A KATP channel deficiency affects resting tension, not contractile force, during fatigue in skeletal muscle

B. Gong1, T. Miki2, S. Seino2, and J. M. Renaud1

1 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5; and 2 Department of Molecular Medicine, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The objective of this study was to determine how an ATP-sensitive K+ (KATP) channel deficiency affects the contractile and fatigue characteristics of extensor digitorum longus (EDL) and soleus muscle of 2- to 3-mo-old and 1-yr-old mice. KATP channel-deficient mice were obtained by disrupting the Kir6.2 gene that encodes for the protein forming the pore of the channel. At 2-3 mo of age, the force-frequency curve, the twitch, and the tetanic force of EDL and soleus muscle of KATP channel-deficient mice were not significantly different from those in wild-type mice. However, the tetanic force and maximum rate of force development decreased with aging to a greater extent in EDL and soleus muscle of KATP channel-deficient mice (24-40%) than in muscle of wild-type mice (7-17%). During fatigue, the KATP channel deficiency had no effect on the decrease in tetanic force in EDL and soleus muscle, whereas it caused a significantly greater increase in resting tension when compared with muscle of wild-type mice. The recovery of tetanic force after fatigue was not affected by the deficiency in 2- to 3-mo-old mice, whereas in 1-yr-old mice, force recovery was significantly less in muscle of KATP channel-deficient than wild-type mice. It is suggested that the major function of the KATP channel during fatigue is to reduce the development of a resting tension and not to contribute to the decrease in force. It is also suggested that the KATP channel plays an important role in protecting muscle function in older mice.

Kir6.2; knockout; mouse; twitch; tetanus; force-frequency curve


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

WHEN THE INTRACELLULAR ATP concentration decreases and/or the intracellular ADP, H+, and lactate concentrations increase (14, 18, 23, 28), the ATP-sensitive K+ (KATP) channel, a voltage-insensitive K+ channel, is activated. There is now excellent evidence that the KATP channels of cardiac and skeletal muscle are activated during ischemia, hypoxia, and metabolic inhibition (6, 8, 9). The KATP channel can thus regulate the electrical activity of the cell membrane in relation to the energy state of the cell.

Noma (18) postulated that the function of the KATP channel is to protect muscle against deleterious energy depletion and irreversible function impairment. To accomplish such function, it has been postulated that the KATP channel reduces force development because muscle activity can increase the metabolic rate 20-fold in slow mammalian muscle and up to 500-fold in amphibian muscle (30). The mechanism of action of the KATP channel involves an increase in K+ conductance, which then contributes to a shortening of the action potential duration (8, 9) and to an increase in the extracellular K+ concentration (4, 8). Greater K+ concentration then depolarizes the cell membrane (10), which increases the proportion of inactivated Na+ channels (1). Together, the shortening of action potential and the inactivation of Na+ channels reduce membrane excitability that leads to a decrease in Ca2+ release (3) and force production (8). The Ca2+-ATPase and myosin ATPase then use less energy because there is less Ca2+ and force.

Muscle fatigue is defined as a decrease in the capacity of a muscle to develop force when it is stimulated repetitively. The decrease in force can be as high as 90% (16). It has, therefore, been suggested that the KATP channel plays an important role in the etiology of muscle fatigue. Indeed, an activation of the KATP channel with pinacidil or levcromakalim increases the rate of fatigue (16, 29), which is evidence that the channel can contribute to the decrease in force. However, most studies have reported that blocking the KATP channel with either glibenclamide (1-100 µM) or tolbutamide (2 mM) does not affect the rate of fatigue (5, 7, 15, 26, 29).

Matar et al. (16) recently showed, with the use of mouse extensor digitorum longus (EDL) and soleus, that 10 µM glibenclamide does not affect the rate of fatigue, but causes large increases in resting tension, which develops when muscles fail to relax between contractions. They suggested that the number of activated KATP channels was too small to affect force development during fatigue. However, the efficiency of glibenclamide to block KATP channels decreases when the ADP concentration increases (27). It is, therefore, possible that the lack of a glibenclamide effect on the rate of fatigue was due to an incomplete block of the KATP channels. Furthermore, glibenclamide at >= 20 µM increases the Ca2+ sensitivity of the contractile apparatus (7, 16). Although this effect is not observed at 10 µM glibenclamide in unfatigued muscle, it is still possible that this effect occurs in fatigued muscle fibers. Consequently, the effect of glibenclamide on the resting tension may be due to a nonspecific effect of glibenclamide, such as the Ca2+ sensitivity of the contractile components.

