K+-induced twitch potentiation is not due to longer action potential

Craig Yensen, Wadih Matar, and Jean-Marc Renaud

Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The objective of this study was to determine whether an increased duration of the action potential contributes to the K+-induced twitch potentiation at 37°C. Twitch contractions were elicited by field stimulation, and action potentials were measured with conventional microelectrodes. For mouse extensor digitorum longus (EDL) muscle, twitch force was greater at 7-13 mM K+ than at 4.7 mM (control). For soleus muscle, twitch force potentiation was observed between 7 and 11 mM K+. Time to peak and half-relaxation time were not affected by the increase in extracellular K+ concentration in EDL muscle, whereas both parameters became significantly longer in soleus muscle. Decrease in overshoot and prolongation of the action potential duration observed at 9 and 11 mM K+ were mimicked when muscles were respectively exposed to 25 and 50 nM tetrodotoxin (TTX; used to partially block Na+ channels). Despite similar action potentials, twitch force was not potentiated by TTX. It is therefore suggested that the K+-induced potentiation of the twitch in EDL muscle is not due to a prolongation of the action potential and contraction time, whereas a longer contraction, especially the relaxation phase, may contribute to the potentiation in soleus muscle.

fatigue; exercise; tetrodotoxin; temperature


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A NET K+ efflux during muscular activity results in an increased extracellular K+ concentration. In general, the K+ concentration in the venous return of exercising muscles increases from the normal 4-4.5 mM to 6-8 mM (20, 21, 38, 41) and can be as high as 9 mM at exhaustion (33). Similar values have been reported in the interstitial space of exercising muscle (23, 24, 28, 31), and in one case a value of 15 mM has been observed (42). Concomitantly with the increase in extracellular K+ concentration, decreases in intracellular K+ concentration between 10 and 70 mM have also been reported depending on the muscle and the type and intensity of the exercise (5, 27, 29, 30, 38).

It is well established that such changes in extracellular and intracellular K+ concentration lead to a depolarization of the cell membrane (1, 25), which in turn inactivates Na+ channels (2) and reduces membrane excitability (34-36). On the basis of these K+ effects and because any decreases in action potential overshoot are expected to result in smaller amounts of Ca2+ released by the sarcoplasmic reticulum, it has been hypothesized that K+ is an important factor responsible for the decrease in force during fatigue development. Indeed, when the extracellular K+ concentration is increased to 10 mM or greater, both the twitch and tetanic force are suppressed in amphibian (36) and mammalian muscle (9, 34, 35).

An increase in extracellular K+ to 9 mM or less potentiates twitch force and submaximal tetanic force by 60-100% in frog sartorius muscle (36) and mammalian muscle (26, 43), but it does not suppress tetanic force. This potentiating effect of K+ suggests that an increase in extracellular K+ does not always cause fatigue and may in fact be beneficial to a muscle during exercise. To further understand the physiological importance of this potentiating effect of K+, one must understand its mechanism.

One mechanism may involve an increased duration of the action potential, resulting in a longer duration and greater amount of Ca2+ release by the sarcoplasmic reticulum (37). For example, the twitch force of frog sartorius muscle is 60% greater and the action potential duration is 65% longer at 9 mM than at 4 mM K+ (36). A 1:1 ratio has been reported between the potentiation of the twitch force and the prolongation of the action potential duration when muscles are exposed to Zn2+ between 1 and 10 µM (40). However, Zn2+ affects more than just the action potential during a contraction (40). Consequently, the correlation between action potential duration and twitch potentiation when muscles are exposed to Zn2+ or high K+ may be causal, as proposed by Sandow et al. (37), or casual.

