AJP - Endo Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 292: E771-E778, 2007. First published November 7, 2006; doi:10.1152/ajpendo.00378.2006
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/3/E771    most recent
00378.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Macdonald, W. A.
Right arrow Articles by Nielsen, O. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Macdonald, W. A.
Right arrow Articles by Nielsen, O. B.

Energy conservation attenuates the loss of skeletal muscle excitability during intense contractions

W. A. Macdonald,1 N. Ørtenblad,2 and O. B. Nielsen1

1Institute of Physiology and Biophysics, University of Aarhus, Aarhus; and 2Institute of Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark

Submitted 28 July 2006 ; accepted in final form 6 November 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
High-frequency stimulation of skeletal muscle has long been associated with ionic perturbations, resulting in the loss of membrane excitability, which may prevent action potential propagation and result in skeletal muscle fatigue. Associated with intense skeletal muscle contractions are large changes in muscle metabolites. However, the role of metabolites in the loss of muscle excitability is not clear. The metabolic state of isolated rat extensor digitorum longus muscles at 30°C was manipulated by decreasing energy expenditure and thereby allowed investigation of the effects of energy conservation on skeletal muscle excitability. Muscle ATP utilization was reduced using a combination of the cross-bridge cycling blocker N-benzyl-p-toluene sulfonamide (BTS) and the SR Ca2+ release channel blocker Na-dantrolene, which reduce activity of the myosin ATPase and SR Ca2+-ATPase. Compared with control muscles, the resting metabolites ATP, phosphocreatine, creatine, and lactate, as well as the resting muscle excitability as measured by M-waves, were unaffected by treatment with BTS plus dantrolene. Following 20 or 30 s of continuous 60-Hz stimulation, BTS-plus-dantrolene-treated muscles showed a 25% lower ATP utilization compared with control muscles. Furthermore, the ability of muscles to maintain excitability during high-frequency stimulation was significantly improved in BTS-plus-dantrolene-treated muscles, indicating a strong link between metabolites, energetic state, and the excitability of the muscle.

metabolites; endurance; adenosine triphosphate

IN SKELETAL MUSCLE, the signal for muscle contraction involves the propagation of action potentials along the sarcolemma and down the transverse tubules (T tubules), where it causes activation of the voltage sensor molecules, subsequent SR Ca2+ release, and ultimately cross-bridge cycling (28), a process known as excitation-contraction coupling. Skeletal muscle fatigue, where the force generating ability of the muscle is compromised, is a complex phenomenon that may occur at any of the above-mentioned steps depending on the type of work and the conditions under which it takes place.

The loss of force in isolated muscles stimulated to contract tetanically is strongly correlated to a reduction in muscle excitability (13, 31), indicating that loss of excitability may be of particular importance in fatigue during intense contractions involving high frequencies of action potentials. The etiology of this reduction in muscle excitability is not fully clear, but it has long been postulated to be related to a loss of intracellular K+ from the muscle fibers, leading to a lowered concentration gradient for K+. Thus, in humans, maximal exercise results in a rapid increase in arterial plasma K+ from 4 to 8 mM (27), which is elevated to 10–12 mM in the muscle interstitium (29). The result is depolarization of the muscle fibers, leading to slow inactivation of the voltage-gated Na+ channels, which prevents the initiation of action potential propagation (6, 12, 37), resulting in muscle fatigue.

Another important consequence of intense contractions is the large change in the metabolic status of the working muscle, with decreased intracellular ATP and phosphocreatine (PCr) and increased creatine (Cr), H+, lactate, ADP, and AMP. There is now a vast amount of evidence implicating the role of metabolites in skeletal muscle fatigue (for reviews, see Refs. 18 and 40), with much of this focusing on the effects of metabolites on the contractile apparatus and on SR Ca2+ cycling. In contrast, very little is known about the role that metabolites play in the loss of muscle excitability. However, since experiments with metabolic poisoning show that the function of many ion channels is sensitive to severe metabolic exhaustion (9, 16, 17), it is possible that metabolically-induced changes in excitability contribute to the development of fatigue during intense contractions.

The aim of this study was, therefore, to examine whether the loss of excitability during intense contractions is influenced by the metabolic status of muscles. Specifically, the effect of energy conservation on the maintenance of muscle excitability was investigated. Here, we reduced muscle ATP utilization using a combination of the cross-bridge cycling blocker N-benzyl-p-toluene sulphonamide (BTS) and the SR Ca2+ release channel blocker Na dantrolene, which reduces SR Ca2+ release and, therefore, activity of the myosin ATPase and SR Ca2+-ATPase. In such muscles, resting excitability was unaffected; however, the maintenance of excitability during high-frequency stimulation was significantly improved.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals, preparation, and incubation of muscles. All handling and use of animals complied with Danish animal welfare regulations, and the euthanasia was approved by the University Animal Welfare Office. Experiments were performed using 4-wk-old Wistar rats, weighing 70–75 g, that were kept in a thermostated environment at 21°C with a 12:12-h light-dark cycle and fed ad libitum. The animals were killed by cervical dislocation, followed by decapitation. Intact extensor digitorum longus muscles (weight 20–25 mg, radius ~0.70 mm) were prepared and incubated in standard Krebs-Ringer bicarbonate buffer (KRB) containing the following (in mM): 122.1 NaCl, 25.1 NaHCO3, 2.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3 CaCl2, and 5.0 D-glucose (pH 7.4). All incubations took place at 30°C under continuous gassing with a mixture of 95% O2 and 5% CO2.

