate of relaxation of the first twitch response when the [ADP] was raised in solutions (this includes the second phase of the response in the 1.0 mM ADP solution) is most likely caused by the markedly increased SR Ca²⁺ leak rate when myoplasmic [Ca²⁺] rises in response to electrical stimulation as shown by evidence provided in Results (Fig. 4). A partly impaired SR Ca²⁺ pump in the presence of ADP (see above) would also maintain for longer an elevated myoplasmic Ca²⁺ and this may facilitate more Ca²⁺ release from the SR via the RyRs by the Ca²⁺-induced Ca²⁺ release mechanism.

Thus, the reduction in SR Ca²⁺ pump combined with the increased SR Ca²⁺ leak in the presence of elevated [ADP], which cause a marked drop in SR Ca²⁺ content at a given myoplasmic pCa, can explain the gradual changes in the twitch parameters upon exposure to ADP and then upon removal of ADP. However, the reduction in the first action potential-induced force response in the presence of elevated [ADP] and the marked recovery of the first twitch response upon removal of ADP (Tables 2 and 4) cannot be explained by marked changes in the SR Ca²⁺ content, as after only a few seconds equilibration in elevated [ADP] or control Twitch₁ solution, the action potential-induced force response is already significantly changed in size. We know from control experiments that it takes at least one order of magnitude longer to markedly change the SR Ca²⁺ content under our conditions.

The most likely explanation for the marked changes in the first twitch response after the addition of ADP or after the removal of ADP is that elevated [ADP] alters the distribution of SR Ca²⁺ pump intermediate states (Inesi & de Meis, 1989), which increases the myoplasmic Ca²⁺ buffering capacity by several tens of µM over the [Ca²⁺] range during a twitch, thus reducing the effectiveness of the SR Ca²⁺ released by binding a larger proportion of the released Ca²⁺. Indeed, we show that a relatively modest 10 µM increase in myoplasmic Ca²⁺ buffering causes a significant reduction in twitch response (Table 5).

Relevance of this study to skeletal muscle fatigue

Despite marked increases in [ADP] during fatigue, ADP has not been generally considered to be a major factor in skeletal muscle fatigue. Here we provided evidence that ADP is likely to play a significant role in metabolic fatigue of skeletal muscle by affecting skeletal muscle E–C coupling in a complex way. In particular, elevated [ADP] within the physiological range markedly depresses action potential-induced force responses, predominantly by affecting Ca²⁺ handling properties of the SR.

Thus, a number of characteristics that occur during metabolic fatigue can be explained by the effects of elevated [ADP], including (i) a reduction in twitch force, (ii) a slowing in the relaxation of the twitch response, and (iii) a rise in the myoplasmic [Ca²⁺]. The ADP-dependent reduction in twitch force is brought about by a gradual, marked, ADP-dependent decrease in SR Ca²⁺ capacity together with an increase in the number and ability of the SR Ca²⁺ pump sites facing the myoplasm to bind Ca²⁺ when it is released from the SR. The slower time course of the twitch is caused at least in part by the ADP-induced reduction in the SR ability to load Ca²⁺ and by the ADP- and Ca²⁺-dependent increased leakage through the pump. Finally, the rise in myoplasmic Ca²⁺ directly follows from the ADP-dependent reduced ability of the SR to sequester Ca²⁺ at given myoplasmic [Ca²⁺]. Under these circumstances the myoplasmic [Ca²⁺] will need to rise to activate the SR Ca²⁺ pump to higher levels in order to compensate for the ADP-dependent increased leakage through the pump and overall ADP-dependent decrease of Ca²⁺-pump activity. The increased myoplasmic [Ca²⁺] may also lead to activation of Ca²⁺-dependent proteases and impaired coupling between the voltage sensors and RyRs (Lamb et al. 1995) and increased Ca²⁺ leakage from the SR (Lamb et al. 2001).

Notwithstanding the effects of ADP on various steps in E–C coupling unveiled in this study, which can explain several important features of metabolic fatigue, one should emphasize that fatigue is a multifactorial process (Stephenson et al. 1998; Allen & Westerblad, 2001).

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