We investigated the role of limitations in aerobic metabolism, glycolysis, and membrane excitability for development of high-frequency fatigue in isolated rat soleus muscle. Muscles mounted on force transducers were incubated in buffer bubbled with 5% CO2 and either 95% O2 (oxygenated) or 95% N2 (anoxic) and stimulated at 60 Hz continuously for 30–120 s or intermittently for 120 s. Cyanide (2 mM) and 2-deoxyglucose (10 mM) were used to inhibit aerobic metabolism and both glycolysis and aerobic metabolism, respectively. Excitability was reduced by carbacholine (10 μM), a nicotinic ACh receptor agonist, or ouabain (10 μM), an Na+-K+ pump inhibitor. Membrane excitability was measured by recording M waves. Intracellular Na+ and K+ contents and membrane potentials were measured by flame photometry and microelectrodes, respectively. During 120 s of continuous stimulation, oxygenated and anoxic muscles showed the same force loss. In oxygenated muscles, cyanide did not alter force loss for up to 90 s, whereas 2-deoxyglucose increased force loss (by 19–69%; P < 0.01) from 14 s of stimulation. In oxygenated muscles, 60 s of stimulation reduced force, M wave area, and amplitude by 70–90% (P < 0.001). Carbacholine or ouabain increased intracellular Na+ content (P < 0.001), induced a 7- to 8-mV membrane depolarization (P < 0.001), and accelerated the rate of force loss (by 250–414%) during 30 s of stimulation (P < 0.001). Similar effects were seen with intermittent stimulation. In conclusion, limitations in glycolysis and subsequently also in aerobic metabolism, as well as membrane excitability but not aerobic metabolism alone, appear to play an important role in the development of high-frequency fatigue in isolated rat soleus muscle.
- Na+-K+ pumps
- skeletal muscle
high-frequency fatigue is induced by continuous, high-frequency stimulation and is characterized by a rapid decline in force during stimulation (half time 5–30 s) (37). The mechanisms contributing to the development of high-frequency fatigue are less clear but may involve ionic perturbations and subsequent loss of membrane excitability (4, 37).
In skeletal muscle, the binding of ACh to the nicotinic ACh receptors (nAChR) induces a membrane depolarization of sufficient magnitude to activate the voltage-gated Na+ channels. The resulting passive Na+ influx is associated with a membrane depolarization or the upward stroke of the action potential, whereas the opening of K+ channels and subsequent passive K+ efflux is associated with a membrane repolarization or the downward stroke of the action potential. These passive ion fluxes are counteracted by activity of the membrane-bound Na+-K+ pumps, which actively transport Na+ out of the cell and K+ back into the cell, allowing the maintenance of membrane excitability (4). However, during periods of frequent action potentials, the accelerated passive Na+ and K+ fluxes overwhelm the capacity of the Na+-K+ pumps, resulting in a net Na+ gain and net K+ loss and an ensuing loss of membrane excitability (8, 32). Repeated muscle contractions (960 stimuli given at 40 Hz) have been shown to increase intracellular Na+ concentration by ∼11 mM in mouse soleus muscle (20), and recent studies that used microdialysis probes inserted into human vastus lateralis muscle suggest that interstitial K+ concentration may reach values as high as 12–14 mM during intense exercise (34). Such large ionic perturbations induce a membrane depolarization (32), leading to slow inactivation of the voltage-gated Na+ channels (31) and a reduced ability to generate the action potentials necessary for repeated muscle contractions.
However, a recent study that used an updated model of A. V. Hill's equation describing oxygen diffusion into cylindrical muscles suggested that studies utilizing electrical stimulation of isolated intact mammalian skeletal muscles may be fundamentally flawed due to the development of central anoxia (2). This model predicts that, for experiments conducted at 30°C and with a contraction duty cycle of 0.25 (contraction duration/interval between the onset of two contractions), intact soleus muscles isolated from 4-wk-old rats may develop central anoxia within 30 s of stimulation (2). Therefore, for experiments involving continuous stimulation, muscles may have developed central anoxia already within the first few seconds of stimulation. Indeed, a recent study showed that, whereas intact muscles fatigued five times faster than single fibers isolated from mouse soleus muscles during intermittent (0.3 contraction duty cycle) high-frequency stimulation, fatigue rates of intact muscles and single fibers were the same when aerobic metabolism was inhibited by cyanide (40). Together, these results suggest that the interpretation of results from experiments using intact muscles and stimulation protocols exceeding this contraction duty cycle and/or stimulation duration may be compromised by the development of central anoxia.
The primary aim of this study was therefore to investigate the role of limitations in aerobic metabolism, glycolysis, and membrane excitability for the development of high-frequency fatigue in skeletal muscle. Here, we provide evidence that limitations in glycolysis and subsequently also in aerobic metabolism, as well as membrane excitability, but not in aerobic metabolism alone, are important for the induction of high-frequency fatigue (induced within 90 s of stimulation) in isolated rat soleus muscle. Part of the data in this paper have been presented in preliminary form (26).