Measurement of force and compound action potentials (M-waves). Muscles were mounted for isometric contractions in thermostated chambers containing standard KRB and adjusted to optimal length for force production. After a 30-min equilibration period, muscles were exposed to field stimulation across the central region through platinum electrodes using 0.5-s trains of 0.02-ms, 12-V pulses at 60 Hz every 20 min. Force was measured using force displacement transducers and recorded with a chart recorder and/or digitally on a computer. Fatiguing stimulation involved stimulating the muscles continuously for 20 or 30 s at 60 Hz, using 0.02-ms trains of 12-V pulses. The mean absolute force produced under control conditions was 0.30 ± 0.03 N (n = 70), with results expressed as a percentage of the control force produced directly prior to treatment.

Immediately following fatiguing stimulation, the chamber was lowered and the muscle detached and frozen in liquid N2 for assessment of metabolites. The resulting metabolite values measured in the muscle represent those at the end of contraction. Control measurements, in which a thermocouple (copper-constantan, od = 0.4 mm; California FineWire, Grover City, CA) was placed in the central part of the muscles, showed that the time that elapsed from the cessation of the stimulation until the core of the muscles was cooled to 0°C was 4.0 ± 0.8 s (n = 8). A sample of the buffer was also frozen for determination of lactate released into the solution during the fatiguing stimulation.

Muscle excitability was determined by recording compound action potential signals (M-waves) from a polyimide-insulated tungsten electrode (TM33B01; World Precision Instruments, Sarasota, FL) placed within the muscle between the innervation zone and the tendon. Signals were sent to a DAM 70 differential amplifier mounted with a low-noise headstage (World Precision Instruments) and then recorded digitally on a computer. The M-wave area was defined as the area between the baseline and the major negative peak of the M-wave trace, whereas the M-wave amplitude was defined as the maximal voltage of the negative peak, as previously described by Overgaard et al. (31). M-wave area and amplitude were analyzed every 2 s during fatiguing stimulation. At each time point, the area and amplitude of five successive M-waves was averaged, with the mean coefficient of variation from successive M-waves being 4.2 ± 0.4 (n = 60) and 3.7 ± 0.5% (n = 60) for M-wave area and M-wave amplitude, respectively. Control experiments were performed and showed that insertion of the M-wave electrode into the muscle did not significantly alter force production (98 ± 4% of controls, P = 0.231, n = 8).

Manipulation of muscle ATP consumption. To alter the ATP consuming processes occurring during muscle contraction, two pharmacological agents were employed. The first, BTS (50 µM), significantly reduces tetanic force production by specifically blocking cross-bridge cycling of myosin type II contractile proteins (10) without altering either Ca2+ transients (15, 7) or muscle excitability (25). The second, Na dantrolene (dantrolene, 25 µM), significantly reduces tetanic force (34, 43) by inhibiting the SR Ca2+ release channels without markedly altering muscle excitability (41), which in turn reduces both the myosin ATPase and the SR Ca2+-ATPase activities.

BTS and dantrolene were used individually or in combination and were added from stocks dissolved in DMSO, with the corresponding amount of DMSO added to controls.

Measurement of muscle metabolites. Muscle metabolites were measured in muscles that were frozen in liquid N2 following the cessation of fatiguing stimulation, whereas resting metabolites were measured from contralateral muscles treated the same, except that they did not undergo fatiguing stimulation. Frozen muscle samples were later freeze-dried, dissected free of nonmuscle tissue, and powdered. Muscle extracts were analyzed for ATP, ADP, AMP, PCr, Cr, lactate, and pyruvate concentrations as previously described (21). To adjust for variability in solid nonmuscle constituents and weighing variability, values were divided by the total amount of Cr (PCr + Cr) and multiplied by the mean of total Cr for the whole material (22). BTS plus dantrolene did not significantly affect total Cr, with total Cr averaging 128 ± 9 and 133 ± 8 mmol/kg dry wt (n = 18, P = 0.159) for control and BTS-plus-dantrolene-treated muscles, respectively. Intracellular pH (pHi) was determined using the equation pHi = 7.06 – 0.00532 ([lactate] + [pyruvate]) (22). ATP utilization was calculated using the following equation: ATP utilization = 2 ({Delta}ATP) + {Delta}PCr + (1.5 {Delta}lactate), where {Delta} refers to the difference between the stimulation value and the resting value from the contralateral muscle under control and BTS-plus-dantrolene conditions, respectively.

Chemicals. All chemicals were of analytical grade. Na dantrolene was obtained from Sigma-Aldrich and BTS from Toronto Research Chemicals (Toronto, ON, Canada).

Statistics. All data were expressed as means ± SD and statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA). The statistical significance of any difference between groups was accepted at P < 0.05, as determined using Student's two-tailed t-test for nonpaired observations or ANOVA, with differences located with Student-Newman-Keuls post hoc test, where appropriate.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Tetanic force and muscle excitability. Because muscle excitability was measured extensively during this study, it was important to first show that BTS and dantrolene did not significantly alter resting muscle excitability while reducing tetanic force and thus also reducing muscle ATP utilization. Figure 1A shows that, over a 120-min period under control conditions, there was no significant alteration in tetanic force (P = 0.075), M-wave area (P = 0.628), or M-wave amplitude (P = 0.233). Figure 1, B–D, shows the effects of 50 µM BTS, 25 µM dantrolene, and a combination of both 50 µM BTS plus 25 µM dantrolene, respectively, on tetanic force and on M-wave parameters. The combination of BTS plus dantrolene induced the fastest and largest reduction in tetanic force to <2% of initial tetanic force at 120 min (P < 0.001; Fig. 1D). Importantly, neither BTS (P = 0.963 and 0.123) nor dantrolene (P = 0.969 and 0.409), nor the combination of BTS plus dantrolene (P = 0.993 and 0.230), significantly reduced M-wave area or M-wave amplitude, indicating that there was no marked effect on muscle excitability.


