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Mechanisms of Tissue Repair
2-adrenoceptor agonist stimulated force recovery in rat skeletal muscle
Department of Physiology and Biophysics, University of Aarhus, Århus, Denmark
Submitted 2 June 2005 ; accepted in final form 22 September 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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10% of the prefatigue level, followed by a spontaneous recovery to 20% in 120–240 min. Loss of force was associated with a decrease in K+ content, gain of Na+ and Ca2+ content, leakage of the intracellular enzyme lactic acid dehydrogenase (10-fold increase), and depolarization (13 mV). Stimulation of the Na+-K+ pump with either the 
2-adrenoceptor agonist salbutamol, epinephrine, rat calcitonin gene-related peptide (rCGRP), or dibutyryl cAMP improved force recovery by 40–90%. The -blocker propranolol abolished the effect of epinephrine on force recovery but not that of CGRP. Both spontaneous and salbutamol-induced force recovery were prevented by ouabain. The salbutamol-induced force recovery was associated with repolarization of the membrane potential (12 mV) to the level measured in unfatigued muscles. In conclusion, in muscles exposed to fatiguing stimulation leading to a considerable loss of force, cell leakage, and depolarization, stimulation of the Na+-K+ pump induces repolarization and improves force recovery, possibly due to the electrogenic action of the Na+-K+ pump. This mechanism may be important for the restoration of muscle function after intense exercise. membrane damage; excitability; Na+-K+ pump; catecholamines; calcitonin gene-related peptide
We have previously shown that in isolated rat skeletal muscle, intermittent electrical stimulation (40 Hz, 10 s on, 30 s off) for 30–60 min elicits cell membrane leakage, as assessed from increased [14C]sucrose space, loss of the intracellular enzyme lactic acid dehydrogenase (LDH), and loss of K+. This was associated with a considerable reduction in tetanic force lasting for at least 240 min (22). In another study on rat muscles, we found that sarcolemmal leaks induced by electroporation and documented by increases in [14C]sucrose space and LDH release were associated with a graded and partly reversible loss of force (11). The force recovery of these electroporated muscles was markedly improved by stimulating the Na+-K+ pumps with epinephrine, norepinephrine, the 2-adrenoceptor agonist salbutamol, or calcitonin gene-related peptide (CGRP). Conversely, inhibition of the Na+-K+ pumps with ouabain suppressed both the spontaneous force recovery and that induced by Na+-K+ pump stimulation. It was surprising that Na+-K+ pump stimulation increased the rate of force recovery even in muscles showing evidence of cellular leakages. In this way, stimulation of the Na+-K+ pumps apparently compensates for the sarcolemmal leakages elicited by electroporation, restoring the resting membrane potential, and improving force development.
Salbutamol has been shown to enhance isotonic contractile properties of rat diaphragm muscle (30). In fatigued diaphragm muscles, force recovery was improved by isoproterenol (18) and selective 2-agonists (1, 12, 29). In the present study, effects of the 2-agonists epinephrine and salbutamol and CGRP on force recovery in fatigued and damaged muscles are investigated. The 2-agonists act via 2-adrenoreceptors and G protein to activate adenylyl cyclase in the cell membrane. Adenylyl cyclase then converts ATP to cAMP, thus increasing the intracellular concentration of cAMP, which in turn stimulates the Na+-K+ pumps via protein kinase A (8). CGRP increases the intracellular concentration of cAMP (8) via binding to specific CGRP receptors. In this way, CGRP stimulates the Na+-K+ pump via the same second messenger as the 2-agonists. Therefore, the effects of CGRP on fatigued muscles are also investigated.
To examine the specificity of the receptors mediating the effects, the -antagonist propranolol is used to block the 2-adrenoceptors in experiments with epinephrine and CGRP. The effect of direct addition of cAMP is tested as well. The dibutyryl-derivative of cAMP (db-cAMP) more readily gains access to the cytoplasm and is used for these experiments.