The objective of this study was therefore to determine whether an increased duration of the action potential contributes to the K+-induced twitch potentiation. The experimental approach consisted of first determining the effect of an increase in K+ concentration on both action potential and twitch contraction in mouse extensor digitorum longus (EDL) and soleus muscle. Then, with the use of tetrodotoxin (TTX), the action potential overshoot and duration were modified to mimic the K+ effects. TTX was used because it is a specific blocker of the Na+ channel and is not expected to affect the Ca2+ released by the sarcoplasmic reticulum or the contractile components other than via an effect on action potential. If the K+-induced potentiation of the twitch force is fully or partially due to a prolongation of action potential, then twitch force should be potentiated in the presence of TTX. The results showed that for EDL muscle a prolongation of the action potential duration is not involved in the twitch potentiation, whereas for soleus muscle a longer twitch contraction may partially contribute to the potentiation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and muscle preparation. The Animal Care Committee of the University of Ottawa approved all experimental procedures. Two- to three-month-old female CD-1 mice weighing 25-30 g were obtained from Charles River and housed according to the guidelines of the Canadian Council for Animal Care. The animals were fed ad libitum. Before any experiment, mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (Somnotol) delivered at a dose of 0.8 mg/10 g body wt. EDL and soleus muscles were excised and both tendons were tied with surgical silk (6.0) to allow attachment of muscle to the experimental apparatus (all mice were killed with an overdose of pentobarbital sodium).

Solutions. Muscles were immersed in physiological saline solution containing (in mM) 118.5 NaCl, 4.7 KCl, 2.4 CaCl2, 3.1 MgCl2, 25 Na2HCO3, 2 NaH2PO4, and 5.5 D-glucose. The solution was continuously bubbled in 95% O2 and 5% CO2, pH 7.4. This solution also represented the control solution. The K+ concentration was increased by adding the proper amount of KCl in the control solution. TTX-containing solutions were obtained by first dissolving TTX-citrate in water (1 mM) and then by adding the proper volume in the control solution. The temperature of all experiments was 37°C.

Force measurements. Muscles were stimulated by passing a current between parallel platinum wires located on opposite sides of the muscle. Twitch contractions were elicited with 0.3-ms-long rectangular pulses of 8 V (supramaximal voltage) every 2 min throughout an experiment. The experimental chamber was as described by Matar et al. (32), except for the following modifications. Twitch force was measured using semiconductor force transducers (model BG-100; Kulite Semiconductors Products). Twitch force was digitized with a Keithley Metrabyte analog-to-digital board (model DAS50) at a sampling rate of 20 kHz. The following parameters were later analyzed on the computer. Twitch force, defined as the maximum force during a twitch, was calculated as the difference between peak force and baseline. Time to peak was calculated as the time interval during which force increased from 5 to 100% of maximum during the contraction phase. Half-relaxation time was calculated as the time interval during which force decreased from 100 to 50% of maximum during the relaxation phase.

Action potential measurements. Membrane potentials were measured by using conventional microelectrodes as described by Matar et al. (32). Briefly, microelectrodes (with tip potentials <5 mV and tip resistance varying between 7 and 15 MOmega ) and reference electrodes (tip resistance of 1 MOmega ) were filled with 3 M KCl. Action potentials were elicited by passing a small current between two fine platinum wires placed along the surface fibers to stimulate a small number of fibers. Action potentials were digitized at a sampling rate of 200 kHz. Resting potential was measured from the baseline of action potential. Overshoot, the membrane potential above 0 mV during the action potential, was measured from the action potential peak. The duration or width of the action potential was calculated as the time interval at 50% of the amplitude.

For each experimental condition, action potentials were measured in four to five fibers per muscle. For each muscle, an average value was calculated for resting potential, overshoot, and width. These averages were then used to calculate the mean values of resting potential, overshoot, and width.

Statistical analysis. Throughout the paper, data are presented as means ± SE. ANOVA was used to determine significant differences. Split plot designs were used when muscles were tested at all levels of a treatment (i.e., time effect after a change in K+). In all other cases, a two-way ANOVA design was used (i.e., dose-response curves of K+ or TTX as different muscles were used for each K+ and TTX concentration). ANOVA calculations were made using the General Linear Model procedures of the Statistical Analysis Software (SAS Institute, Cary, NC). When a main effect or an interaction was significant, the least-square difference was used to locate significant differences (39). Differences of P < 0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of K+

Twitch contraction. The twitch force of both EDL and soleus muscle increased significantly when the extracellular K+ concentration was raised from 4.7 (control) to 9 mM (Fig. 1, A and B). Increases in twitch force were significant within 10 min for soleus and 20 min for EDL muscles when twitch forces were, respectively, 137 and 121% of the initial force (Fig. 1C). Twitch force continued to increase until it reached a maximum of 164-168% in 90 min for EDL and a maximum of 197-202% in 60 min for soleus. When the extracellular K+ concentration was returned from 9 to 4.7 mM, the twitch force of both EDL and soleus muscle decreased back toward their initial value. After 80 min at 4.7 mM K+, the twitch forces of EDL and soleus muscles were, respectively, 92 and 88% of the initial twitch force.