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. The effect of N-benzyl-p-sulfonamide (BTS) and dantrolene on tetanic force and muscle excitability (M-waves) under control conditions. Simultaneous recordings of tetanic force (bullet), M-wave area ({circ}), and M-wave amplitude ({blacktriangledown}) measured every 20 min, during 60-Hz, 0.5-s tetanic contractions in control (A), 50 µM BTS- (B), 25 µM dantrolene- (C), and 50 µM BTS + 25 µM dantrolene-treated muscles (D). BTS and dantrolene were added at time 40 min. Data points are means ± SD and are normalized to pretreatment responses (n = 4). *Significant difference (P < 0.001) from pretreatment (40 min) responses.

 
Force production during fatiguing stimulation. Representative traces of the effect of BTS, dantrolene, and the combination of BTS plus dantrolene on force production during 30-s, 60-Hz continuous stimulation are shown in Fig. 2A. The combination of BTS plus dantrolene was by far the most effective at reducing force production and, therefore, probably ATP utilization. Control muscles and those treated with a combination of BTS plus dantrolene were thus compared in all subsequent experiments. Summarized data showing force production during 30-s, 60-Hz continuous stimulation of control and BTS-plus-dantrolene-treated muscles are shown in Fig. 2B. In control muscles, force was maintained for the first 10 s of stimulation (all P > 0.05) before steadily declining to 20% of initial force at the end of stimulation (P < 0.001). The combination of BTS plus dantrolene prevented >95% of the force production during 30-s, 60-Hz continuous stimulation, with the force being significantly lower than in control muscles throughout the stimulation period (all P < 0.001).


Figure 2
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 2. The effect of BTS and dantrolene on force production during 30-s, 60-Hz continuous stimulation. A: representative traces of control muscles and those treated with 50 µM BTS, 25 µM dantrolene (dan), or 50 µM BTS + 25 µM dantrolene (BTS + dan) as indicated in the figure. Similar results were observed in 5 other muscles with corresponding treatments. B: summarized data from 6 muscles showing force production in control (bullet) and 50 µM BTS + 25 µM dantrolene-treated muscles ({circ}). For the sake of clarity, data points are means ± SD and are normalized to pretreatment responses (n = 6). *P < 0.001; #P < 0.01 from controls. BTS and dantrolene were added 60 min prior to stimulation.

 
Muscle metabolism. Figures 3, 4, and 5 show that the resting values for ATP, PCr, Cr, and lactate are unchanged by 60-min incubation with BTS plus dantrolene (P = 0.947, 0.349, 0.349, and 0.249, respectively). In control muscles, stimulation for 20 or 30 s resulted in a significant reduction in ATP, by 50 (P < 0.001) and 55% (P < 0.001), respectively (Fig. 3). By comparison, in the presence of BTS plus dantrolene, ATP was reduced by only 7 (P = 0.004) and 15% (P < 0.001) at 20 and 30 s, respectively. After 20 and 30 s of continuous 60-Hz stimulation, the PCr levels in control muscles were significantly reduced by 65 (P < 0.001) and 67% (P < 0.001), respectively, and were not altered by the presence of BTS plus dantrolene (20 s, 61%, P < 0.001; 30 s, 69%, P < 0.001; Fig. 4A). In line with the large reductions in PCr during stimulation, there was a large, significant increase in Cr at both 20 (P < 0.001) and 30 s (P < 0.001) for controls and BTS-plus-dantrolene-treated muscles (P < 0.001 and P < 0.001; Fig. 4B). The effect of continuous 60-Hz stimulation on lactate production is shown in Fig. 5. Following 20- and 30-s stimulation, lactate significantly increased from ~1 to ~60 (P < 0.001) and ~80 mmol/kg dry wt (P < 0.001) in control muscles, whereas in BTS-plus-dantrolene-treated muscles, lactate significantly increased to ~40 (P < 0.001) and 55 mmol/kg dry wt (P < 0.001), respectively. Table 1 shows that there was a small but significant (all P < 0.02) release of lactate from the muscle into the buffer during the continuous stimulation. These values are included in the lactate values in Fig. 5. Resting pyruvate levels were the same in control and BTS-plus-dantrolene-treated muscles (P = 0.101), with large, significant stimulation-induced increases in pyruvate in both control (P < 0.001) and BTS-plus-dantrolene-treated muscles (P < 0.001). Using the values for lactate and pyruvate, pHi was estimated following stimulation, and, as indicated in Table 1, pHi fell to a lower level in control muscles than in BTS-plus-dantrolene-treated muscles. Table 1 also shows that there were significant stimulation-induced increases in ADP in both control (P < 0.001) and BTS-plus-dantrolene-treated muscles (P < 0.001). There were no significant alterations to the AMP levels during 20- or 30-s continuous stimulation (all P > 0.05) in control or BTS-plus-dantrolene-treated muscles.