Furthermore, the influence of salbutamol on sarcolemmal integrity evidenced by the release of LDH is investigated. The focus of this paper is the force loss after fatiguing electrical stimulation, the subsequent recovery of force, and the extent to which stimulation of the Na+-K+ pump can compensate the commonly occurring muscle fatigue seen after heavy workload or exercise, and restore force. Cell membrane leakages frequently arise in skeletal muscles during strenuous work, but also after bruises or electrical shocks. It was of general interest, therefore, to test the working hypothesis that the Na+-K+ pumps contribute to the restoration of contractility/force generation in muscles that have lost cell membrane integrity or have a functional defect after fatiguing stimulation.
Some of the results of this study have been presented in a preliminary version (21).
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MATERIALS AND METHODS |
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All experiments were carried out using fed 4-wk-old female or male Wistar rats (own breed) weighing 60–70 g. Animals of this size were chosen to obtain muscles of a relatively small size to improve diffusion and oxygenation during incubation. The rats had free access to food and water and were maintained at a constant temperature (21°C) with constant day length (12 h). The animals were handled and maintained in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The animal facilities were checked by the Danish Inspectorate for Experimental Animals and the Animal Welfare Officer of the Medical Faculty of the University of Aarhus. All handling and use of animals complied with APS Guiding Principles in the Care and Use of Animals.
Muscle Preparation and Incubation
Animals were killed by cervical dislocation followed by decapitation and intact extensor digitorum longus (EDL) muscles, weighing 20–30 mg, were excised, as previously described (7). The standard incubation medium was a Krebs-Ringer bicarbonate buffer (pH 7.2–7.4) containing (in mM) 122.1 NaCl, 25.1 NaHCO3, 2.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3 CaCl2,and 5.0 D-glucose. After preparation, the muscles were mounted for isometric contractions on muscle holders with electrodes on either side of the central part of the muscle. The buffer was continuously gassed with a mixture of 95% O2-5% CO2. All muscles were equilibrated for a minimum of 30 min in the standard medium before further treatment. This procedure has been shown to allow the maintenance of constant membrane potential and Ca2+, Na+, and K+ contents for several hours in vitro (11, 13). Incubations took place at 30°C in a volume of 5, 8, or 23 ml of buffer.
Fatiguing Protocol
EDL muscles were exposed to fatiguing stimulation using a protocol previously shown to elicit muscle cell damage and long-lasting loss of force (22). This standard fatiguing stimulation protocol was applied in all experiments. The muscles were stimulated intermittently (10 s on, 30 s off) at 40 Hz (1-ms pulses of 10 V). Stimulation was applied through two platinum electrodes on either side of the central part of the muscle. Different groups of muscles were stimulated for 0, 5, 10, 30, or 60 min. Because the distance between the stimulation electrodes was 0.4 cm, 10-V pulses correspond to an electrical field of 25 V cm–1.
Force Measurement
The experimental setup used for force measurements allowed for the simultaneous stimulation of four muscles in incubation chambers containing 8 or 23 ml buffer. EDL muscles were mounted on a force displacement transducer (Grass FTO3; W. Warwick, RI) and during repeated stimulation with single pulses adjusted to optimal length for twitch force development. Force was recorded with chart recorders (Servogor; Kolding, Denmark) and a PowerLab data acquisition system (ADI Instruments). Before stimulation, all four muscles were tested at 90 Hz (1-ms pulses of 10 V) for 0.5 s to achieve maximal tetanic activation of all muscle fibers. Testing was done three times at 15-min intervals before the fatiguing stimulation, and the mean of these force determinations was defined as the initial force level. The mean initial force was 452 mN [59 (SD), n = 72]. Force recovery in each group of muscles is given as %initial force of the same muscles. The muscles were then allowed to rest or were fatigued for 5, 10, 30, or 60 min. Force development was recorded during the fatiguing stimulation and, as previously described (22), declined by 88% within 10 min (data not shown). Subsequently, force recovery was tested at 90 Hz (0.5-s pulse trains) at intervals up to 240 min after cessation of stimulation. Force was on average 12% immediately after 30-min fatiguing stimulation and 8% immediately after 60-min fatiguing stimulation. At different time points, epinephrine (10–8 M-10–5 M), salbutamol (10–5 M), db-cAMP (1 mM), or CGRP (10–7 M) were added to evaluate the effect of Na+-K+ pump stimulation on force recovery. In some experiments, propranolol (10–5 M) or ouabain (10–3 M) were added before these agents to test their specificity. In experiments with epinephrine at concentrations <10–5 M, ascorbic acid (1 mM) and EDTA (0.05 mM) were added to the buffer to prevent metabolic degradation of epinephrine. These agents were also added to the controls.