Animals and preparation of muscles.
Experiments were carried out with 4-wk-old Wistar rats, weighing 60–70 g. Rats of this age were used because the relatively small size of their muscles (∼20–25 mg) minimizes the diffusional barriers to substrates, ions, and oxygen to the cell surface. Some experiments were also carried out with 12-wk-old Wistar rats weighing 230–250 g. All animals were fed ad libitum and were maintained in a temperature-controlled environment (21°C) with constant day length (12 h). All handling and use of animals complied with Danish animal welfare regulations. The animals were killed by cervical dislocation, followed by decapitation, with intact soleus muscles (predominantly slow-twitch fibers) dissected out as previously described (27).
Muscles were equilibrated for 30 min at 30°C in standard Krebs-Ringer (KR) bicarbonate buffer (pH 7.4) containing the following (in mM): 122.1 NaCl, 25.1 NaHCO3, 2.8 KCl, 1.2 KH2PO4, 1.2 MgSO2, 1.3 CaCl2, and 5.0 d-glucose. Samples were bubbled continuously with a mixture of 5% CO2 and 95% O2 or 95% N2, depending on the experiment.
Muscles were mounted vertically with their tendons intact on electrodes for isometric contractions in thermostatically controlled chambers. Isometric force development was measured with a force displacement transducer (Grass FTO3Fig. 1); therefore, a stimulation of 2 s was used to ensure the development of peak absolute tetanic force. Incubation with carbacholine or ouabain did not alter the time course at which maximum force was obtained. After incubation in KR buffer bubbled with 95% O2 (oxygenated) or with 95% N2 (anoxia), without or with carbacholine, tubocurarine, ouabain, cyanide, or 2-deoxyglucose, as described below, muscles were either checked again for tetanic force or were stimulated continuously for between 30 and 120 s at 60 Hz (0.2 ms, 12 V). The stimulation frequency of 60 Hz was chosen to induce high-frequency fatigue because this is the frequency at which maximum tetanic force is achieved in the soleus muscle of adult rats in vivo (18). Figure 1 demonstrates that, in isolated soleus from 4-wk-old rats, tetanic fusion occurred at 60 Hz but was incomplete at 30-Hz stimulation and that tetanic force was not elevated by increasing stimulation frequency from 60 to 80 Hz. These results indicate that, similar to results shown in adult rats, maximum tetanic force in 4-wk-old rats is also obtained at 60 Hz at 30°C. For experiments for measurement of M waves, it was necessary to use a shorter pulse duration (0.02 ms) to minimize the interference from stimulus artifacts. This shorter pulse duration caused no significant reduction in peak tetanic force (0.02 ms, 0.33 ± 0.03 N; 0.2 ms, 0.35 ± 0.02 N; P = 0.61, n = 8–12) compared with those stimulated with a pulse duration of 0.2 ms. Force was measured by force displacement transducers and recorded with a chart recorder and/or digitally on a computer, with results expressed in absolute terms (N) or in relative terms as a percentage of the initial force produced (fatiguing stimulation experiments).
Additional experiments were conducted to investigate the role of limitations in aerobic metabolism alone, glycolysis as well as aerobic metabolism, and membrane excitability in the development of skeletal muscle fatigue during high-frequency intermittent stimulation. After equilibration, muscles were stimulated intermittently (0.5 s on, 1.5 s off; 0.25 contraction duty cycle) for 120 s at 60 Hz (0.2 ms, 12 V).
To investigate the effects of oxygenation on contractile endurance, muscles were stimulated as described above in each of the following oxygenation conditions: oxygenated (bubbled with 95% O2-5% CO2) or anoxic (bubbled with 95% N2-5% CO2). All muscles were initially equilibrated for 30 min in standard KR buffer bubbled with 95% O2-5% CO2. Muscles were then incubated in buffer that had been pregassed for 30 min with either 95% O2 and 5% CO2 (oxygenated) or 95% N2 and 5% CO2 (anoxic). The buffer was then bubbled continuously with the same gas mixture; after a 5-min rest to ensure maximum diffusion of gases, muscles were electrically stimulated.
Inhibition of aerobic metabolism with cyanide.
Muscles were incubated for 5 min in KR buffer without (control) or with 2 mM cyanide, to inhibit mitochondrial respiration (21, 22, 28, 38). As discussed by Zhang et al. (39), the inhibition constant value for cyanide-induced inhibition of oxygen consumption is ∼8–10 μM in isolated mouse quadriceps muscle (22, 38) and 5–10 μM in human skeletal muscle (21). Furthermore, oxygen consumption is almost completely inhibited (∼2–10% of control) with 35–40 μM cyanide (21, 22) and is completely inhibited with 1 mM cyanide (28). Together, these results suggest that cyanide at a concentration of 2 mM should be sufficient to fully inhibit aerobic metabolism in the present preparation. In the cyanide experiments, the buffer was bubbled continuously with 95% O2-5% CO2.
