repair of regenerating rat skeletal muscle after injury ...

β2-Adrenoceptor agonist fenoterol enhances functional repair of regenerating rat skeletal muscle after injury

Felice Beitzel, Paul Gregorevic, James G. Ryall, David R. Plant, Martin N. Sillence and Gordon S. Lynch J Appl Physiol 96:1385-1392, 2004. First published 7 November 2003; doi: 10.1152/japplphysiol.01081.2003 You might find this additional info useful... This article cites 45 articles, 12 of which you can access for free at: http://jap.physiology.org/content/96/4/1385.full#ref-list-1 This article has been cited by 12 other HighWire-hosted articles: http://jap.physiology.org/content/96/4/1385#cited-by

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J Appl Physiol 96: 1385–1392, 2004. First published November 7, 2003; 10.1152/japplphysiol.01081.2003.

␤2-Adrenoceptor agonist fenoterol enhances functional repair of regenerating rat skeletal muscle after injury Felice Beitzel,1 Paul Gregorevic,1 James G. Ryall,1 David R. Plant,1 Martin N. Sillence,2 and Gordon S. Lynch1 1 Department of Physiology, The University of Melbourne, Melbourne, Victoria 3010, Australia; and 2School of Agriculture, Charles Sturt University, Wagga Wagga 2678, New South Wales, Australia

Submitted 6 October 2003; accepted in final form 4 November 2003

severely as a consequence of direct trauma such as crush, contusion, and laceration; from exposure to temperature extremes associated with severe burns or freezing; as a result of ischemia; and even by their own contractions, especially when activated and then stretched forcibly (7, 20, 26). Regardless of the injury, the subsequent degeneration and extensive spontaneous regeneration of skeletal muscle always follows a characteristic sequence to regain functional capacity (7). Myotoxic injury provides a suitable model for studying muscle regeneration. When injected into skeletal muscle, bupivacaine hydrochloride (bupivacaine), causes rapid degeneration by disrupting intracellular Ca2⫹ homeostasis (14, 36, 44). Bupivacaine does not damage elements that influence the rate and extent of muscle regeneration, including endomysium,

basal lamina, vascular supply, intramuscular nerves, and muscle satellite cell population (4, 12, 19, 46). Thus, after bupivacaine treatment, rapid myofiber regeneration within the preexisting basal lamina proceeds (3, 35) and the chronology of subsequent events has been well documented (3, 13, 29, 35). Pharmacological agents that promote muscle protein accretion have clinical potential for improving muscle regeneration after injury. Synthetic ␤2-adrenoceptor agonists (␤2-agonists), such as clenbuterol and fenoterol, were initially developed for acute asthma treatment, to facilitate bronchiolar smooth muscle dilation (45). Further investigations found that, when administered at higher doses, ␤2-agonists have an anabolic effect on skeletal muscle (9). Studies have shown that prolonged ␤2agonist administration produces muscle hypertrophy and often increases maximum force-producing capacity (8, 22, 31, 37, 43). This increase in muscle mass is due to ␤2-adrenoceptormediated protein accretion via increases in intracellular cAMPpromoting both an increase in protein synthesis and a decrease in degradation (27). Examinations of the mechanisms influenced by ␤2-agonist treatment on normal skeletal muscle, particularly protein metabolism, suggest that these compounds have the potential to enhance regeneration of skeletal muscle after injury. However, the responses of regenerating skeletal muscles to ␤2-adrenergic stimulation have not been studied extensively. Previous investigations, using a muscle transplant model of regeneration, have suggested that improvements in regeneration with isoprenaline or clenbuterol may involve a ␤2-adrenergic effect on muscle vasculature, fiber hypertrophy, and possibly satellite cell proliferation (32, 33). Thus, in the presence of a population of functional receptors, ␤2-adrenoceptor-mediated mechanisms have the potential to promote muscle hypertrophy and enhance muscle repair. No information exists regarding the ␤2-adrenoceptor population in regenerating muscles; therefore, it is also essential to establish whether ␤2-adrenoceptors are present in regenerating muscle fibers in sufficient numbers to mediate a response and whether stimulation of these receptors can enhance functional repair after injury. Fenoterol is a full agonist of ␤1- and ␤2-adrenoceptors (5) that produces dramatic increases in mass and force-producing capacity of skeletal muscles, suggesting clinical potential with application for promoting regeneration of skeletal muscle after trauma (37). We tested the hypothesis that ␤2-adrenoceptors would be present in regenerating muscles and that stimulation of these receptors with fenoterol would hasten the recovery of

Address for reprint requests and other correspondence: G. S. Lynch, Dept. of Physiology, The Univ. of Melbourne, Victoria, 3010 Australia (E-mail: [email protected]).