Membrane Potential Recordings
Resting membrane potentials were recorded using standard electrophysiological techniques as previously described in detail (25). In brief, each muscle was impaled by a glass microelectrode filled with 3 M KCl (tip resistance, 15–35 M
) and the potential, recorded via an Axoclamp-2A amplifier, was displayed on an oscilloscope and a chart recorder. The muscle bath was grounded with an Ag/AgCl wire. In each muscle, 10 fibers were impaled. To avoid measuring from the same fiber twice, the electrode was moved a small distance across the muscle between each impalement. EDL muscles were exposed to 30 min of fatiguing stimulation. Immediately after the end of stimulation, the muscles were moved to the membrane potential measurement setup and mounted at resting length. The chamber was perfused with Krebs-Ringer bicarbonate buffer, continuously gassed with a mixture of 95% O2-5% CO2. The first membrane potential recording was performed 15 min after the end of stimulation. Contralateral control muscles were mounted in the transducers, and force was tested, but they were not fatigued before the recording of membrane potential. The effect of Na+-K+ pump stimulation was examined in both resting and fatigued muscles by the addition of salbutamol (10–5 M) 130 min after the muscles had been removed from the force transducer.
LDH Release
As an indicator of cellular integrity, the release of LDH from the muscles into the incubation medium was determined, as previously described (15). In short, after being mounted on muscle holders in 5-ml chambers but before measurement, the muscles were prewashed 4 x 30 min to remove LDH released from cells damaged during excision of the muscles. Buffer samples for determination of the level of LDH release before the fatiguing stimulation were taken from the last of the prewash tubes. During recovery after the fatiguing stimulation, the muscles were moved to new tubes every 30 min. Buffer samples were taken immediately after removal of the muscle, and BSA was added to a final concentration of 0.1%. A 250-µl sample was mixed with 2.65 ml phosphate buffer (0.1 M K2HPO4 titrated with KH2PO4 to pH 7.0) containing NADH (0.3 mM) and pyruvate (0.8 mM), and the decline in the absorbance of the substrate NADH was monitored at 340 nm (Lambda 20, Perkin Elmer) at 30°C. Activity of LDH was expressed as units per gram wet weight per 30 min, 1 unit being the amount of enzyme that catalyzes the use of 1 µmol substrate/min.
Ca2+, Na+, and K+ Contents
At the end of the experiment, the tendons were removed and the muscles were blotted, weighed, and soaked overnight in 3 ml 0.3 M TCA to extract all ions. Previous studies showed that this procedure was as efficient in extraction of ions as homogenization and subsequent centrifugation of the TCA extract (10, 15). Ca2+ content was determined by atomic absorption spectrophotometry (Solaar AAS; Thermo Elemental, Franklin, MA) using 1.5 ml of the TCA extract mixed with 150 µl of 0.27 M KCl. The muscle extracts were measured against a blank and standards containing 12.5 or 25 µM Ca2+ and the same amount of TCA and KCl. Na+ and K+ contents of the TCA extracts were determined with the use of a FLM3 flame photometer (Radiometer, Copenhagen, Denmark) with lithium as the internal standard. For each 0.5-ml sample of the TCA extract, 1.5 ml of 5 mM LiCl and 0.5 ml of 0.3 M TCA were added.