Inhibition of glycolysis with 2-deoxyglucose.
Muscles were incubated in glucose-free KR buffer with the glucose analog 2-deoxyglucose (10 mM). 2-Deoxyglucose is phosphorylated by hexokinase to 2-deoxyglucose-6-phosphate but inhibits glycolysis by not being metabolized any further (16) and by inhibiting glycogen phosphorylase (11). This inhibition of glycolysis will therefore also lead to an inhibition of aerobic metabolism. The uptake of 2-deoxyglucose in skeletal muscle is quite slow, requiring several hours (16), and is increased by insulin (15, 16). Therefore, to ensure that sufficient uptake of 2-deoxyglucose had occurred to inhibit glycolysis during the fatiguing stimulation protocol, muscles were incubated with 2-deoxyglucose without or with insulin (10 mU/ml), stimulated for 2 s every 10 min for 120 min, and then stimulated continuously or intermittently for 120 s at 60 Hz. Importantly, there was no effect of insulin alone on force production during 120 s of continuous 60-Hz stimulation, with a pilot study demonstrating no significant difference in the force production at any time point between muscles preincubated for 120 min in glucose-free KR buffer without or with insulin (10 mU/ml) (n = 4, all P > 0.05, data not shown). In these experiments, buffer was bubbled continuously with 95% O2-5% CO2.
Measurement of M waves.
Muscle excitability was determined by recording compound action potentials (M waves) in oxygenated muscles. These recordings were made by inserting a polyimide-insulated tungsten electrode (TM33B01, World Precision Instruments, Sarasota, FL) into the muscle between the innervation zone and the tendon, and a reference electrode (Ag/AgCl) was placed in the standard KR buffer at a distance of a few centimeters from the muscle. Signals were led through a low-noise head stage, processed by a DAM 70 differential amplifier (World Precision Instruments) and recorded digitally on a computer. Indirect stimulation was performed via the nerve endings using pulses of 0.02-ms duration. During fatiguing stimulation, M waves were analyzed every 5 s; at each of these time points, the area and amplitude of five successive M waves were averaged. 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 in studies from this laboratory (30). M wave duration was defined as the interval between the start and end of the negative trace relative to the baseline. The mean coefficient of variation in these successive M wave recordings was 4.7 ± 0.4% (n = 138), 3.9 ± 0.3% (n = 138), and 5.1 ± 0.7% (n = 138) for area, amplitude, and duration, respectively. The mean initial (at 1 s of stimulation) force, M wave area, amplitude, and duration were 0.32 ± 0.02 N, 0.035 ± 0.007 mV/s, 4.5 ± 0.6 mV, and 1.35 ± 0.14 ms, respectively (all n = 6).
Manipulation of passive and active Na+ and K+ fluxes.
To elevate passive Na+ and K+ fluxes, oxygenated muscles were incubated for 15 min in KR buffer with 10 μM carbacholine, to activate the nAChR (19). We have previously shown that carbacholine increases Na+ influx in rat soleus muscle (23). Preincubation for 15 min with tubocurarine (10 μM) was used to block the nAChR (19). To reduce active Na+-K+ transport, muscles were preincubated for 30 min in KR buffer without (control) or with 10 μM ouabain. At 10 μM ouabain, we have shown that, after 30 min, ∼80% of Na+-K+ pumps in rat soleus muscle have bound ouabain (6). Some experiments were also carried out to investigate the effect of ouabain on force production in soleus muscles obtained from adult 12-wk-old animals. In these experiments, muscles were preincubated for 15 min in KR buffer without (control) or with 10 μM ouabain.
Measurement of intracellular Na+ and K+ contents.
After incubation, oxygenated muscles were immediately transferred to ice-cold Na+-free Tris-sucrose buffer and underwent a 4 × 15-min washout to remove extracellular Na+. After washout, muscles were blotted, tendons were cut off, muscle wet weight was determined, and the muscles were soaked overnight in 0.3 M TCA to give complete extraction of ions from the tissue (5). The Na+ and K+ contents in the TCA extract was measured by flame photometry (FLM3, Radiometer, Copenhagen, Denmark) with lithium as internal standard. Values for Na+ content were then multiplied by 1.46 to correct for the loss of intracellular Na+ during the ice-cold washout (12). In contrast, the loss of K+ during the washout was minimal (12).
Measurement of resting membrane potential.
The resting membrane potential was measured by use of standard electrophysiological techniques, as described previously (24). Briefly, a microelectrode was filled with 3 M KCl (18–20 MΩ) and placed in an individual fiber in the middle third region of the oxygenated muscle. For each measurement of membrane potential in a muscle, 10 insertions were made over a 5- to 10-min period. The mean coefficient of variation of these 10 insertions was 4.5 ± 0.6% (n = 24). To avoid measurement of the same muscle fiber, the electrode was moved a small distance across the muscle between each of the 10 insertions.