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muscle regeneration; ␤2-agonist; bupivacaine; muscle function SKELETAL MUSCLES CAN BE INJURED

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Beitzel, Felice, Paul Gregorevic, James G. Ryall, David R. Plant, Martin N. Sillence, and Gordon S. Lynch. ␤2-Adrenoceptor agonist fenoterol enhances functional repair of regenerating rat skeletal muscle after injury. J Appl Physiol 96: 1385–1392, 2004. First published November 7, 2003; 10.1152/japplphysiol.01081.2003.— ␤2-Adrenoceptor agonists such as fenoterol are anabolic in skeletal muscle, and because they promote hypertrophy and improve forceproducing capacity, they have potential application for enhancing muscle repair after injury. No previous studies have measured the ␤2-adrenoceptor population in regenerating skeletal muscle or determined whether fenoterol can improve functional recovery in regenerating muscle after myotoxic injury. In the present study, the extensor digitorum longus (EDL) muscle of the right hindlimb of deeply anesthetized rats was injected with bupivacaine hydrochloride, which caused complete degeneration of all muscle fibers. The EDL muscle of the left hindlimb served as the uninjured control. Rats received either fenoterol (1.4 mg䡠kg⫺1 䡠day⫺1) or an equal volume of saline for 2, 7, 14, or 21 days. Radioligand binding assays identified a ⬃3.5-fold increase in ␤2-adrenoceptor density in regenerating muscle at 2 days postinjury. Isometric contractile properties of rat EDL muscles were measured in vitro. At 14 and 21 days postinjury, maximum force production (Po) of injured muscles from fenoterol-treated rats was 19 and 18% greater than from saline-treated rats, respectively, indicating more rapid restoration of function after injury. The increase in Po in fenoterol-treated rats was due to increases in muscle mass, fiber cross-sectional area, and protein content. These findings suggest a physiological role for ␤2-adrenoceptor-mediated mechanisms in muscle regeneration and show clearly that fenoterol hastens recovery after injury, indicating its potential therapeutic application.

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FENOTEROL IMPROVES FUNCTION OF REGENERATING MUSCLE

functional properties of regenerating skeletal muscle after myotoxic injury. MATERIALS AND METHODS