Chemicals
All chemicals were of analytical grade. Salbutamol (racemic), db-cAMP, and ouabain were purchased from Sigma (St. Louis, MO); propranolol (racemic) was from Ferrosan (Soeborg, Denmark); NADH and pyruvate were from Boehringer-Mannheim (Mannheim, Germany). (R-isomer) Epinephrine was from Pharmacies of Danish Hospitals; rCGRP was from Bachem AG (Bubendorf, Switzerland). Ascorbic acid was from Merck (Albertslund, Denmark).
Statistics
Results are given as mean values with SD. The statistical significance of any difference between groups was ascertained using Student's two-tailed t-test for unpaired observations. All comparisons made were between separate groups of muscles. When required, a one-way ANOVA and a post hoc Student's t-test were performed to ascertain the significance of the differences between the groups.
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RESULTS |
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Effect of 2-agonists.
As shown in Fig. 1, 30 min of fatiguing stimulation caused a drop in tetanic force to 12% of the initial level followed by a slow spontaneous recovery to 20–25%. Addition of salbutamol (10–5 M) after a 140-min recovery period improved tetanic force by an average of 77% (P = 0.007, at 180- to 240-min recovery) compared with the controls (Fig. 1A). Control experiments with unfatigued muscles showed that the same dose of salbutamol (10–5 M) induced no significant change in tetanic force (90 Hz, 0.5 s, n = 4 vs. 4 muscles, data not shown). The role of the Na+-K+ pump was examined using the Na+-K+ pump inhibitor, ouabain. Ouabain (10–3 M), added at 115-min recovery completely abolished spontaneous force recovery in all muscles, and the stimulating effect of salbutamol (10–5 M, added at 130-min recovery) was alleviated. As shown in Fig. 1B, epinephrine (10–5 M) added to fatigued muscles after 130-min recovery improved tetanic force by an average of 90% (P = 0.0004, at 180- to 240-min recovery).
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-antagonist propranolol was added 15 min before epinephrine to block the -adrenoceptors.
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-adrenoceptors.
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-adrenoceptors.
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As shown in Fig. 8, the membrane potential of the resting controls remained stable around –73 mV for 240 min. In the salbutamol-treated group, the resting membrane potential was a little lower (–75 mV) before salbutamol treatment. Salbutamol caused no significant hyperpolarization. After 30 min of fatiguing stimulation, the first membrane potential recordings (at 15 min) showed values around –62 (control group) and –60 mV (salbutamol group), representing stimulation-induced depolarizations of 11 and 15 mV, respectively. Without treatment, the muscles showed no spontaneous repolarization. After the addition of salbutamol (10–5 M at 130-min recovery), a significant repolarization of the membrane potential of the stimulated muscles to –72 mV (at 240 min; P = 0.01; n = 4–5) was observed. This is an improvement of 12 mV returning the membrane potential of the stimulated muscles to values not significantly different from the resting controls.
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After 30–60 min of fatiguing stimulation, the cell membranes are leaky, as evidenced by an increased resting uptake of 45Ca, resting [14C]sucrose space, and release of LDH (22). The present results indicate that despite the damaged membranes, stimulation of the Na+-K+ pump by hormones, a 2-agonist and their second messenger cAMP, improves recovery of tetanic force. It was of interest whether stimulation of the Na+-K+ pump by salbutamol would also have effects on membrane integrity.
After 60 min of fatiguing stimulation, release of the intracellular enzyme LDH was followed for 4 h. As shown in Fig. 9, 60 min of fatiguing stimulation caused a large increase in LDH release beginning during the first 30 min of recovery. Addition of salbutamol (10–5 M at 120-min recovery) had no effect on the LDH release. Thus the force recovery induced by salbutamol cannot be attributed to resealing of the cell membrane.