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Fig. 1.   Effect of 9 and 15 mM K+ on the twitch contraction of extensor digitorum longus (EDL) and soleus muscles. Twitch contractions of EDL (A) and soleus (B) muscles at 4.7 (a) and 9 mM K+ (b). Calibration bars represent 2 N/cm2 and 20 ms. Changes in twitch force over time are shown after an increase to 9 mM (C) and to 15 mM K+ (D). Twitch force is defined as the maximum force developed during a twitch contraction and is expressed as a percentage of the twitch force measured at 4.7 mM K+ (control). Experimental temperature was 37°C. open circle , EDL; , soleus. The dashed horizontal line represents 100%. The hatched horizontal bar represents the time muscles were exposed to 9 or 15 mM K+. Data represent means ± SE of 5 muscles (error bars not shown if smaller than symbol). * Time when mean twitch force became significantly different from mean value at time 0; ANOVA and least-square difference (LSD), P < 0.05.

Twitch force also increased when the extracellular K+ concentration was raised from 4.7 to 15 mM (Fig. 1D). However, the increase was transient because twitch force soon decreased, with decreases being faster in soleus than EDL muscle. After 100 min, the twitch forces of EDL and soleus muscles were, respectively, 10 and 6%. When the extracellular K+ concentration was returned from 15 to 4.7 mM, twitch force recovered to 100% for the EDL and to 89% for the soleus muscle.

Steady-state potentiation or depression by K+ was measured once twitch force had reached a new steady state, which is defined as the time period when twitch force no longer changed for 20 min (10 contractions) after an increase or decrease in extracellular K+ concentration. Note that the time to reach steady state varied between muscles and K+ concentrations as shown in Fig. 1, C and D. For the EDL muscle, significant potentiation of the twitch force was observed between 7 and 13 mM K+ (Fig. 2A). The largest potentiation was between 8 and 10 mM K+ as twitch force was 176-181% of the force measured at 4.7 mM K+. The twitch force of EDL muscle was suppressed only at 15 mM K+. For the soleus muscle, twitch potentiation was observed only between 7 and 11 mM K+ with a maximum at 9 mM when twitch force was 196% of the force measured at 4.7 mM K+. Contrary to the EDL muscle, 12 and 13 mM K+ depressed twitch force in soleus muscle.


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Fig. 2.   Effect of K+ on twitch force (A) and half-relaxation time (B) in EDL and soleus muscles. Twitch force and half-relaxation time were measured after a change in extracellular K+ concentration once twitched force had reached a steady state defined as the time period during which force remained constant for 20 min (10 contractions). Twitch force and time to peak are expressed as a percentage of the value measured at 4.7 mM K+ (control). Experimental temperature was 37°C. Half-relaxation times are shown only for the range of K+ concentrations that potentiated twitch force (i.e., 6-12 mM). open circle , EDL; , soleus. Data represent means ± SE of 3-5 muscles (error bars not shown if smaller than symbol). The dashed horizontal line represents 100%. * Mean twitch force or half-relaxation time was significantly different from the mean value at 4.7 mM K+; ANOVA and LSD, P < 0.05.

In EDL muscle, potentiation of the twitch force at 9 mM K+ was not associated with a prolongation of the twitch contraction (Fig. 1A). On an average basis, neither the time to peak (data not shown) nor the half-relaxation time changed significantly between 4.7 and 12 mM K+ (Fig. 2B). In soleus muscle, both the time to peak and the relaxation phase became longer at 9 mM K+ (Fig. 1B). Increase in time to peak was significant only at 9 mM K+ and was 144% of the time measured at 4.7 mM K+ (data not shown). Significant increases in half-relaxation time, on the other hand, were observed at 8, 9, 10, and 11 mM K+ with a maximum value of 164% at 9 mM K+ (Fig. 2B).