Classical KATP channels comprise two subunits, the pore-forming subunit Kir6.2 and a sulfonylurea receptor (SUR1 or SUR2) (2, 22). Whereas pancreatic beta -cell KATP channels comprise Kir6.2 and SUR1 (12), skeletal muscle comprise Kir6.2 and SUR2A (11, 22). Recently, a KATP channel-deficient mouse (Kir6.2-/-) was generated by disrupting the Kir6.2 gene in a C57Bl6/J mouse (defined here as the wild-type or Kir6.2+/+ mouse) (17). The skeletal muscle of a Kir6.2-/- mouse has no KATP channel activity in its cell membrane (Seino, unpublished data). Thus the Kir6.2-/- mouse gives a new approach to the study of the physiological role of the KATP channel.

The first objective of this study was, therefore, to determine whether a KATP channel deficiency affects the kinetics of fatigue. Matar et al. (16) determined the effects of glibenclamide on the EDL and soleus muscle of 2- to 3-mo-old mice. We therefore used muscle from mice of the same age to compare the effects of the KATP channel deficiency with those of glibenclamide. The second objective of this study was to determine whether a KATP channel deficiency adversely affects muscle function over time. For this, we compared the capacity to generate tetanic force (defined as the maximum force a muscle can develop) and the kinetics of fatigue in EDL and soleus muscle of 2- to 3-mo-old and 1-yr-old mice. The results showed 1) the KATP channel reduces the development of resting tension during fatigue but does not affect the rate of fatigue; and 2) the capacity to generate tetanic force in both EDL and soleus decreases in KATP channel-deficient mice to a greater extent than in wild-type mice when the animals get older.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Kir6.2+/+ (or wild-type) and Kir6.2-/- mice were obtained as previously described by Miki et al. (17). Mice were fed ad libitum and housed according to the guidelines of the Canadian Council for Animal Care. Kir6.2-/- mice did not require any special care. The Animal Care Committee of the University of Ottawa also approved all experimental procedures.

Force Measurement

Force measurement was as described by Matar et al. (16). Briefly, mice were anesthetized with an intraperitoneal injection of pentobarbitol sodium (Somnotol) delivered at a dose of 0.8 mg/10 g body wt. EDL and soleus muscle were excised, and both tendons were tied with surgical silk to allow attachment of muscle to the experimental apparatus. Muscles were constantly immersed in physiological saline solution that contained (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 with 95% O2-5% CO2 and had a pH of 7.4. The temperature of all experiments was 37°C.

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). Tetanic contractions were elicited with a 200-ms-long train of pulses at 140 Hz for soleus muscle and 200 Hz for EDL muscle. Force was measured with a Kulite semiconductor strain gauge (model BG100) and digitized with a Keithley Metrabyte analog-to-digital board (model DAS50). Sampling rates were 20 kHz for twitch contractions and 5 kHz for tetanic contractions. Twitch or tetanic force was defined as the force that developed during stimulation. It was calculated as the difference between the maximum force during contraction and the force (or tension) measured 5 ms before the contraction (see Fig. 1A for tetanic force). A resting tension developed when muscles failed to fully relax between contractions during fatigue development. The development of resting tension was measured as the difference between the tension measured 5 ms before a contraction and the tension measured 5 ms before the first contraction of the fatigue period (Fig. 1). Time to peak during a twitch was calculated as the time interval between the time 5% of the maximum twitch force had developed and the time the maximum force was reached (this calculation was used to avoid differences related to the latent period). The half-relaxation time of a twitch was obtained from the time interval between the time to peak and the time force had decreased by 50% during relaxation. For the tetanus, the half-relaxation time was obtained from the time interval between the last stimulus of a train of pulses and the time force had decreased by 50%. The maximum rate of force development was obtained by measuring the slope of the force change over time at every 10 data points.