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As shown in Table 1, Na+ content was significantly increased, and K+ content was significantly decreased after 60-min stimulation and 240-min recovery. None of these changes were affected by the addition of salbutamol (10–5 M at 120-min recovery). Likewise, the Ca2+ content of these muscles was significantly increased, and no change was observed when adding salbutamol.
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DISCUSSION |
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Force recovery elicited by epinephrine is seen down to the concentrations measurable in plasma during running exercise (27). The effect is abolished by propranolol, indicating that it is mediated via -adrenoreceptors. The control force is not affected by propranolol as seen in Figs. 5 and 6. Because the 2-agonist salbutamol (10–5 M) induces almost the same maximum force recovery as epinephrine, it is reasonable to assume that the effect of salbutamol is mediated via 2-adrenoreceptors. In contrast, the force recovery induced by rCGRP is not affected by propranolol, indicating that the effect of this peptide hormone is mediated via its specific receptors. Finally, cAMP, which is second messenger for the actions of epinephrine and CGRP, caused a similar, albeit slightly later, force recovery.
The long-lasting loss of force induced by fatiguing stimulation may have several causes. Depletion of metabolizable substrates, glycogen, creatine phosphate, and ATP has repeatedly been proposed (for a review, see Ref. 14). It is unlikely, however, that these stores can be repleted by stimulating active Na+-K+ transport, which, in itself, is an energy-consuming process. Accumulation of lactic acid is another frequently mentioned cause of fatigue (14). In the present experimental situation with a large volume of incubation medium, however, the lactic acid formed is not likely to be retained for several hours in vitro. Moreover, catecholamines induce an increase in lactic acid production, which cannot explain the force recovery observed. Finally, lactic acid causes no inhibition of contractile force in isolated rat skeletal muscle (23).
Long-term fatigue may also be related to impairment of the excitation-induced increase in cytoplasmic Ca2+ (6, 32, 33). There is no evidence, however, that stimulation of the Na+-K+ pumps can improve the release of Ca2+ from SR or the subsequent rise in cytoplasmic Ca2+.
On the other hand, it has repeatedly been shown that fatiguing stimulation leads to depolarization of muscle cells, both in vivo (20) and in vitro (2, 17). This may be due to rundown of the transmembrane concentration gradients for K+ and the ensuing reduction in the equilibrium potential for K+. In the present study, fatiguing stimulation for 30 min caused no significant decrease in the K+ content of the muscles, whereas 60 min of stimulation gave a significant decrease. However, after 30 min of fatiguing stimulation, the muscles underwent a depolarization of 11–15 mV, showing no spontaneous repolarization. If salbutamol (10–5 M) was added 130 min after the end of stimulation, a rapid repolarization (12 mV) was observed returning the membrane potential to levels not significantly different from the controls.
It was recently shown that electroporation of isolated muscles led to a large depolarization of the membrane potential. This is most likely due to the increased permeability of the membrane allowing flux of Na+ and K+ down to their electrochemical gradients. After electroporation, there was a large spontaneous repolarization of the membrane potential. However, salbutamol (10–5 M) not only increased the rate of recovery of the membrane potential but also the extent of the recovery. Salbutamol also improved the rate and extent of force recovery after electroporation (11).
The depolarization induced by fatiguing stimulation may also be caused by sarcolemmal damage, an interpretation supported by previous observations of increased [14C]sucrose space and release of LDH (22). Also in the present study, muscles exposed to 60 min of fatiguing stimulation showed a marked increase in LDH-release. Even in these damaged muscles, salbutamol induced a significant recovery of force. This was not associated with any restoration of total Na+ or K+ contents or changes in LDH release, indicating that stimulation of the electrogenic Na+-K+ pump in the remaining intact sarcolemma or T-tubular membranes might be sufficient to restore excitability. The restoration of excitability by the addition of salbutamol seems to be achieved by compensation of the cell membrane leakages, because no effect on membrane integrity was observed. This suggests that the stimulation of the Na+-K+ pump is a local effect that acts to reestablish the ionic gradients and the membrane potential without changing the global content of Na+ and K+ in the muscle fibers.