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Animals. The Animal Experimentation Ethics Committee of The University of Melbourne approved all experiments, which were conducted in accordance with the guidelines for Care and Use of Experimental Animals, as described by the National Health and Medical Research Council of Australia. Adult (300–350 g) male SpragueDawley rats (n ⫽ 73) were bred and housed in the Biological Research Facility at The University of Melbourne. They were maintained under a 12:12 h light-dark cycle (light 0600–1800), with access to standard rat chow and water ad libitum. Experimental injury. All rats were deeply anesthetized with pentobarbital sodium (Nembutal, Rhone Merieux, Pinkenba, Queensland, Australia; 60 mg/kg body mass) via intraperitoneal injection, such that they were unresponsive to tactile stimuli. The right extensor digitorum longus (EDL) muscle was surgically exposed, with care taken to avoid damaging its nerve and blood supplies, and then injected to maximal holding capacity with 0.5% bupivacaine hydrochloride (bupivacaine) (Marcain, Astra, North Ryde, NSW, Australia), through several injections in the distal, proximal, and midbelly regions of the muscle, using a 26-gauge needle. This procedure ensured the degeneration of all fibers in the injected muscle (11). The left EDL muscle served as an uninjured control and was not injected with vehicle because previous studies have shown that injection with saline does not affect the morphological, structural, or functional characteristics of skeletal muscle (15, 36). After the intramuscular injections, the skin incision was closed by using Michel clips (Aesculap, Tuttlingen, Germany) and swabbed with povidone iodine solution. After the surgical procedure, rats were randomly assigned to receive daily treatment with either fenoterol (1.4 mg䡠kg⫺1 䡠day⫺1 dissolved in 1 ml isotonic saline ip; Sigma-Aldrich, Castle Hill, NSW, Australia) or an equal volume of saline, for either 2, 7, 14, or 21 days. Contractile properties. Muscle function was assessed at 7, 14, and 21 days after myotoxic injury. Function was not assessed at 2 days because muscle degeneration is maximal at this time (35). At the completion of the treatment period, rats were anesthetized deeply with pentobarbital sodium (60 mg/kg body mass ip), and supplemental doses were administered to maintain a depth of anesthesia such that there was no response to tactile stimuli. The EDL muscle was surgically exposed, and proximal and distal tendons of each muscle were tied with braided silk suture (3-0, Pearsalls Sutures, Somerset, UK). Nerve and blood supplies were intact throughout dissection, to ensure optimum muscle viability when transferred to an organ bath for the measurement of contractile properties. The custom-built Plexiglas bath was filled with Ringer solution (in mM: 1.37 NaCl, 24 NaHCO3, 11 D-glucose, 5 KCl, 2 CaCl2, 1 NaH2PO4 䡠H2O, 0.487 MgSO4 䡠7H2O, 0.293 d-tubocurarine chloride; pH 7.4) that was gassed with Carbogen (5% CO2 in 95% O2, BOC Gases, Preston, Victoria, Australia) and thermostatically maintained at 25°C. These experimental conditions facilitate optimal oxygen diffusion throughout the muscle and stability of functional measures in vitro (37, 39). In the bath, each muscle was aligned horizontally and tied between a fixed pin and the lever arm of a dual-mode servomotor (305-LR, Aurora Scientific, Aurora, Ontario, Canada). Two platinum-plate electrodes flanked the length of the muscle preparation. The muscle was stimulated with supramaximal square-wave pulses (0.2-ms duration) amplified (Ebony, power amplifier EP500B, Audio Assemblers, Campbellfield, Victoria, Australia) to ensure sufficient current intensity to produce a maximum isometric tetanic contraction. The servomotor and stimulation operations were controlled by custom-written applications (D. R. Stom Software Solutions, Ann Arbor, MI) of LabView software (National Instruments, Austin, TX), driving a personal computer with on-board controller (PCI-MIO-16XE-10, National Instruments) interfaced with