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Fig. 1.   Measurement of tetanic force and resting tension during fatigue. The traces are tetanic contractions before (A) and after 90 s (B) and 180 s (C) of stimulation to fatigue (soleus muscle from 1-yr-old Kir6.2-/- mouse). Tetanic force (upward arrows) was calculated as the difference in force between the maximum force during contraction and the force (or tension) measured 5 ms before the contraction (A, dashed line; B and C, dotted line). The development of resting tension (downward arrows) during fatigue was measured as the difference in tension measured 5 ms before a contraction (B and C, dotted line) and the tension measured 5 ms before the first contraction (A, dashed line).

Experimental Protocol

Two groups of mice were tested. The first group consisted of six 1-yr-old wild-type and six 1-yr-old Kir6.2-/- mice (3 males and 3 females in each case). Muscle length was adjusted, and the 8-V stimulation was tested for maximal tetanic force. Muscles were then allowed to equilibrate for 30 min. During that time, one tetanic contraction was elicited every 2 min. Fatigue was elicited with one tetanic contraction every second for 3 min. After fatigue, muscles were stimulated at 10, 20, 100, and 200 s; 5 min; and every 5 min thereafter until 30 min to measure the recovery of tetanic force. The second group consisted of 10 2- to 3-mo-old female wild-type and 10 2- to 3-mo-old female Kir6.2-/- mice. The tetanic force and the fatigue kinetics were measured in muscles from five wild-type and five Kir6.2-/- mice as described for the 1-yr-old mice. For the other five wild-type and five Kir6.2-/- mice, muscle length and stimulation were adjusted for maximal twitch force (this second approach was chosen to measure twitch force without a potentiation artifact caused by tetanic contractions). After a 30-min equilibrium period, a force-frequency curve (between 1 and 200 Hz) was measured (the duration of the train of pulses was increased to 500 ms for soleus muscle only for these measurements). Fatigue was then elicited with tetanic contractions as described for the other muscles.

Hematoxylin and Eosin Staining

The second EDL and soleus muscle of the same mice used for the contractile measurements were frozen in isopentane precooled in liquid nitrogen. Cross sections, 10 µm thick, were postfixed with 10% Formalin (pH 7.0) before being stained for 10 min in 4.7% (wt/vol) hematoxylin solution that contained 2% (vol/vol) glacial acetic acid. After the excess hematoxylin was washed off in water, cross sections were dipped in acidic 70% (vol/vol) alcohol and then in 0.2% (vol/vol) ammonia solution. Cross sections were counterstained in 4.3% (wt/vol) eosin-phloxine solution dissolved in 70% alcohol followed by a wash in 95% alcohol.

Statistical Analysis

ANOVA was used to determine significant differences. Two-way ANOVA were used to determine the significant differences for body and muscle weight, as well as the parameters of the twitch and tetanic contractions. Split-plot ANOVA designs were used for the fatigue measurements because muscles were tested at all levels of time during fatigue and recovery. 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 significant difference was used to locate the significant differences (24). The word "significant" refers only to a statistical difference (P < 0.05).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Weight and Contractile Characteristics

The body and muscle weight (Table 1), maximum force, time to peak, and half-relaxation time of the twitch (Table 2), as well as the force-frequency curve (data not shown) of EDL and soleus muscle of Kir6.2-/- mice, were not significantly different from those of wild-type mice. There was also no difference for the tetanic force, half-relaxation time, and maximum rate of force development between wild-type and Kir6.2-/- mice for the 2- to 3-mo-old group (Table 3).