All chemicals were of analytical grade. Sodium cyanide, 2-deoxyglucose, carbamylcholine chloride (carbacholine), tubocurarine, and ouabain were purchased from Sigma (St. Louis, MO). Insulin (porcine) was a gift from Novo-Nordisk (Copenhagen, Denmark).
All data are presented as means ± SE. The statistical significance of the difference between the force decline of two or more groups was analyzed by two-way ANOVA with repeated measures on both factors (time, treatment). The statistical significances of the differences between force, intracellular Na+ and K+ contents, resting membrane potential, and stimulation times for force, M wave area, and amplitude were analyzed with a one-way ANOVA. Differences were located with a Student-Newman Keuls post hoc test. Correlations were determined by least squares linear regression. Significance was accepted at P < 0.05.
Comparison of peak absolute tetanic force and of endurance between oxygenated and anoxic muscles.
To assess the importance of oxygenation for the development of high-frequency fatigue, the force produced during 120 s of 60-Hz stimulation by oxygenated muscles was compared with that of anoxic muscles. Before this continuous stimulation, there was no significant difference in peak absolute tetanic force between oxygenated and anoxic muscles (P = 0.15; Table 1). During 120 s of continuous 60-Hz stimulation, there was no significant difference in the force production at any time point between oxygenated and anoxic muscles (Fig. 2).
Effect of cyanide on peak absolute tetanic force and on endurance in oxygenated muscles.
Cyanide was used to inhibit aerobic metabolism in oxygenated muscles to assess the importance of aerobic metabolism for fatigue induced during 120 s of continuous 60-Hz electrical stimulation. Before continuous stimulation, peak absolute tetanic force showed no significant difference between those incubated without or with cyanide (5 min at 2 mM) (control, 0.34 ± 0.04 N; with cyanide, 0.37 ± 0.04 N, n = 7–9; P = 0.54). During the first 90 s of continuous 60-Hz stimulation, there was no significant effect of cyanide on the force production compared with controls (Fig. 3). However, after 95 s of stimulation, muscles incubated with cyanide produced significantly less force than controls (11–14%; all P < 0.05; Fig. 3).
Effect of 2-deoxyglucose on peak absolute tetanic force and on endurance in oxygenated muscles.
2-Deoxyglucose was used to assess the importance of inhibition of glycolysis and therefore also aerobic metabolism for fatigue induced during 120 s of continuous 60-Hz electrical stimulation in oxygenated muscles. Before continuous stimulation, peak absolute tetanic force showed no significant difference between those incubated in glucose-free KR buffer with 2-deoxyglucose (10 mM) and without or with insulin (10 mU/ml) (2-deoxyglucose, 0.34 ± 0.04 N; 2-deoxyglucose + insulin, 0.39 ± 0.01 N; n = 4–6; P = 0.53). Preincubation with insulin (Fig. 4) was used to increase the uptake of 2-deoxyglucose to reach an accumulation of 2-deoxyglucose-6-phosphate sufficient to inhibit glycolysis during the fatiguing stimulation protocol (120 s of continuous 60-Hz stimulation). Muscles incubated with 2-deoxyglucose + insulin produced significantly less force from 14 s of fatiguing stimulation than those incubated with 2-deoxyglucose alone (by 19–69%; all P < 0.01; Fig. 4).
Effect of 60 s of 60-Hz stimulation on force, M wave area, and M wave amplitude in oxygenated muscles.
M waves, a measurement of compound action potentials, were recorded to assess whether loss of membrane excitability was a physiological phenomenon occurring with high-frequency fatigue induced by 60 s of continuous 60-Hz electrical stimulation. The pulse duration was reduced to 0.02 ms to achieve indirect stimulation via the nerve endings. Only 60 s of stimulation was used because M waves became too small to be accurately measured after this duration. This is demonstrated in Fig. 5, which shows representative traces of the force response to this stimulation paradigm, as well as representative M waves at the beginning (Fig. 5a) and end of stimulation (Fig. 5b). The force production was significantly reduced after 15 s of stimulation (P < 0.04) and at 30 and 60 s of stimulation and was 23% (P < 0.001) and 69% lower than initial force (P < 0.001), respectively (Fig. 5). Thus a faster force decline was found when muscles were stimulated with a pulse duration of 0.02 ms (Fig. 5) compared with 0.2 ms, as shown in Fig. 2. This discrepancy most likely reflects the involvement of neuromuscular fatigue, which would be induced with a pulse duration of 0.02 ms but not 0.2 ms (which directly activates the voltage-gated Na+ channels). M wave amplitude was significantly reduced at 25 s of stimulation (P < 0.04) and was 64% (P < 0.001) and 92% (P < 0.001) lower than initial values at 30 and 60 s of stimulation, respectively (Fig. 5). After a slight nonsignificant initial potentiation, M wave area was significantly reduced at 30 s of stimulation (35% lower than initial; P < 0.001) and at 60 s of stimulation was 86% lower than initial values (P < 0.001; Fig. 5). M wave duration was significantly increased at 16 s of stimulation (P < 0.01), peaked at 30 s of stimulation (168% longer than initial; P < 0.001), and was 138% longer than initial at 60 s of stimulation (P < 0.001, data not shown). Thus changes in M wave area reflect changes in both M wave amplitude and duration.