Figure 3
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 3. ATP at rest and following 20 and 30 s of 60-Hz continuous stimulation. Filled bars, control muscles; open bars, muscles treated with 50 µM BTS + 25 µM dantrolene for 60 min prior to stimulation. To adjust for variability in solid nonmuscle constituents and weighing variability, values were divided by the total amount of creatine [phosphocreatine (PCr) + creatine (Cr)] and multiplied by the mean of total creatine for the whole material. Values are means ± SD for 6 muscles. *P < 0.001 from rest; $P = 0.004 from rest; #P = 0.002 from 20 s; §P < 0.001 from control 20 s; £P < 0.001 from control 30 s.

 

Figure 4
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 4. PCr (A) and Cr (B) at rest and following 20 and 30 s of 60-Hz continuous stimulation. Filled bars, control muscles; open bars, muscles treated with 50 µM BTS + 25µM dantrolene for 60 min prior to stimulation. To adjust for variability in solid nonmuscle constituents and weighing variability, values were divided by the total amount of Cr (PCr + Cr) and multiplied by the mean of total Cr for the whole material. Values are means ± SD for 6 muscles. *P < 0.001 from rest.

 

Figure 5
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 5. Lactate at rest and following 20 and 30 s of 60-Hz continuous stimulation. Filled bars, control muscles; open bars, muscles treated with 50 µM BTS + 25 µM dantrolene for 60 min prior to stimulation. To adjust for variability in solid nonmuscle constituents and weighing variability, values were divided by the total amount of Cr (PCr + Cr) and multiplied by the mean of total Cr for the whole material. Values are means ± SD for 6 muscles. *P < 0.001 from rest; #P < 0.001 from 20 s; §P < 0.001 from control 20 s; £P < 0.001 from control 30 s.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Muscle metabolites following 60-Hz continuous stimulation

 
Using these values, it was possible to estimate the ATP utilization during 30-s, 60-Hz stimulation in control and BTS-plus-dantrolene-treated muscles. Control muscles that underwent continuous 60-Hz stimulation for 30 s had an ATP utilization of 221 ± 12 mmol/kg dry wt, which was significantly higher (P < 0.001) than that of BTS-plus-dantrolene-treated muscles (165 ± 22 mmol/kg dry wt), demonstrating a 25% conservation of energy in BTS-plus-dantrolene-treated muscles.

Muscle excitability during fatiguing stimulation. The overall excitability of the muscles during the 30 s of 60-Hz continuous stimulation was assessed by measurements of M-wave area and amplitude. As shown in Fig. 6, the area of the M-wave in control muscles increased progressively during the first 16 s of stimulation before rapidly declining to 15% of initial level at 30 s of stimulation. A similar, two-phased development was observed in the M-wave amplitude, which showed a slow decline during the first 10 s of stimulation before steadily decreasing at a faster rate to 10% of initial level at 30 s of stimulation (Fig. 6B). In BTS-plus-dantrolene-treated muscles, M-wave area increased for the first 20 s of stimulation (all P > 0.05) before declining to <50% of initial value at 30 s (P < 0.001). Due to the later onset of the decline, the M-wave area was significantly larger in BTS-plus-dantrolene-treated muscles than in controls from 20 s of stimulation and onward (all P < 0.05). Similarly, the onset of the fast decline in M-wave amplitude was delayed in muscles treated with BTS plus dantrolene, leading to a significantly larger M-wave amplitude in BTS-plus-dantrolene-treated than control muscles from 18 s and onward during the 30-s, 60-Hz stimulation (all P < 0.05).


Figure 6
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 6. Muscle excitability (M-waves) during 30-s, 60-Hz continuous stimulation. M-wave area (A) and M-wave amplitude (B) in control (bullet) and 50 µM BTS + 25 µM dantrolene-treated muscles ({circ}). BTS plus dantrolene were added 60 min prior to stimulation. For the sake of clarity, data points are means ± SD and are normalized to pretreatment responses (n = 6). *P < 0.001; #P < 0.01; §P < 0.05 from controls.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major observation of this study was that, in fast-twitch muscles, preincubation with BTS plus dantrolene significantly reduced ATP utilization (conserved energy status) during continuous high-frequency stimulation and at the same time improved the maintenance of excitability, indicating that the loss of muscle excitability during intense exercise is hastened if the muscles experience metabolic exhaustion.

BTS plus dantrolene and force production. Individually, both BTS (10, 15, 25) and dantrolene (34, 43) have previously been shown to reduce tetanic force in fast-twitch skeletal muscle; however, this is the first study to combine these two compounds. In the present study, BTS and dantrolene alone were effective at reducing tetanic force during short contractions; however, the increase in force observed in Fig. 2A after 5 s of continuous stimulation demonstrates that the inhibition of the myosin ATPase by BTS was partially overcome during longer contractions. Even more striking was that, during longer contractions, the effect of dantrolene-induced inhibition of SR Ca2+ release appears to be removed, resulting in a substantial increase in force production (Fig. 2A). The exact mechanisms for these losses of inhibition are not clear, although, in the case of dantrolene, it is possibly via a Ca2+-induced activation of the SR Ca2+ release channels.