the servomotor control-feedback position controller hardware (6650LR Dual-Mode Lever System, Aurora Scientific). The optimal muscle length for contraction (Lo) was determined by stimulating the muscle to produce an isometric twitch response and adjusting muscle length to produce a maximum isometric twitch force (Pt). Optimum fiber length (Lf) was calculated by using the previously determined Lf/Lo ratio of 0.44 for the EDL muscle (6), which accounts for the pennation of fibers within the muscle. The degeneration and regeneration induced by bupivacaine does not affect this ratio, because new fibers grow within the preexisting basal laminae, and so the angle of muscle fiber insertion is not altered (11, 36). The frequency-force relationship of the muscle was determined by stimulating at frequencies of 10, 30, 50, 80, 100, 120, and 150 Hz, with a 2-min rest period between each stimulus to prevent fatigue. The maximal isometric tetanic force (Po) of the EDL muscle was determined from the plateau of the frequency-force relationship. Immediately after the functional measurements, the muscles were carefully blotted on filter paper (Whatman no. 1 filter paper, Maidstone, UK), trimmed of tendons and any adherent nonmuscle tissue, and weighed. Muscle cross-sectional area (CSA) was calculated taking into account muscle mass, Lf, and 1.06 g/m3, the density of mammalian skeletal muscle (25). Specific or “normalized” force (sPo) was expressed as the maximum force per CSA of the muscle. Immediately after weighing, each muscle was tied to a glass capillary tube at Lo and then snap-frozen in isopentane cooled in liquid nitrogen. Muscles were stored at ⫺80°C for later histological analysis, biochemical analysis and ␤2-adrenoceptor density measurements. After the surgical procedures, the thoracic region was exposed and rats were killed by cardiac excision. Histological analysis. Histological procedures were performed to examine the effects of fenoterol on general tissue morphology of both injured and uninjured muscles, after 2, 7, 14, and 21 days of treatment. Transverse 8-␮m-thick sections were cut from as close to the midbelly of each muscle as possible, using a cryostat microtome at ⫺20°C (CTI cryostat, IEC, Needham Heights, MA). Two serial sections of both the left and right EDL from each animal were placed onto silane-coated glass slides and stained with hematoxylin and eosin. Digital images of muscles were obtained by using a camera (Spot model 1.3.0, Diagnostic Instruments, Sterling Heights, MI) attached to an upright microscope (Olympus BX51 light microscope, Olympus, Tokyo, Japan) at ⫻10 magnification. Analysis of images was performed in a single-blinded manner, using an appropriately calibrated Analytical Imaging Station (AIS, v6.0, Imaging Research, Ontario, Canada). The mean CSA of individual fibers was calculated by interactive determination of the circumference of at least 150 adjacent fibers from the center of each muscle section. Biochemical analysis. Muscle protein content was assessed in both injured and uninjured muscles, after treatment for 2, 7, 14, and 21 days. Approximately 5–10 mg of each muscle were placed separately in a buffer solution (buffer A; composition in mM: 50 KCl, 10 KH2PO4, 2 MgCl2 䡠6H2O, 0.5 EDTA, and 2 dithiothreitol in 1:50 wet weight/volume) and homogenized by using a glass tissue grinder. The muscle sample, buffer, and tissue grinder were kept ice cold at all times. Crude homogenates (⬃40 ␮l) of individual muscle samples were set aside for determination of total protein concentration. The remainder of the homogenate was centrifuged at 5°C for 10 min at 1,000 g (centrifuge 5417 R, Eppendorf, Hamburg, Germany, using F 45-30-11 rotor). The supernatant, containing cytosolic proteins, was discarded, and the pellet, containing contractile proteins, was resuspended in ice-cold buffer (buffer B; buffer A containing 1% Triton X-100 in 1:20 wet weight/volume). Protein concentration was determined by using a Bradford protein assay (Bio-Rad protein assay, Bio-Rad Laboratories, Hercules, CA) with BSA standards. Radioligand binding assays. The characterization of the muscle ␤2-adrenoceptor population utilized procedures previously described by Sillence et al. (41) and more recently, by Ryall et al. (37). The


Table 1. Muscle protein content of uninjured and injured EDL muscles from rats treated with saline or fenoterol for 2, 7, 14, or 21 days Saline Treatment Duration

8 22.0⫾1.2 147.1⫾5.6 37.4⫾0.9

Injured

Uninjured

8 8 14.4⫾1.1* 23.1⫾0.8 78.3⫾3.8* 156.8⫾4.5 41.7⫾2.0 35.8⫾1.1

10 10 9 22.6⫾2.0 13.2⫾0.8* 24.8⫾1.7 129.2⫾7.8 105.0⫾5.6* 128.4⫾7.1 38.0⫾1.8 35.2⫾1.4 33.4⫾1.2

Injured

8 14.0⫾1.1* 84.8⫾7.1* 41.2⫾2.0 9 11.1⫾0.8* 79.5⫾3.9*† 36.0⫾2.7

10 10 9 9 23.1⫾1.3 19.6⫾0.9* 30.2⫾1.1* 21.9⫾1.6 122.8⫾5.3 103.2⫾4.7* 134.4⫾4.0 105.5⫾7.2* 34.5⫾1.9 37.3⫾2.0 33.6⫾2.4 34.3⫾2.2 10 10 9 9 26.3⫾1.1 23.8⫾0.9 29.3⫾0.9 31.7⫾1.9*† 126.5⫾3.6 104.9⫾5.5* 124.4⫾3.3 124.5⫾7.4† 32.4⫾1.5 35.3⫾2.2 37.0⫾2.3 38.0⫾1.8