Thus the twitch force is potentiated when the extracellular K+ concentration is increased from 4.7 mM up to 11 mM for the soleus muscle and up to 13 mM for the EDL muscle. The twitch potentiation is associated with a prolongation of the contraction and relaxation time in soleus but not in EDL muscle.

Action potential. Measurements of action potentials were limited to the K+ concentrations that potentiated twitch force. In EDL muscle, an increase in the extracellular K+ concentration from 4.7 mM to 9 and 11 mM had three major effects: 1) depolarization of the cell membrane, 2) reduction of the action potential overshoot, and 3) prolongation of the action potential duration (Fig. 3, A and B). On average, depolarization of the cell membrane was 35.6 mV per 10-fold increase in K+ concentration (Fig. 3C). The overshoot at 4.7 mM K+ was 26.9 mV and decreased significantly to 22.4 mV at 7 mM and 8.0 mV at 11 mM K+. Although action potential became longer, significant increases were observed only at 10 and 11 mM K+ (Fig. 3D) compared with 7-8 mM for the resting potential and overshoot (Fig. 3C). At 10 and 11 mM K+, the action potential widths were, respectively, 17 and 29% longer than at 4.7 mM K+. The increase in width was due to an increased duration of both the depolarization and repolarization phases (Fig. 3B).


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Fig. 3.   Effect of K+ on resting and action potential of EDL muscle. A: action potential at 4.7 (a) and 9 mM K+ (b). B: action potential at 4.7 (a) and 11 mM K+ (b). Calibration bars represent 40 mV and 0.5 ms. C: effects of K+ on resting membrane potential (open circle ) and overshoot of action potential (). Overshoot is defined as the membrane potential at the peak of the action potential. D: effect of K+ on the width of action potential (triangle ). Width was calculated as the time interval at 50% of the action potential amplitude. Experimental temperature was 37°C. Data represent means ± SE of 11 muscles (35 fibers) at 4.7 mM K+ and 4-5 muscles (20-27 fibers) for each of the other K+ concentrations (error bars not shown if smaller than symbol). * Mean resting potential, overshoot, or width was significantly different from the mean value at 4.7 mM K+; ANOVA and LSD, P < 0.05.

Resting and action potentials were also measured in soleus muscle, although in fewer fibers. The depolarization of the cell membrane and decrease in overshoot at 9 mM K+ was greater in soleus than in EDL muscle fibers (Table 1). Taking all data between 4 and 10 mM K+, depolarization of the cell membrane was 44.5 mV per 10-fold increase in extracellular K+ concentration (43 fibers from 12 muscles, data not shown). Although on an absolute scale the increase in action potential width was greater in soleus than in EDL fibers (Table 1), on a relative scale the increase was quite similar, being 13% for EDL and 15% for soleus.

                              
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Table 1.   Effects of [K+] on action potential and the change in resting potential in mouse EDL and soleus

Effect of TTX

Twitch contraction. TTX had no effect on the twitch force of EDL muscle between 10 and 50 nM, whereas it significantly suppressed force at 100 and 150 nM (Fig. 4A). TTX, between 10 and 100 nM, also had no effect on the time to peak (data not shown) and the half-relaxation time (Fig. 4B). The TTX effect on twitch force at 100 and 150 nM was partially reversible because twitch force returned to 77 ± 6% of its initial value upon washout of TTX (data not shown).


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Fig. 4.   Effect of tetrodotoxin (TTX) on twitch force (A) and half-relaxation time (B) of EDL muscle. Twitch force and time to peak are expressed as a percentage of the value measured at 0 nM TTX. Experimental temperature was 37°C. Data represent means ± SE of 3-5 muscles (error bars not shown if smaller than symbol). * Mean twitch force or time to peak was significantly different from the mean value at 0 nM TTX; ANOVA and LSD, P < 0.05.

Action potential. Exposing EDL muscle to 10 nM TTX caused a small hyperpolarization from -78.5 to -82.3 mV; similar hyperpolarizations were observed at 25, 50, and 100 nM TTX (Fig. 5A). TTX, at a concentration as small as 10 nM, significantly reduced the overshoot from 28.7 to 21.4 mV (Fig. 5A), whereas significant prolongations of the action potential width were observed only at 50 and 100 nM TTX (Fig. 5B).