Linear regression analysis was performed to assess the correlation between force and both M wave amplitude and area during 60 s of continuous 60-Hz electrical stimulation. A significant positive correlation was found between relative force (%initial) and relative M wave amplitude (%initial) (y = 1.6x + 46.7, where y = force and x = M wave; n = 13; r2 = 0.92, P < 0.001) and relative M wave area (%initial) (y = 1.4x + 48.1; n = 13; r2 = 0.90, P < 0.001).
Effect of carbacholine and ouabain on endurance in oxygenated muscles.
The effect of experimentally increasing passive Na+-K+ fluxes and of reducing active Na+-K+ transport on force production during 30 s of continuous 60-Hz electrical stimulation was investigated using carbacholine (15 min at 10 μM) and ouabain (30 min at 10 μM, see ⇓Fig. 7), respectively, with representative traces shown in Fig. 6. Only 30 s of stimulation was used, since the above results demonstrate that losses of membrane excitability but not limitations in aerobic metabolism limit the development of high-frequency fatigue during this period. Furthermore, this stimulation period is in line with the time course of the development of high-frequency fatigue, occurring with a stimulation half time of 5–30 s (37). Before continuous stimulation, there was no significant change in peak absolute tetanic force with either carbacholine (P = 0.17), carbacholine with tubocurarine (P = 0.90), or ouabain (P = 0.16, Table 1).
During continuous 60-Hz stimulation, carbacholine significantly reduced force production from 10 s of stimulation, compared with controls (P < 0.04; Fig. 7A). At the end of stimulation (30 s), the force production in muscles incubated with carbacholine was 26% lower than that of control muscles (P < 0.001), and the average rate of force decline was 250% greater with carbacholine (P < 0.0001; Fig. 7A). The reduction in force production induced by carbacholine was completely abolished by preincubation with the nAChR blocker tubocurarine (10 μM; Fig. 7A). Incubation with ouabain significantly reduced force production from 5 s of stimulation (P < 0.04), with force production at the end of stimulation being 66% lower than controls (P < 0.001; Fig. 7B). The average rate of force decline was 414% greater with ouabain (P < 0.0001; Fig. 7B). Importantly, the inhibitory effects of carbacholine and ouabain on peak absolute tetanic force were completely reversed when muscles were returned to normal KR buffer for 60 min after the cessation of fatiguing stimulation (control, 98.4 ± 3.5; with carbacholine, 98.3 ± 2.7; and with ouabain, 99.0 ± 6.5% of initial force; n = 4–5; P = 0.98).
Other experiments were carried out on soleus muscle from 12-wk-old rats to investigate whether the inhibitory effects of ouabain on force production were also seen in mature rats. Before continuous stimulation, there was no significant difference in peak absolute tetanic force between muscles incubated without or with ouabain (control, 0.59 ± 0.12 N; with ouabain, 0.62 ± 0.13 N; n = 6–8; P = 0.86). During 120 s of continuous 60-Hz stimulation, ouabain significantly reduced force production from 16 to 110 s of fatiguing stimulation, compared with controls (by 13–60%; all P < 0.05; data not shown). The average rate of force decline was greater with ouabain from 8 to 45 s of stimulation (by 90–442%; all P < 0.05; data not shown).
Effects of carbacholine, tubocurarine, and ouabain on intracellular Na+ and K+ contents and on resting membrane potential in oxygenated muscles.
To assess whether the inhibitory effects of carbacholine and ouabain on force production during fatiguing stimulation involved altered Na+ and K+ fluxes leading to a loss of membrane excitability, the effects of these compounds on the intracellular contents of Na+ and K+ and on resting membrane potential were examined. Because of the neural depolarization induced by ouabain and the effects of carbacholine on the motor end plate, we were unable to stimulate via the nerve and, as such, unable to record M waves for these muscles.
Carbacholine (15 min at 10 μM) increased intracellular Na+ content by 21% (P < 0.001; Fig. 8A), caused no significant change in intracellular K+ content (P = 0.36; Fig. 8B), and induced a 7-mV membrane depolarization (P < 0.001; Fig. 9). The increase in intracellular Na+ content induced by carbacholine was completely blocked by preincubation with tubocurarine (15 min at 10 μM; P = 0.09 vs. control; Fig. 8A), which also increased intracellular K+ content by 9% (P < 0.04; Fig. 8B) and induced a small (2 mV) but significant membrane hyperpolarization, compared with control muscles (P < 0.04; Fig. 9). The preincubation with tubocurarine as such caused no decrease in force, indicating that the stimulation of the muscle fibers was direct. Incubation with ouabain (30 min at 10 μM) increased intracellular Na+ content by 107% (P < 0.001; Fig. 8A), reduced intracellular K+ content by 14% (P < 0.001; Fig. 8B), and induced a 8-mV membrane depolarization (P < 0.001, Fig. 9).