                              
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Table 1.   Effect of KATP channel deficiency on body and muscle weight


                              
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Table 2.   Effect of KATP channel deficiency on the twitch contraction of 2- to 3-mo-old mice


                              
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Table 3.   Effect of KATP channel deficiency on the tetanic contraction

Significant differences were, however, observed for the parameters of the tetanic contraction for the 1-yr-old mice. First, the tetanic force and the maximum rate of force development were significantly less (24-40%) in the EDL and soleus muscle of 1-yr-old mice than 2- to 3-mo-old Kir6.2-/- mice. For the wild-type mice, the differences between the 2- to 3-mo-old and 1-yr-old groups were smaller (7-17%) and not significant. Second, the tetanic force and the maximum rate of force development of the EDL muscle of 1-yr-old Kir6.2-/- mice were also significantly less (21-23%) than those from age-matched wild-type mice. Third, the half-relaxation times of soleus muscle of 1-yr-old Kir6.2-/- mice were not different from those of 2- to 3-mo-old mice, whereas they were significantly slower for the 1-yr-old wild-type mice.

Cross sections of three EDL and two soleus muscles from 1-yr-old wild-type and Kir6.2-/- mice were stained with hematoxylin and eosin. There was no evidence of connective tissue infiltration or muscle fibers with central nuclei in either group of mice (data not shown). Thus the greater decrease in tetanic force in Kir6.2-/- muscle when mice got older was not associated with any apparent muscle damage.

Kinetics of Fatigue

Tetanic force. When EDL muscles from 2- to 3-mo-old wild-type mice were stimulated with one tetanic contraction every second, the tetanic force decreased to 26.7 and 8.8% of the prefatigue force after 30 and 60 s of stimulation, respectively (Fig. 2A). Thereafter, the tetanic force decreased very little and was 6.0% after 180 s of stimulation. For the EDL muscle, there was no significant difference in the rate of fatigue between wild-type and Kir6.2-/- mice at both 2-3 mo and 1 yr of age.


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Fig. 2.   Effect of ATP-sensitive K+ (KATP) deficiency on the tetanic force during fatigue in extensor digitorum longus (EDL; A) and soleus (B) muscle. Tetanic force is defined as the maximum force developed during a contraction and is expressed as a percentage of the tetanic force measured before fatigue. Tetanic contractions were elicited with a 200-ms-long train of pulses at 140 Hz for soleus and 200 Hz for EDL muscle. Fatigue was elicited with 1 tetanic contraction every second, but data are shown every 30 s for clarity. The experimental temperature was 37°C. open circle , 2- to 3-mo-old wild-type mice; , 2- to 3-mo-old Kir6.2-/- mice; , 1-yr-old wild-type mice; , 1-yr-old Kir6.2-/- mice. Vertical bars represent SE of 10 muscles for the 2- to 3-mo-old female mice and 6 muscles for the 1-yr-old mice (3 males and 3 females). *Significantly different mean tetanic force of muscle from wild-type and age-matched Kir6.2-/- mice; ANOVA, least significant difference (LSD), P < 0.05.

The soleus muscle had greater fatigue resistance than the EDL muscle. For example, the tetanic forces after 60 s of stimulation were 53.2% of the prefatigue force in soleus compared with 8.8% in EDL (2- to 3-mo-old wild-type mice). For the 1-yr-old group, the decreases in tetanic force during fatigue were the same in wild-type and Kir6.2-/- mice. For the 2- to 3-mo-old group, the decreases in tetanic force were less for the Kir6.2-/- mice than for wild-type soleus. However, the differences were observed only between the 60th and 120th min of stimulation and ranged only between 6.0 and 10.2%. Thus in most cases and most of the time, the KATP channel deficiency had no effect on the rate of fatigue in EDL and soleus muscle of 2- to 3-mo-old and 1-yr-old mice.

Resting tension. During fatigue development, muscles eventually failed to fully relax between contractions, and that gave rise to an increase in resting tension. The increase in resting tension was small and not significantly different from zero in EDL muscle of wild-type mice (Fig. 3A). In EDL muscle of Kir6.2-/- mice, the increase in resting tension occurred sooner and was significantly larger than in the muscle of wild-type mice. After 30 s of stimulation, the resting tensions of EDL muscle of 2- to 3-mo-old and 1-yr-old Kir6.2-/- mice were, respectively, 5.6 and 8.7% of the prefatigue tetanic force. At the end of the fatigue period, resting tensions were 8.4-8.8% for the Kir6.2-/- mice, compared with only 1.6-2.2% for the wild-type mice.