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Fig. 5.   Effect of TTX on resting and action potential of EDL muscle. A: effects on resting potential (open circle ) and action potential overshoot (). B: effect on action potential width (triangle ). Experimental temperature was 37°C. Data represent means ± SE of 11 muscles (41 fibers) at 0 nM TTX and 3-5 muscles (14-17 fibers) for each of the other TTX concentrations (error bar not shown if smaller than symbol). * Mean overshoot or width was significantly different from the mean value at 0 nM TTX; ANOVA and LSD, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of this study are that, at 37°C, 1) the twitch force of EDL and soleus muscles increased 1.6- to 1.9-fold despite significant decreases in the action potential overshoot, when the extracellular K+ concentration was increased from 4.7 mM (control) to 8-11 mM; 2) twitch force was suppressed only when the extracellular K+ concentration exceeded 11 mM for the soleus and 13 mM for the EDL muscle; 3) the K+-induced depolarization of the resting membrane potential was small, being only 35.6 and 44.5 mV per 10-fold increase in extracellular K+ concentration; and 4) at 10-50 nM, TTX significantly reduced the action potential overshoot but had no effect on twitch force.

K+-twitch force relationship. The dual effect of K+ on twitch force (i.e., the potentiation with small increases in extracellular K+ concentration and the subsequent depression with further increases in K+ concentration) is in agreement with previous studies in which amphibian (6, 11, 18, 36) and mammalian muscle (10, 26, 35) have been used. A comparison between these studies, however, shows that temperature has two effects on the K+-twitch force relationship. The first effect is on the magnitude of twitch potentiation. For mouse EDL and soleus muscle, the potentiation reaches 80-90% at 37°C (Fig. 2) compared with a maximum of 10-20% at 25°C (10). The potentiation in guinea pig EDL and soleus muscles is almost twofold at 37°C (26), whereas in rat soleus it is 30% at 30°C (35). The second effect of temperature is on the extracellular K+ concentration at which twitch force becomes suppressed. At 37°C, this K+ concentration is 15 mM K+ for the EDL and 12 mM for the soleus (Fig. 2A). High K+ concentrations are also necessary to suppress twitch force in rat gastrocnemius at 37°C because a 61% potentiation has been observed at 13-14 mM K+ (43). At 25-30°C, twitch force is suppressed at the much lower K+ concentrations of 9-11 mM (4, 10, 12, 13).

Temperature effect on the magnitude of potentiation cannot be elucidated from the results obtained in this study, but the effect on the K+ concentration at which twitch force is suppressed can be explained. If the relationship between the resting membrane potential (Em) and K+ concentration (Fig. 3) is extrapolated to 15 mM K+, then twitch force of EDL muscle becomes suppressed when Em is about -62 mV. If the same extrapolation is done for the soleus muscle (Table 1), an Em of -58 mV is obtained (12 mM K+). At 25°C, twitch force is suppressed at -58 mV for both EDL and soleus muscle. Thus the Em at which twitch force decreases is the same at 25°C and 37°C, which is in agreement with the suggestion of Cairns et al. (10) that Em and not the extracellular K+ concentration appears as the most important parameter determining the condition at which twitch force is suppressed. This study also shows that higher extracellular K+ concentrations are necessary to suppress twitch force at 37°C than at 25°C because the K+-induced depolarization of the cell membrane is smaller at 37°C than at 25°C.

K+-resting membrane potential relationship. For the EDL muscle, a 10-fold increase in extracellular K+ caused a 35.6-mV depolarization of the cell membrane (Table 1). This value is similar to the value reported for rat red sternomastoid muscle at 37°C (i.e., 28 mV) (15) but smaller than the value reported for mouse EDL at 25°C (51.0 mV) (10). For the soleus muscle, the values are 44.5 mV at 37°C (Table 1) and 48.1 mV at 25°C (10). Thus, for the EDL muscle, the K+-induced depolarization is smaller at 37°C than at 25°C, and it is also smaller in EDL than in soleus at 37°C. Although these differences cannot be explained from our results, at least two factors can be proposed.