Muscle metabolites. There was no difference in resting metabolites of control or BTS-plus-dantrolene-treated muscles. Importantly, our resting metabolite results are similar to those of adult rat extensor digitorum longus muscles, determined by freeze-clamping with aluminium tongs cooled in liquid N2 (36). However, following continuous stimulation for 20 or 30 s at 60 Hz, muscles pretreated with BTS plus dantrolene had a smaller reduction in ATP and increase in lactate and, therefore, a lower ATP utilization than controls. Rather surprisingly, in control muscles the ATP and PCr levels observed after 30-s stimulation were not lower than after 20-s stimulation. However, the lactate was considerably elevated. In the BTS-plus-dantrolene muscles, 30-s stimulation reduced ATP to a lower level than after only 20 s, although there was also a large increase in lactate from 20 to 30 s. Despite the clear conservation of ATP in BTS-plus-dantrolene-treated muscles, the depletion in PCr, as well as the concomitant increase in Cr, was the same in both control and BTS-plus-dantrolene-treated muscles. It is important to consider that some metabolism may still be occurring in the muscles during the time from the cessation of stimulation to the muscle being frozen. Despite this, it is clear that the addition of BTS plus dantrolene substantially conserves the energy status of the muscle.

Muscle excitability. Importantly, neither BTS nor dantrolene alone, nor the combination of BTS plus dantrolene, altered resting muscle excitability (Fig. 1). This is in agreement with a previous study showing that BTS does not markedly alter M-waves or intracellularly recorded action potentials (25). Dantrolene has also been shown to have little effect of muscle excitability, since it does not alter resting membrane potential or action potential parameters (41), although it has been shown to cause a small increase in the rheobase current required to trigger an action potential (30).

In accord with other studies on muscles exposed to high-frequency stimulation (13, 31), M-wave area and amplitude were, in the present study, well maintained during the first seconds of 60-Hz stimulation (Fig. 6) but then declined at a fast rate as stimulation was continued. A similar, two-phased development was observed in the force production of control muscles (Fig. 2), and Fig. 7 shows that, in these muscles, force was significantly correlated to both M-wave area (r2 = 0.61, P < 0.001) and M-wave amplitude (r2 = 0.89, P < 0.001) during the 30-s of 60-Hz continuous stimulation. Thus, as shown in other studies on isolated muscles (13, 31), the loss of excitability significantly contributes to the fatigue development in these muscles. Since muscles treated with BTS plus dantrolene produced very little force, it was not possible to examine the correlation between excitability and fatigue in these muscles, but the main observation in this study was that BTS-plus-dantrolene-treated muscles, which have a conserved energy status, also show a marked delay in the fast loss of excitability late in the experiment (Fig. 6). Thus, after 20 s of stimulation in control muscles, there was a large decrease in ATP and increase in lactate, with corresponding reductions in M-wave area and amplitude of 50 and 70%, respectively. At the same 20-s time point, BTS-plus-dantrolene-treated muscles had much higher ATP and lower lactate than controls and showed no reduction in M-wave area and only a 30% decrease in M-wave amplitude. These results strongly suggest that the loss of muscle excitability during intense exercise is hastened if muscles experience metabolic exhaustion. In support of this conclusion, previous observations point to several mechanisms that may convey a link between energy status and excitability in skeletal muscles.


Figure 7
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 7. Correlation between force and M-wave parameters during 30-s, 60-Hz continuous stimulation of control muscles. Corresponding data points of tetanic force and M-wave area (A) and tetanic force and M-wave amplitude (B) were correlated by linear regression lines of the equation Y = Y0 + {alpha}X, where Y0 = 15.9 and {alpha} = 1.12, r2 = 0.61, in A, and Y0 = –10.0 and {alpha} = 0.97, r2 = 0.89, in B, respectively. P < 0.001 for A and B. Dashed lines represent 95% confidence intervals. Tetanic force, M-wave area, and M-wave amplitude are shown as % initial control value (n = 6).

 
Generally, the loss of muscle excitability during intensive contractions has been related to the loss of intracellular K+ and the accumulation of extracellular K+ (see Refs. 11 and 37). The resulting membrane depolarization leads to slow inactivation of the voltage-gated Na+ channels (35) and eventually loss of muscle excitability. The concomitant increase in intracellular Na+ will also reduce the driving force for the inward Na+ current, which will further reduce action potential amplitude (1). Comparison of sarcolemmal action potentials, before and after fatiguing stimulation of frog skeletal muscle, shows a 10- to 20-mV membrane depolarization, a 20- to 40-mV decrease in action potential amplitude, and an approximate doubling in the duration of the action potential (2, 24). However, in agreement with the maintenance of M-wave area and amplitude during the first seconds of 60-Hz stimulation shown in Fig. 6, studies in which the concentration gradients for K+ and Na+ have been changed in isolated muscles (8, 37) show that a large safety margin exists that allows a substantial reduction in the concentration gradients of the two ions to take place before excitability is lost. Recently, it was shown (33) that this tolerance of muscles to a reduction in the concentration gradients for Na+ and K+ is sensitive to changes in resting membrane conductance, with a smaller loss of excitability at reduced membrane conductance. In this context, improved maintenance of excitability in BTS-plus-dantrolene-treated muscles can be envisaged to involve effects of metabolites on the loss of intracellular K+ and on the resting membrane conductance.