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Fig. 3.   Effect of KATP deficiency on the development of resting tension during fatigue in EDL (A) and soleus (B) muscle. Resting tension is defined as the force measured 5 ms before a tetanic stimulation and is expressed as a percentage of the tetanic force measured before fatigue. The experimental temperature was 37°C. Fatigue was elicited with 1 tetanic contraction every second, but data are shown every 30 s for clarity. open circle , 2- to 3-mo-old wild-type mice; , 2- to 3-mo-old Kir6.2-/- mice; , 1-yr-old wild-type mice; , 1-yr-old Kir6.2-/- mice. Vertical bars represent SE of 10 muscles for the 2- to 3-mo-old female mice and 6 muscles for the 1-yr-old mice (3 males and 3 females). *Significantly different mean resting tensions of muscle from Kir6.2-/- mice and age-matched wild-type mice; ANOVA, LSD, P < 0.05. §Times when resting tensions became significantly >0; ANOVA, LSD, P < 0.05. dagger Significantly different mean resting tensions of muscle from 1-yr-old mice and 2- to 3-mo-old mice; ANOVA, LSD, P < 0.05.

The increase in resting tension during fatigue was significantly greater in soleus than EDL muscle (Fig. 3B). For example, the resting tension after 180 s of stimulation was 10.7% of the prefatigue tetanic force in soleus muscle, compared with 2.2% in EDL muscle (2- to 3-mo-old wild-type mice). At 2-3 mo of age, the increase in resting tension was not significantly different between wild-type and Kir6.2-/- mice. For the wild-type mice, the development of resting tension during fatigue was significantly less in soleus muscle of 1-yr-old mice than 2- to 3-mo-old mice, whereas for the Kir6.2-/- mice, the increase in resting tension was greater in the older mice. So, at the age of 1 yr, soleus muscle of wild-type mice developed significantly less resting tension than the soleus muscle of Kir6.2-/- mice. Thus a KATP channel deficiency always resulted in greater development of resting tension during fatigue in EDL muscle, as well as in soleus muscle of older mice.

Recovery of tetanic force. After fatigue, tetanic force increased back toward its prefatigue levels. For the 2- to 3-mo-old mice, the recovery of tetanic force was not significantly different between wild-type and Kir6.2-/- EDL muscle (data not shown). For the 1-yr-old group, the recovery of tetanic force was significantly greater in EDL and soleus muscle of wild-type mice than Kir6.2-/- mice (Fig. 4). The difference between wild-type and Kir6.2-/- mice was especially large for soleus muscle, which was 20.5% after 5 min of recovery and 14.2% after 30 min.


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Fig. 4.   Effect of KATP deficiency on the recovery of tetanic force after fatigue in EDL (A) and soleus (B) muscle. Tetanic contractions were elicited with a 200-ms-long train of pulses at 140 Hz for soleus and 200 Hz for EDL muscle. Fatigue was elicited with 1 tetanic contraction every second for 3 min. The experimental temperature was 37°C. Tetanic force is expressed as a percentage of the tetanic force measured before fatigue. , 1-yr-old wild-type mice; , 1-yr-old Kir6.2-/- mice. Vertical bars represent SE of 6 muscles for 1-yr-old mice (3 males and 3 females). *Significantly different mean tetanic force of muscles from age-matched wild-type mice and Kir6.2-/- mice; ANOVA, LSD, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It has been hypothesized that the KATP channel plays an important role in protecting muscle against function impairment (18). In an attempt to further test this hypothesis, we compared the contractile and fatigue characteristics of EDL and soleus muscle between wild-type and Kir6.2-/- mice, the latter being KATP channel-deficient mice (17). Compared with muscles from wild-type mice, we found that KATP channel-deficient muscles had 1) less capacity to relax between contractions as they developed greater resting tension during fatigue; 2) smaller capacity to recover tetanic force after fatigue (1-yr-old mice only); and 3) greater loss of tetanic force when mice get older.