Effect of carbacholine and ouabain on peak absolute tetanic force and on endurance in anoxic muscles.
The effects of carbacholine and ouabain on the force production during fatiguing stimulation in anoxic muscles were investigated to assess whether the inhibitory effects of carbacholine and ouabain on force in oxygenated muscles were dependent on oxygenation. In anoxic muscles, there was no significant effect of either carbacholine (P = 0.15) or tubocurarine and carbacholine on peak absolute tetanic force before fatiguing stimulation (P = 0.15), whereas ouabain reduced peak absolute tetanic force by 26% compared with controls (P < 0.01; Table 1). During continuous 60-Hz electrical stimulation, carbacholine significantly reduced force production after only 4 s of stimulation (P < 0.01), with the force production at the end of stimulation being 57% lower than controls (P < 0.001) and the average rate of force decline being 377% greater with carbacholine (P < 0.0001; Fig. 10A). This effect of carbacholine was completely blocked by preincubation with tubocurarine (Fig. 10A). Anoxic muscles incubated with ouabain demonstrated a significantly lower force production than controls after only 5 s of stimulation (P < 0.04), with the force production at the end of stimulation being 72% lower than controls (P < 0.001; Fig. 10B). The average rate of force decline was 329% greater with ouabain (P < 0.0001; Fig. 10B).
High-frequency intermittent stimulation.
Additional experiments were conducted to investigate the effects of each of anoxia, cyanide, 2-deoxyglucose, carbacholine, and ouabain on fatigue induced during 120 s of high-frequency (60-Hz) intermittent (0.5 s on, 1.5 s off) stimulation. Thus the intermittent stimulation protocol elicited the same number of action potentials (1,800) as 30 s of continuous stimulation. For control muscles, force production at the end of stimulation was 7% (P = 0.02) lower with 120 s of intermittent stimulation (Fig. 11) than with 30 s of continuous stimulation (Fig. 7).
As shown in Fig. 11A, there was no significant difference in the force production at any point during intermittent stimulation between oxygenated and anoxic muscles. Similarly, inhibition of aerobic metabolism with cyanide (5 min at 2 mM) had no significant effect on force production compared with controls (Fig. 11B). In contrast, when the uptake of 2-deoxyglucose was stimulated with insulin (10 mU/ml) to inhibit glycolysis and subsequently also aerobic metabolism, force production was significantly smaller than that shown when muscles were incubated with 2-deoxyglucose alone from 28 to 120 s of stimulation (by 27–39%; P < 0.05 to P < 0.001; Fig. 11C). Increasing passive Na+-K+ fluxes using carbacholine (15 min at 10 μM) and reducing active Na+-K+ transport using ouabain (30 min at 10 μM) reduced force production compared with controls, from 92 (by 12–15%; P < 0.05 to P < 0.01) and 46 s of stimulation, respectively (by 11–19%; P < 0.05 to P < 0.001; Fig. 11D). The average rate of force decline compared with controls was 160–195% (all P < 0.001) and 181–233% (all P < 0.001) greater with carbacholine and ouabain, respectively (Fig. 11D). Force production at the end of stimulation for carbacholine-treated muscles was not significantly different between muscles stimulated intermittently (65% of initial force; Fig. 11D) or continuously (66% of initial force; Fig. 7; P = 0.92). In contrast, force production at the end of stimulation for ouabain-treated muscles was 33% (P < 0.0001) greater with intermittent (Fig. 11D) than with continuous stimulation (Fig. 7).
The present study finds that, in intact soleus muscles isolated from 4-wk-old rats, inhibition of glycolysis and subsequently also aerobic metabolism and alterations in Na+ and K+ homeostasis, leading to a loss of membrane excitability, are important for the development of fatigue induced by high-frequency electrical stimulation. In contrast, the development of this high-frequency fatigue does not appear to involve significant limitations in aerobic metabolism alone within 90 s.
Loss of membrane excitability accelerates force loss during 30 s of continuous high-frequency stimulation in isolated rat soleus muscle.
A loss of membrane excitability is a physiological phenomenon occurring during high-frequency electrical stimulation in isolated rat soleus muscle, with this and earlier studies showing that the loss of force elicited by continuous 60-Hz stimulation is associated with progressive declines in M wave area and amplitude (8). Importantly, significant correlations were found between this loss of force and the loss of M wave amplitude and area. Furthermore, incubation with carbacholine, an nAChR agonist, or ouabain, an inhibitor of the Na+-K+ pump, markedly accelerates the decline in force production during 30 s of 60-Hz stimulation. These results are in keeping with an earlier study where aconitine, an activator of the voltage-gated Na+ channels, and ouabain each accelerated the decline in force during continuous 90-Hz stimulation in rat soleus muscle (17).