The first factor is the Na+-K+ pump. Activation of the Na+-K+ pump with insulin, calcitonin gene-related peptide (CGRP), or salbutamol reduces the extent of the force depression at 10 mM K+ (4, 12) and the rate at which force is suppressed at 12.5 mM (4, 13). The hormones also produced a reasonable recovery of force after it had been depressed at 12.5 mM K+ (4, 12). This effect of the pump involves a repolarization of the cell membrane, which results in a recovery of its excitability (14, 34). Furthermore, the contribution of the pump to the resting membrane potential increases with temperature (3, 8, 22). Thus the smaller K+-induced depolarization of the cell membrane of EDL muscle fibers at 37°C compared with 25°C may in part be due to a greater activity of the Na+-K+ pump at 37°C.

Although the difference in slope for the K+ effect on Em between the two temperatures was 16 mV for the EDL muscle, it is <4 mV for soleus muscle. Perhaps the second factor, Cl-, may also play a role. Cl- reduces both the rate and extent of the K+-induced membrane depolarization in mammalian muscle (16). Furthermore, the Cl- conductance is 2.4 times greater in mouse EDL than in soleus muscle (7). Thus Cl- may be more effective (concomitantly with the Na+-K+ pump) in reducing the extent of the K+-induced depolarization in EDL muscle compared with soleus muscle at 37°C.

Action potential and twitch force potentiation. The main objective of this study was to determine whether a prolongation of the action potential duration contributes to the K+-induced twitch potentiation. The notion was based on studies showing a good correlation between the magnitude of the twitch potentiation and action potential duration in the presence of 1-10 µM Zn2+ (37, 40). However, as mentioned in the Introduction, Zn2+ affects not only the action potential but also other events in the contractile cycle. Therefore, to test whether a prolongation of the action potential contributes to the K+-induced twitch potentiation, we exposed EDL muscles to TTX to modify and especially to mimic the action potential observed at high extracellular K+ concentration.

Several parameters of the action potential were similar between 9 mM K+ and 25 nM TTX, as well as between 11 mM K+ and 50 nM TTX (Table 2). The major difference between the K+ and TTX effects was the lack of a depolarization in the presence of TTX. Although the K+ and TTX effects on the action potential were quite similar, 9 and 11 mM K+ caused, respectively, a 68 and 56% increase in twitch force, whereas 25 and 50 nM TTX caused a small decrease (<5%) in twitch force. Reducing the extracellular Na+ concentration by >60 mM also fails to reduce twitch force in amphibian (6) and mammalian muscle (S. P. Cairns and J.-M. Renaud, unpublished results), despite a reduction in action potential overshoot. These results, thus, do not support the notion that a prolongation of the action potential duration is responsible for the potentiation of the twitch force between 8 and 11 mM K+ in EDL muscle. This conclusion is further supported by the fact that twitch potentiation was significant at 7 mM K+ (Fig. 2), whereas significant prolongations of the action potential are observed only at 10 mM or greater K+ concentration (Fig. 3B).

                              
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Table 2.   A comparison of the effect of K+ and TTX on membrane potential

An increase in twitch force can also occur if the contraction time is longer. For EDL muscle, neither the time to peak (data not shown) nor the half-relaxation time changes when muscles are exposed to 8-11 mM K+ (Fig. 2B). However, for soleus muscle, longer time to peak (data not shown) and half-relaxation time (Fig. 2B) were observed. It is therefore proposed that the K+-induced potentiation of the twitch force in EDL muscle is not due to longer action potential or twitch contraction, whereas for soleus muscle a longer contraction may contribute to the potentiation.

Significance of the K+ effects to the etiology of exercise and fatigue. It has long been suggested that the increase in extracellular K+ concentration (concomitantly with the decrease in intracellular K+ concentration) is an important factor contributing to the depolarization of the cell membrane, which then reduces membrane excitability and force development (i.e., fatigue). However, this concept is an oversimplification because of the K+-induced potentiation and the conditions that must be met to suppress force.

The K+-induced potentiation observed in this study is not limited to the twitch contraction because submaximal tetanic force (40 Hz) can also be potentiated twofold (26). Although it can be argued that the time to reach maximum potentiation is quite long (i.e., 60-80 min; Fig. 1), it is important to take into account the time the K+ must diffuse throughout the entire muscle and perhaps throughout t tubules where diffusion is considered limited due to the small diameter. Furthermore, a significant potentiation (~20%) is observed within 10 min after an increase in extracellular K+ concentration. Finally, in the intact working muscle, it can be assumed that the rise in extracellular K+ develops within a few seconds in the relevant area. It is thus suggested that the potentiating effect of K+ is important during exercise, especially during mild and long-term exercise such as running long distances.