One possibility is that the loss of muscle excitability in muscles with reduced energy status is accelerated by the opening of ATP-sensitive K+ (KATP) channels that are in the closed state at resting levels of ATP and open state at low ATP. Davies et al. (14) showed that KATP channels were virtually inactive at 3 mM ATP and exhibited only limited activity at 1 mM ATP; however, decreasing pHi from 7.2 to 6.3 resulted in significant channel activity. However, Standen et al. (39) found that KATP channels could be activated by lowering pHi to 6.45, without reducing ATP, suggesting that a reduction in pHi alone can also open KATP channels. The ATP level in control muscles was markedly reduced (Fig. 3), and the large increase in lactate (Fig. 4) was associated with a drop in pHi to 6.63 (Table 1), making it possible that KATP channels are activated. Indeed, in frog skeletal muscle that has undergone chemically-induced metabolic exhaustion, there is an increase in K+ conductance that can be inhibited by the KATP channel blocker glibenclamide (9). Opening of KATP channels would cause an increase in both the K+ conductance and the K+ efflux. In accord with this, pharmacological opening of KATP channels with pinacidil reduces action potential overshoot and slows the time course of the action potential (20). Furthermore, in isolated muscles, pinacidil has been shown to decrease time to fatigue (26) and accelerate the run down in M-waves associated with fatiguing stimulation (20). Although it is clear that the opening of KATP channels increases the rate of fatigue, the exact role of KATP channels during contractions is not so obvious. Blocking KATP channels with glibenclamide does not alter action potential parameters or prolong the time to fatigue of isolated muscles (19).

Another possible mechanism for the improved maintenance of excitability in BTS-plus-dantrolene-treated muscles could be that the conserved energy status allows for a larger active reuptake of K+ via the Na+-K+-ATPase, which would limit the loss of intracellular K+ during contractions by improving the leak pump ratio for the ion (23). However, the global ATP values indicated in Fig. 3 should be sufficient to supply the Na+-K+-ATPase, since it has a Km for ATP of 0.5 mM (18). It is also conceivable that a large reduction in pHi will inhibit the Na+-K+-ATPase (38), although de Paoli et al. (15a) reported no alteration to Na+-K+-ATPase activity when pHi was decreased to 6.8.

Ca2+-activated K+ Formula channels, which open in the presence of high intracellular Ca2+, may also provide a link between energy status and muscle excitability. As with KATP channels, opening of KFormula channels would lead to a loss of muscle excitability. In metabolically exhausted frog muscles, Fink and Lüttgau (16) showed a large increase in membrane K+ conductance that could be prevented by the Ca2+-chelating agent EGTA, suggesting a role for KFormula channels. They suggested that metabolically exhausted fibers lose the ability to control myoplasmic Ca2+ and, furthermore, that the reduced energy status may itself increase the membrane conductance via opening of KFormula channels. It is possible that dantrolene prevents myoplasmic Ca2+, increasing enough to activate KFormula channels, and that the maintenance of muscle excitability we observed is related to a lower myoplasmic Ca2+ level. However, Fig. 2 shows that muscles treated with dantrolene alone still produce ~70% of control force 10 s into the stimulation, indicating that, despite the presence of dantrolene, there must be a significant elevation in myoplasmic Ca2+. It is therefore unlikely that the maintenance of muscle excitability we observed is associated with the lack of opening of the KFormula channels.

Finally, Bennetts et al. (5) have suggested that the main skeletal muscle Cl– channel, the ClC-1 channel, is sensitive to the energetic state of muscle fibers, and that ATP depletion increases its open probability. Such a mechanism would result in a high-membrane conductance in metabolically exhausted fibers, which is in accord with studies on frog muscle (16) showing that inhibition of ATP production causes the resting Cl– conductance to increase.

Implications for muscle function. It has long been suggested (6, 11, 37) that loss of muscle excitability plays a role in skeletal muscle fatigue, particularly during intensive work involving high frequencies of action potentials. The present study shows that if the work performed also causes a substantially reduced energy status of the muscle, the loss of excitability is hastened. Since the preparation used was an isolated muscle without blood circulation that was stimulated to contract tetanically for 30 s, this conclusion is relevant to work involving static, intense contractions. The impedence of blood flow during such contractions mimics the conditions of the isolated muscle in two important aspects. First, the buildup of K+ in the extracellular space of the muscle depends only on the balance between the loss and the uptake of K+ from the muscle fibers. Second, the energy requirements of the muscle fibers must mainly be met by anerobic metabolism. During dynamic work, where oxygen delivery to the muscles is increased, the maintenance of the energy status is supported by oxidative ATP production, and the energy status may never decrease to reach levels that would cause a loss of excitability. A recent study modeling oxygen consumption in contracting isolated muscles (3) suggests that, even when oxygen is available, anerobic metabolism is able to supply the extra energy requirements of contraction for at least the first 10 s. Therefore, it is also plausible that intense exercise in vivo may cause a reduction in muscle excitability, even though the contractions are dynamic.

An important implication for the role of excitability in skeletal muscle fatigue is that, if a muscle is allowed to contract, even after energy status has started to decrease, then severe and irreversible damage may occur as a consequence of a reduced energy status and an inability to effectively control cytosolic Ca2+ levels. When muscle excitability is reduced, muscle activation is shut down at a very early stage in the excitation-contraction coupling process, which effectively reduces not only the amount of ATP utilized by the contractile apparatus but also ATP used for Ca2+ recycling. Indeed, Thabet et al. (42) observed severe damage to type IIb fibers following treadmill running of mice deficient in KATP channels. Thus a link between metabolites and muscle excitability may provide a feedback mechanism that effectively protects muscle energy status and thus muscle integrity during intense exercise.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from the Aarhus Universitets Forskningsfond, The Lundbeck Foundation, and the Danish Medical Research Council (j.n.r. 272-05-0304).