The inhibitory effect of carbacholine on muscle function is not due to blockade of the neuromuscular junction, since muscles are stimulated with pulses of 0.2 ms to directly activate the voltage-gated Na+ channels. Furthermore, preincubation with the nAChR antagonist tubocurarine, which would also cause neuromuscular junction blockade, produces no inhibition of force but completely prevents the inhibitory effects of carbacholine. Importantly, peak absolute tetanic force is not reduced by exposure to either carbacholine (15 min) or ouabain (30 min) in oxygenated muscles. With carbacholine (10 μM), it takes 20–30 min to reach steady-state force levels (23); with ouabain (10 μM), it takes 60 min to reach complete occupancy of ouabain binding sites in rat soleus muscle (6). However, incubation durations of 15 and 30 min were used here for carbacholine and ouabain, respectively, to minimize any reduction in peak absolute tetanic force before fatiguing stimulation because a large reduction in absolute force may compromise the results obtained during stimulation. Furthermore, it is unlikely that the diffusion of these compounds would change significantly during the 30 s of continuous stimulation. The inhibitory effects of carbacholine and ouabain most likely reflect the resulting increase in passive Na+-K+ fluxes and reduction in active Na+-K+ transport, respectively. Indeed, both carbacholine and ouabain altered the intracellular contents of Na+ and K+, resulting in a significant membrane depolarization. It is expected that, given their diffusional limitations, the effects of changes in Na+ and K+ fluxes on intraluminal Na+ and K+ concentrations would be amplified in the T tubules, resulting in an even larger membrane depolarization (4, 32). In keeping with this, the considerably faster force loss during high-frequency stimulation in the extensor digitorum longus vs. in the soleus muscle (8, 40) is associated with larger passive Na+ and K+ fluxes and a greater loss of membrane excitability (8). Because of neural depolarization induced by ouabain and the effects of carbacholine on the motor end plate, we were unable to stimulate via the nerve endings and, as such, record M waves for these muscles. Nonetheless, the membrane depolarization induced by these compounds would induce slow inactivation of the voltage-gated Na+ channels (31), leading to a reduced ability to generate the action potentials required for sarcoplasmic reticulum Ca2+ release and hence contractile activity and, as such, to the development of muscle fatigue. Although these results are found in muscles taken from younger (4-wk-old) animals, the important role that a loss of membrane excitability appears to play for the development of muscle fatigue is also expected in mature animals, since inhibitory effects of ouabain on force production are also seen in older (12-wk-old) animals.
Although the inhibitory effects of carbacholine and ouabain on force decline are quite pronounced, they are completely reversible, indicating that they are not due to cellular damage. Importantly, the inhibitory effects of carbacholine and ouabain are seen in both oxygenated and anoxic muscles. The rate of force loss induced with carbacholine is faster in anoxic muscles than in oxygenated muscles. This is probably because of inhibition of carbacholine-induced Na+-K+ pump stimulation (23) as a consequence of the reduction in ATP availability in anoxic muscles. Together, these results suggest that, for up to 90 s, limitations in oxygenation are not important factors for fatigue induced by high-frequency stimulation in isolated rat soleus muscle. Moreover, the accelerations of force decline with carbacholine and ouabain do not reflect a faster development of central anoxia. Indeed, if oxygen consumption were limiting for muscle function, ouabain would be expected to delay, rather than accelerate, force decline during stimulation because ouabain reduced oxygen consumption during electrical stimulation in mouse diaphragm muscle (28).
Limitations in aerobic metabolism do not contribute to force loss during 30 s of continuous high-frequency stimulation in isolated rat soleus muscle.
Muscles incubated in buffer bubbled with nitrogen (anoxic) would intuitively have impaired oxygen supply. However, there was no difference in the force production during 120 s of continuous 60-Hz stimulation between oxygenated and anoxic muscles. In support of these findings, using cyanide to block aerobic metabolism (21, 22, 28, 38), we found no effect on the force production during the first 90 s of continuous 60-Hz stimulation, although thereafter force was reduced compared with controls. Importantly, because 1 mM cyanide completely inhibited oxygen consumption in mouse diaphragm muscle (28), cyanide at a concentration of 2 mM should be sufficient to fully inhibit aerobic metabolism in the present preparation (39). Because anoxia and cyanide, by suppressing energy supplies, might be expected to impair active Na+-K+ transport, these observations compared with those with ouabain are surprising. However, neither the replacement of oxygen with nitrogen nor the addition of cyanide (2 mM) alters Na+-K+ pump activity in the isolated soleus muscle from 4-wk-old rats, as demonstrated by an unchanged rate coefficient of 22Na efflux (7). Together, these results indicate that, during continuous high-frequency stimulation of isolated rat soleus muscle, aerobically derived ATP is not limiting for contractile endurance during the first ∼90 s of stimulation. In contrast, ATP supplied via glycolysis and subsequently also aerobic metabolism (inhibited with 2-deoxyglucose) appears to be limiting for contractile endurance. This effect may involve insufficient ATP supply for Na+-K+ pump activity. Indeed, glycolysis has been shown to preferentially provide ATP for the Na+-K+ pump (14, 29), and, as such, when ATP supply is inhibited with 2-deoxyglucose, Na+-K+ pump activity is reduced (14). This would induce a membrane depolarization that, as described here, would lead to the loss of contractile endurance that is seen with 2-deoxyglucose.