All of our experiments were performed at 30°C, a temperature that ascertains optimum stability (19, 26). Because the Na+-K pump has a Q10 of 2.3, elevations of the temperature cause marked increase in the pumping rate. Heating of muscles might therefore contribute to the force recovery after fatiguing stimulation.
Burniston et al. (4) have shown a myotoxic effect in vivo of the 2-agonist clenbuterol administered in doses down to 0.1 mg/kg. Assuming an extracellular volume of 20%, this dose would give a concentration in plasma of 1.6 x 10–6 M. In our studies, we have not observed an increase in LDH leakage with salbutamol treatment; however, we cannot rule out that high doses of salbutamol may be myotoxic as well. It should be noted that CGRP induced a significant improvement of force recovery. This is of particular interest because CGRP is stored in nerve endings in skeletal muscle from where it can be released and stimulate the Na+-K+ pump (9).
In conclusion, fatiguing stimulation leads to severe functional impairment, depolarization, and loss of cellular integrity. Perhaps the most interesting observation of this study is that even in "leaky" muscles, stimulation of the Na+-K+ pumps with salbutamol induces repolarization, but no recovery of integrity or Na+ and K+ contents. This is accompanied by a partial, but significant, restoration of contractile force, which is likely to reflect improved excitability.
Perspectives
Vigorous physical activity often leads to long-lasting and considerable loss of force. This may in part be attributed to loss of excitability due to depolarization or sarcolemmal leakage. The present results indicate that the ensuing impairment of contractility may be compensated by catecholamines or CGRP, agents that are both available in plasma and skeletal muscles. This information may be used for a therapeutic restoration of muscle performance after intense exercise or muscle damage due to trauma. Moreover, treatment with 2-agonists is widely used by asthmatic patients as bronchodilators to improve respiration. Because these patients often perform fatiguing or exhausting work with their respiratory muscles, the 2-agonists may in addition, compensate the ensuing fatigue in these muscles (1, 12, 30).
A possible ergogenic effect of 2-agonists in athletes has been highly disputed, and the results are inconsistent. Signorile et al. (28) found that acute inhalation of albuterol had an ergogenic effect on short-term power output on a cycle ergometer. Peak power was increased, and fatigue was reduced. With respect to endurance exercise, results are conflicting. Norris et al. (24) found no effect of salbutamol inhalation on time needed to complete a 20-km time trial. Likewise, Goubault et al. (16) found no effect on endurance time at 85% of 
O2 max in a cycle ergometer. On the other hand, Bedi et al. (3) showed that sprint time until exhaustion after 1 h of heavy exercise was increased by pretest albuterol inhalation. In contrast, a study by Carlsen et al. (5) showed reduced running time until exhaustion after salbutamol inhalation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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rhus C, Denmark (e-mail: tc{at}fi.au.dk)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2-agonist stimulated force recovery in rat (Abstract). Physiologist 47: 307, 2004.2-agonist. J Physiol 551: 277–286, 2003.This article has been cited by other articles:
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  D. N. French, W. J. Kraemer, J. S. Volek, B. A. Spiering, D. A. Judelson, J. R. Hoffman, and C. M. Maresh Anticipatory responses of catecholamines on muscle force production J Appl Physiol, January 1, 2007; 102(1): 94 - 102. [Abstract] [Full Text] [PDF]
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 H. Scholz Unraveling the basic principles Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2006; 290(6): R1485 - R1487. [Full Text] [PDF]
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(n = 3) and 
(n = 3), salbutamol (10–5 M) was added at 140-min of recovery to 
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