   ACKNOWLEDGMENTS
 
We thank Marianne Stürup Johansen and Chris Christensen for skilled technical assistance. We also thank Kent Sahlin for helpful discussions and comments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. A. Macdonald, Institute of Physiology and Biophysics, Univ. of Aarhus, DK-8000, Aarhus C, Denmark (e-mail: wmd{at}fi.au.dk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Baker PF, Hodgkin AL, Shaw TI. The effects of changes in internal ionic concentrations on the electrical properties of perfused giant axons. J Physiol 164: 355–374, 1962.[Free Full Text]
  2. Balog EM, Thompson LV, Fitts RH. Role of sarcolemmal action potentials and excitability in muscle fatigue. J Appl Physiol 76: 2157–2162, 1994.[Abstract/Free Full Text]
  3. Barclay C. Modelling diffusive O2 supply to isolated preparations of mammalian skeletal and cardiac muscle. J Muscle Res Cell Motil 26: 225–235, 2005.[CrossRef][Web of Science][Medline]
  4. Barclay C, Loiselle DS. Can activation account for 80% of skeletal muscle energy use during isometric contraction? Am J Physiol Cell Physiol 291: C612, 2006. First published April 5, 2006; 10.1152/ajpcell.00122.2006.
  5. Bennetts B, Rychkov GY, Ng HL, Morton CJ, Stapleton D, Parker MW, Cromer BA. Cytoplasmic ATP-sensing domains regulate gating of skeletal muscle ClC-1 chloride channels. J Biol Chem 280: 32452–32458, 2005.[Abstract/Free Full Text]
  6. Bigland-Ritchie B, Jones DA, Woods JJ. Excitation frequency and muscle fatigue: electrical responses during human voluntary and stimulated contractions. Exp Neurol 64: 414–427, 1979.[CrossRef][Web of Science][Medline]
  7. Bruton J, Pinniger GJ, Lannergren J, Westerblad H. The effects of the myosin-II inhibitor N-benzyl-p-toluene sulphonamide on fatigue in mouse single intact toe muscle fibres. Acta Physiol (Oxf) 186: 59–66, 2006.
  8. Cairns SP, Buller SJ, Loiselle DS, Renaud JM. Changes of action potentials and force at lowered [Na+]o in mouse skeletal muscle: implications for fatigue. Am J Physiol Cell Physiol 285: C1131–C1141, 2003.[Abstract/Free Full Text]
  9. Castle NA, Haylett DG. Effect of channel blockers on potassium efflux from metabolically exhausted frog skeletal muscle. J Physiol 383: 31–43, 1987.[Abstract/Free Full Text]
  10. Cheung A, Dantzig JA, Hollingworth S, Baylor SM, Goldman YE, Mitchison TJ, Straight AF. A small-molecule inhibitor of skeletal muscle myosin II. Nat Cell Biol 4: 83–88, 2002.[CrossRef][Web of Science][Medline]
  11. Clausen T. Na+-K+ pump regulation and skeletal muscle contractility. Physiol Rev 83: 1269–1324, 2003.[Abstract/Free Full Text]
  12. Clausen T, Nielsen OB. The Na+,K(+)-pump and muscle contractility. Acta Physiol Scand 152: 365–373, 1994.[Web of Science][Medline]
  13. Clausen T, Overgaard K, Nielsen OB. Evidence that the Na+-K+ leak/pump ratio contributes to the difference in endurance between fast- and slow-twitch muscles. Acta Physiol Scand 180: 209–216, 2004.[CrossRef][Web of Science][Medline]
  14. Davies NW, Standen NB, Stanfield BR. The effect of intracellular pH on ATP-dependent potassium channels of frog skeletal muscle. J Physiol 445: 549–568, 1992.[Abstract/Free Full Text]
  15. Dentel JN, Blanchard SG, Ankrapp DP, McCabe LR, Wiseman RW. Inhibition of cross-bridge formation has no effect on contraction-associated phosphorylation of p38 MAPK in mouse skeletal muscle. Am J Physiol Cell Physiol 288: C824–C830, 2005.[Abstract/Free Full Text]
  16. de Paoli F, Overgaard K, Nielsen OB. Protective effects of acidosis and Na+-K+ pump activation on force in K+-depressed skeletal muscle. J Physiol 544: 82P, 2002.
  17. Fink R, Lüttgau HC. An evaluation of the membrane constants and the potassium conductance in metabolically exhausted muscle fibres. J Physiol 263: 215–238, 1976.[Abstract/Free Full Text]
  18. Fink R, Hase S, Lüttgau HC, Wettwer E. The effect of cellular energy reserves and internal calcium ions on the potassium conductance in skeletal muscle of the frog. J Physiol 336: 211–228, 1983.[Abstract/Free Full Text]
  19. Fitts RH. Cellular mechanisms of muscle fatigue. Physiol Rev 74: 49–94, 1994.[Abstract/Free Full Text]
  20. Gramolini A, Renaud JM. Blocking ATP-sensitive K+ channel during metabolic inhibition impairs muscle contractility. Am J Physiol Cell Physiol 272: C1936–C1946, 1997.[Abstract/Free Full Text]
  21. Gong B, Legault D, Miki T, Seino S, Renaud JM. KATP channels depress force by reducing action potential amplitude in mouse EDL and soleus muscle. Am J Physiol Cell Physiol 285: C1464–C1474, 2003.[Abstract/Free Full Text]
  22. Harris RC, Hultman E, Nordesjö LO. Glycogen, glycolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values. Scand J Clin Lab Invest 33: 109–120, 1974.[Web of Science][Medline]
  23. Harris RC, Sahlin K, Hultman E. Phosphagen and lactate contents of m. quadriceps femoris of man after exercise. J Appl Physiol 43: 852–857, 1977.[Abstract/Free Full Text]