In contrast to these findings, a recent study found evidence for a limiting role of aerobic metabolism for muscle function during brief, fatiguing stimulation in intact mouse soleus muscle (40). Incubation with cyanide significantly reduced force production after only 20 s of intermittent (0.3 contraction duty cycle) stimulation (40). Furthermore, whereas the rate of force decline during stimulation was five times greater in intact muscles than in single fibers isolated from mouse soleus, fatigue rates were the same when both preparations were incubated with cyanide (40). Therefore, in contrast to the present findings in rat soleus muscle, these results suggest that aerobic metabolism is important for fatigue and that oxygen diffusion may be rate limiting during brief, intermittent high-frequency electrical stimulation in intact mouse skeletal muscle. This difference between studies may reflect the two types of stimulations used: continuous vs. intermittent stimulation. Studies in both humans and rodents have reported a 24–42% greater energy expenditure (ATP utilization/work) during intermittent than during continuous contractions (3, 9, 33). The greater ATP utilization during intermittent contractions most likely reflects the enhanced Ca2+-ATPase activity necessary for relaxation and also the increased actomyosin ATPase activity associated with each contraction. As such, aerobic metabolism may be more important during intermittent than continuous contractions and may in part explain the effect of cyanide being inhibitory for muscle function during intermittent (40) but not during continuous high-frequency stimulation, as found in the present study. Because mouse muscles have a higher energy turnover than rat muscles (13), the rundown of ATP content may also be faster. Furthermore, the difference between the results may reflect differences in fiber-type composition between the rat and mouse, with mouse soleus muscle containing more fast-twitch oxidative fibers than the rat soleus muscle (10, 35).
High-frequency intermittent stimulation.
The conclusions made using continuous high-frequency stimulation in the present study are relevant for work involving static, intense contractions where maximal contractions are sustained without blood circulation. However, intermittent high-frequency stimulation is more representative of the physiological working patterns observed in vivo. Similar to continuous stimulation, we find that the development of fatigue in rat soleus muscle during high-frequency intermittent (0.25 contraction duty cycle) electrical stimulation appears to depend on limitations in glycolysis and subsequently also in aerobic metabolism, as well as membrane excitability, but not on aerobic metabolism. Interestingly, whereas muscles incubated with carbacholine show the same force production at the end of stimulation with intermittent and continuous stimulation, with both stimulation protocols eliciting the same number of action potentials, muscles incubated with ouabain show greater force production with intermittent stimulation. The reasons for this discrepancy are unknown but require further investigation.
A recent study that used an updated model of A. V. Hill's equation describing oxygen diffusion into cylindrical mammalian muscles concluded that intact rat skeletal muscles produce central anoxia during almost any level of steady activity and, as such, would compromise the interpretation of the results (2). To circumvent such a problem, the author suggested that intact muscles should only be used when temperature and contraction duty cycles are low (2). However, results from the present study demonstrate that, in intact soleus muscles from 4-wk-old rats, total cessation of oxygen supply does not compromise the maintenance of contractile force during 90 s of stimulation at 30°C. This indicates that diffusion of O2 from the buffer into the muscles is not important for the maintenance of force during this period. Thus a discrepancy exists between experimental data and those obtained via modeling. This may be explained by the assumptions that are required for the modeling process, with perhaps the most pertinent being that oxidative processes were the sole mechanisms of regenerating high-energy phosphates (2). In fact, during the first 30 s of high-intensity contractile activity, high-energy phosphates are predominantly synthesized via anaerobic processes in human muscle (1, 25, 36).
In conclusion, alterations in trans-sarcolemmal Na+ and K+ gradients leading to a loss of membrane excitability appear to play an important role in the development of high-frequency fatigue in isolated rat soleus muscle. In contrast, experiments where aerobic metabolism was blocked by cyanide indicate that this source of energy is not a limiting factor for muscle function during continuous high-frequency electrical stimulation of up to 90-s duration.
This study was supported by grants from the Velux and the Lundbeck Foundations, The Danish Medical Research Council (j.n.r. 22-04-0241), and the Karen Elise Jensen Foundation.
We thank Ann-Charlotte Andersen, Marianne Stürup Johansen, Tove Lindahl Andersen, and Vibeke Uhre for skilled technical assistance. We also thank William Macdonald for helping to perform the membrane potential and M wave measurements.
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