In regard to the conditions that are necessary to suppress force, this study raises two important issues. The first is in regard to the effect of depressing action potential overshoot on force development during a twitch. A plot of mean twitch forces vs. mean overshoots from the TTX experiments shows that the twitch force of EDL muscle remains unchanged, while the overshoot decreases from 30 to 10 mV (Fig. 6). A similar relationship has been observed when soleus muscles are exposed to low extracellular Na+ concentrations instead of TTX (S. P. Cairns and J. M. Renaud, unpublished results). More importantly, the twitch force-overshoot relationship from the data of the K+ effects suggests that an increase in extracellular K+ concentration may help in delaying the onset of fatigue because, at a given overshoot, twitch force is greater at high K+ concentration than in the presence of TTX.


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Fig. 6.   Twitch force-overshoot relationship. open circle , Values from experiments on the effects of K+ on twitch force (Fig. 2A) and overshoot (Fig. 3C); , values from experiments on the effects of TTX on twitch force (Fig. 4A) and overshoot (Fig. 5A).

The second issue is in regard to the changes in extracellular K+ concentration during fatigue and the expected effect on force development. Most studies have shown that the K+ concentration in the venous return (20, 21, 33, 38, 41) and interstitial space (19, 23, 24, 28, 31) of exercising muscles ranges between 6 and 10 mM K+. These values are less than the 12 mM (soleus) and 15 mM (EDL) concentrations that are required for K+ to suppress twitch force. These findings do not exclude a role for K+ in the etiology of muscle fatigue, but they further support the suggestion of Bouclin et al. (6) that, by itself, K+ is not a major factor responsible for the decrease in force and that other changes, such as a decrease in Na+ concentration gradient, are necessary for K+ to affect force.

In summary, this study has shown that, at 37°C, there is a large potentiation (1.8- to 1.9-fold) of twitch force in unfatigued mouse EDL and soleus muscle when the extracellular K+ concentration is increased. The potentiation is observed at extracellular K+ concentrations as high as 11 mM for soleus and 13 mM for EDL muscle. For the EDL muscle, potentiation was not due to a longer action potential or twitch contraction, whereas a longer twitch contraction may have partially contributed to the potentiation in soleus muscle.

Perspective

This study raises two major issues. The first is the mechanism by which K+ potentiates force development. Because a prolongation of the action potential is not involved, other mechanisms will have to be studied. One mechanism may involve the depolarization of the cell membrane because Ba+ induces depolarization and twitch potentiation (17). Perhaps the K+-induced depolarization of the cell membrane affects the dihydropyridine receptor or voltage sensor in the t tubules and/or causes an increase in inositol 1,4,5-triphosphate levels, which allows for faster and greater Ca2+ release by the sarcoplasmic reticulum. The second issue is how muscles determine whether K+ potentiates or suppresses force development. One can argue that, early during exercise, the main effect of K+ is one of potentiation, whereas, during fatigue, development of the K+ effect is one of suppression. One factor that may be involved in the switch from potentiation to suppression is the energy status of the fiber. Although ATP levels decrease very little during fatigue, there are large increases in H+, lactic acid, and adenosine, three factors that activate the ATP-sensitive K+ channel allowing for greater K+ efflux, which in the t tubules may reach a concentration high enough to suppress force.


    ACKNOWLEDGEMENTS

We thank Dr. Eva Chin for comments on the manuscript.


    FOOTNOTES

This study was supported by a grant from National Sciences and Engineering Research Council (J. M. Renaud).

Address for reprint requests and other correspondence: J. M. Renaud, Dept. of Cellular and Molecular Medicine, Univ. of Ottawa, Ottawa Ontario, Canada K1H 8M5 (E-mail: jmrenaud{at}uottawa.ca).

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.

First published February 20, 2002;10.1152/ajpcell.00549.2001

Received 15 November 2001; accepted in final form 15 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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