Comparison of the Effects of Glibenclamide With Those of a KATP Channel Deficiency on the Kinetics of Fatigue

Matar et al. (16) studied the role of the KATP channel during fatigue in EDL and soleus muscle of 2- to 3-mo-old mice with the use of glibenclamide, a KATP channel blocker. As described in the Introduction, the glibenclamide effects can be influenced by its efficiency to block the channels when the ADP concentration increases (27) and by its effect on the Ca2+ sensitivity of the contractile apparatus (7, 16). Although drug specificity and efficiency are not considered when KATP channel-deficient mice are used, these mice can develop compensations that mask the effect of no KATP channel in the cell membrane. To determine whether any compensation, drug specificity, and/or efficiency are important to consider, we can compare the effects of the KATP deficiency from the data from 2- to 3-mo-old mice obtained from this study with those of glibenclamide from Matar et al. (16).

First, neither glibenclamide nor a KATP channel deficiency affects the rate of fatigue [Fig. 2 in this study; Fig. 4 in Matar et al. (16)] and the capacity to recover tetanic force [for 2- to 3-mo-old mice, data not shown; Fig. 6 of Matar et al. (16)] after fatigue in EDL and soleus muscle. Second, the resting tension increases very little during fatigue in EDL muscle of wild-type mice, which was 1-2% of the prefatigue tetanic force after 180 s of stimulation [Fig. 3A in this study; Fig. 5A in Matar et al. (16)]. In the presence of glibenclamide, resting tension increases significantly to 4.6-4.9% within 60 s [Fig. 5A in Matar et al. (16)], which is comparable to the 7.8-8.4% values observed in Kir6.2-/- EDL muscle (Fig. 3A). Third, the difference in resting tensions between wild-type and Kir6.2-/- soleus muscle (2- to 3-mo-old group) was not significant throughout the fatigue stimulation (Fig. 3B), and the same was observed between control and glibenclamide-exposed soleus muscle [Fig. 5B of Matar et al. (16)].

Thus considering all these similarities, we suggest 1) the lack of a glibenclamide effect on the decrease in tetanic force during fatigue was not due to a decreased efficiency of glibenclamide to block KATP channels; 2) the glibenclamide effect on resting tension in EDL muscle is due to a blocking of KATP channels and not to an increase in Ca2+ sensitivity of the contractile apparatus; and 3) there is no evidence that the Kir6.2-/- mice compensate for the KATP channel deficiency (at least for the fatigue kinetics).

Role of the KATP Channel in Skeletal Muscle During Fatigue

As described in the introduction, the postulated mechanism of action of the KATP channel is to reduce force development to protect against irreversible function impairment. Our results do not support this mechanism, at least during muscle fatigue, because a lack of KATP channel in the cell membrane had no effect on the rate of fatigue in EDL muscle from mice of all ages and in soleus muscle from 1-yr-old mice. Although an effect was observed in soleus muscle of 2- to 3-mo-old Kir6.2-/- mice, the effect was small (<11%) and did not persist throughout the fatigue period. Our finding is also in agreement with other studies in which blocking the KATP channels with tolbutamide or glibenclamide had no effect on the rate of fatigue (5, 7, 15, 16, 26, 29). Matar et al. (16) suggested that the lack of an effect of KATP channels during fatigue was because not enough channels were activated. However, muscle fatigue constitutes an appreciable metabolic stress: it causes large decreases in tetanic force, exceeding 70% in soleus and 90% in EDL muscle (Fig. 2), as well as several changes in metabolite, such as a large depletion in phosphocreatine content, large increases in lactic acid, and decreases in intracellular pH (16, 19). Thus contrary to the hypothesis that in cardiac muscle KATP channels contribute to a decrease in force during metabolic stress (18), we now propose that in skeletal muscle KATP channels do not contribute to the decrease in force during fatigue development.