  24. Harrison AP, Nielsen OB, Clausen T. The role of Na+-K+ pump and Na+ channel concentrations in the contractility of rat soleus muscle. Am J Physiol Regul Integr Comp Physiol 272: R1402–R1408, 1997.[Abstract/Free Full Text]
  25. Lannergren J, Westerblad H. Force and membrane potential during and after fatiguing, continuous high-frequency stimulation of single Xenopus muscle fibres. Acta Physiol Scand 128: 369–378, 1986.[Web of Science][Medline]
  26. Macdonald WA, Pedersen TH, Clausen T, Nielsen OB. N-Benzyl-p-toluene sulphonamide allows the recording of trains of intracellular action-potentials from nerve-stimulated intact fast-twitch skeletal muscle of the rat. Exp Physiol 90: 815–826, 2005.[Abstract/Free Full Text]
  27. Matar W, Nosek TM, Wong D, Renaud J. Pinacidil suppresses contractility and preserves energy but glibenclamide has no effect during muscle fatigue. Am J Physiol Cell Physiol 278: C404–C416, 2000.[Abstract/Free Full Text]
  28. Medbø JI, Sejersted OM. Plasma potassium changes with high intensity exercise. J Physiol 421: 105–122, 1990.[Abstract/Free Full Text]
  29. Melzer W, Herrmann-Frank A, Lüttgau HC. The role of Ca2+ ions in excitation-contraction coupling of skeletal muscle fibres. Biochim Biophys Acta 1241: 56–118, 1995.
  30. Nordsborg N, Mohr M, Pedersen LD, Nielsen JJ, Langberg H, Bangsbo J. Muscle interstitial potassium kinetics during intense exhaustive exercise: effect of previous arm exercise. Am J Physiol Regul Integr Comp Physiol 285: R143–R148, 2003.[Abstract/Free Full Text]
  31. Oba T, Hotta K. Transient effect of intracellular dantrolene on E-C coupling in skeletal muscle. Eur J Pharmacol 51: 81–84, 1978.[CrossRef][Web of Science][Medline]
  32. Overgaard K, Nielsen OB, Flatman JA, Clausen T. Relations between excitability and contractility in rat soleus muscle: role of the Na+-K+ pump and Na+/K+ gradients. J Physiol 518: 215–225, 1999.[Abstract/Free Full Text]
  33. Pedersen TH, de Paoli F, Nielsen OB. Increased excitability of acidified skeletal muscle: role of chloride conductance. J Gen Physiol 125: 237–246, 2005.[Abstract/Free Full Text]
  34. Reading SA, Murrant CL, Barclay JK. Increased cAMP as a positive inotropic factor for mammalian skeletal muscle in vitro. Can J Physiol Pharmacol 81: 986–996, 2003.[CrossRef][Web of Science][Medline]
  35. Ruff RL. Sodium channel slow inactivation and the distribution of sodium channels on skeletal muscle fibres enable the performance properties of different muscle fibre types. Acta Physiol Scand 156: 159–168, 1996.[CrossRef][Web of Science][Medline]
  36. Sahlin K, Gorski J, Edström L. Influence of ATP turnover and metabolite changes on IMP formation and glycolysis in rat skeletal muscle. Am J Physiol Cell Physiol 259: C409–C412, 1990.[Abstract/Free Full Text]
  37. Sejersted OM, Sjøgaard G. Dynamics and consequences of potassium shifts in skeletal muscle and heart during exercise. Physiol Rev 80: 1411–1481, 2000.[Abstract/Free Full Text]
  38. Skou JC. Effects of ATP on the intermediary steps of the reaction of the (Na+ + K+)-ATPase. IV. Effect of ATP on K05 for Na+ and on hydrolysis at different pH and temperature. Biochim Biophys Acta 567: 421–435, 1979.[Medline]
  39. Standen NB, Pettit AI, Davies NW, Stanfield PR. Activation of ATP-dependent K+ currents in intact skeletal muscle fibres by reduced intracellular pH. Proc Biol Sci 247: 195–198, 1992.
  40. Stephenson DG, Lamb GD, Stephenson GM. Events of the excitation-contraction-relaxation (E-C-R) cycle in fast-twitch muscle fibres relevant to muscle fatigue. Acta Physiol Scand 162: 229–245, 1998.[CrossRef][Web of Science][Medline]
  41. Takauji M, Takahashi N, Nagai T. Effect of dantrolene sodium on excitation contraction coupling in frog skeletal muscle. Jpn J Physiol 25: 747–758, 1975.[Web of Science][Medline]
  42. Thabet M, Miki T, Seino S, Renaud JM. Treadmill running causes significant fiber damage in skeletal muscle of KATP channel-deficient mice. Physiol Genomics 22: 204–212, 2005.[Abstract/Free Full Text]
  43. Wendt IR, Barclay JK. Effects of dantrolene on the energetics of fast- and slow-twitch muscles of the mouse. Am J Physiol Cell Physiol 238: C56–C61, 1980.[Free Full Text]



This article has been cited by other articles:


Home page
 J. Physiol.Home page
S. P. Cairns and M. I. Lindinger
Do multiple ionic interactions contribute to skeletal muscle fatigue?
J. Physiol., September 1, 2008; 586(17): 4039 - 4054.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/3/E771    most recent
00378.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Macdonald, W. A.
Right arrow Articles by Nielsen, O. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Macdonald, W. A.
Right arrow Articles by Nielsen, O. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.