One consistent effect of the lack of KATP channel activity in the cell membrane is greater increases in resting tension during fatigue compared with control. This effect was observed in EDL muscle of both 2- to 3-mo-old and 1-yr-old mice and in soleus muscle of 1-yr-old mice (Fig. 3). Greater increases in resting tension were also observed when the channel was blocked with glibenclamide (16). The activity of KATP channels was not measured in this study because it is not possible to measure membrane potential with microelectrodes while muscles are contracting and because glibenclamide had very little effect on membrane potential and K+ efflux (16). However, considering the similarities in the effects of glibenclamide and KATP channel deficiency, we now suggest that the major function of the KATP channel during fatigue is to reduce the development of resting tension. The capacity of the KATP channel to modulate the resting tension during fatigue is further supported by the fact that an activation of the channel with pinacidil completely abolishes the development of resting tension in both EDL and soleus muscle (16).

There is evidence that the KATP channels in human suffering of hypokalemic periodic paralysis (HOPP) have abnormal characteristics (25), even though the mutation has been identified on the L-type calcium channel (13). Associated with the KATP channel abnormality is a tendency to have a depolarized cell membrane (20). Also, the cell membrane of frog sartorius muscle fibers does not depolarize during metabolic inhibition unless the KATP channels are blocked with glibenclamide. The depolarizations in the presence of glibenclamide exceeded 30 mV (9), which is large enough to activate L-type Ca2+ channels and to allow for an increase in Ca2+ influx and eventually the development of resting tension. It can then be suggested from these studies that KATP channels are important in maintaining resting potential by providing a hyperpolarizing current and that this effect is important to prevent a large development of resting tension.

Although such a mechanism may be important during metabolic inhibition, it remains to be determined whether it is also important during fatigue. Matar et al. (16) showed that blocking KATP channels only causes a 3-mV depolarization, which is too small to consider an activation of L-type Ca2+ channels unless the depolarization measured at the surface membrane with microelectrodes does not reflect the extent of the depolarization in T tubules. Thus further studies will be necessary to determine whether an effect of KATP channels on resting potential between contractions is the mechanism by which these channels affect resting tension.

A deficiency in KATP channels had no significant effect on the twitch and tetanic force (Tables 2 and 3), as well as on the recovery of force, after fatigue in 2- to 3-mo-old mice (data not shown). However, the situation was different for the muscles of 1-yr-old mice. First, the tetanic forces were significantly less in EDL and soleus muscles of 1-yr-old mice than in 2- to 3-mo-old Kir6.2-/- mice, whereas the differences in tetanic force were not significant in muscles of wild-type mice (Table 3). Second, the capacity to recover tetanic force after fatigue was greater in muscles from 1-yr-old wild-type than Kir6.2-/- mice (Fig. 4). These results suggest that a KATP deficiency causes some loss of muscle function with age. It is interesting to note that the effects of a KATP channel deficiency on muscle function was observed in mice that were mostly sedentary. In consideration of the effect of this deficiency on the resting tension during fatigue, it will therefore be interesting to determine in future studies whether the deterioration of muscle function is accentuated when Kir6.2-/- mice are subjected to chronic exercise.

As discussed above, HOPP patients have abnormal KATP channel characteristics (25). HOPP symptoms also include periodic paralyses and the apparition of central nuclei and vacuoles in affected muscle (21). So far, Kir6.2-/- mice have not shown any sign of periodic paralyses, and our histological studies on muscle from 1-yr-old mice did not reveal any vacuoles or centrally located nuclei (data not shown). Thus the HOPP symptoms do not appear to be solely due to the abnormality observed for the KATP channels.

In summary, this study provides evidence that the major function of the KATP channel is to reduce the increase in resting tension during fatigue and not to contribute to a decrease in force, as originally postulated. The study also shows that the capacity of skeletal muscle to generate force and to recover force after fatigue decreases with aging to a greater extent in KATP channel-deficient mice than in wild-type mice, which suggests that the KATP channel is important in protecting muscle function.


    ACKNOWLEDGEMENTS

This study was supported by a grant from the National Science and Engineering Research Council of Canada (to J. M. Renaud) and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan (to S. Seino).


    FOOTNOTES

Address for reprint requests and other correspondence: J. M. Renaud, Dept. of Cellular and Molecular Medicine, Univ. of Ottawa, 451 Smyth Rd., 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.

Received 12 March 2000; accepted in final form 24 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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