Values are means ⫾ SE; n, no. of animals in each group. EDL, extensor digitorum longus; [protein], protein concentration. *Significantly different from time-matched saline-uninjured (control) muscles; †significant difference between time-matched saline-injured and fenoterol-injured muscles, P ⬍ 0.05.

remaining portions of the EDL muscles were pooled in pairs according to treatment and injury state and placed in 2 ml ice-cold buffer [buffer C; in mM: 50 Tris (pH 7.0), 250 sucrose, and 1 EGTA; pH 7.4 at 4°C]. Each sample was homogenized (Polytron, PT 2100, Kinematica, Luzernerstrasse, Switzerland) for ⬃30 s. Cell membrane fragments were prepared by centrifugation at 4°C. Homogenates were centrifuged for 10 min at 1,000 g (Avanti J-251 centrifuge, Beckman Instruments, Palo Alto, CA; using a JA-17 rotor), from which the pellet was discarded, and the supernatant was filtered through two layers of surgical gauze (Smith and Nephew, Victoria, Australia) and centrifuged for a further 15 min at 10,000 g (Centrifuge 5417 R, Eppendorf; using an F 45-30-11 rotor). The supernatant was then transferred to ultracentrifuge tubes (Polyallomer centrifuge tubes, Beckman Instruments), which were filled with ice-cold buffer C and ultracentrifuged for 30 min at 100,000 g (L7 Ultracentrifuge, Beckman; using an SW4ITI 6 bucket rotor). The pellets obtained were resuspended in 1 ml ice-cold buffer [buffer D; in mM: 50 Tris (pH 7.6), 10 MgCl2 䡠6H2O, and 150 NaCl; pH 7.4 at 4°C] by using a Pasteur pipette before storage at ⫺80°C. Frozen cell membrane samples were thawed and vortexed (Vortex mixer, Ratek Instruments, Victoria, Australia) for 30 s. Protein concentration of the membrane suspension was determined using a Bradford protein assay with BSA standards, ensuring that the sample concentration was within the concentration range of 0.05–0.3 mg/ml, in which the binding of the radioligand to the ␤2-adrenoceptor sites has been shown to be linear (41). Because of the limited amount of protein that could be obtained from the small muscles, single-point saturation assays were performed, by incubating 400 ␮l of the cell membrane suspension with 50 ␮l of the radioligand [125I]iodocyanopindolol (ICYP; 135 pM) and 50 ␮l of either buffer D (to determine total counts of ICYP bound to ␤2-adrenoceptors) or the nonselective ␤-antagonist dl-propanolol (2 ␮M; to determine nonspecific binding of ICYP to the membrane) in polyethylene tubes (12 ⫻ 75 mm). The tubes were incubated for 90 min in a shaking water bath set at 37°C and 120 cycles/min. To separate the bound from free radioligand, the contents of each tube were filtered through glass fiber filter paper J Appl Physiol • VOL

(Whatman GF-C filter paper, Maidstone, UK) and rinsed three times with 7 ml of ice-cold buffer D, by using a cell harvester (Brandel M-48R cell harvester, Biomedical Research and Development Laboratories, Gaithersburg, MD). Radioactivity remaining on the filters was determined in a gamma counter (1470 Wizard-automatic gamma counter, Wallac OY, Turku, Finland) at a counting efficiency of 78%. Results were expressed as gamma radiation counts per minute, and a conversion was applied to determine the concentration of ␤-adrenoceptors (fmol per mg of protein) on the basis of the specific activity of the radioligand. Rat EDL muscle contains predominantly ␤2-adrenoceptors, with ␤1-adrenoceptors usually undetectable by this technique (41). Hence ␤-adrenoceptors measured were designated as ␤2-adrenoceptors. Statistical analysis. All values are expressed as means ⫾ SE (unless otherwise stated). Experimental groups from each time point were compared by using a two-way ANOVA for influences of treatment (saline vs. fenoterol) and injury (injured vs. uninjured), by using Fisher’s least significant difference post hoc multiple-comparison procedure. In all cases, differences between groups were considered significant when P ⬍ 0.05. RESULTS

Effects of myotoxic injury. Local myotoxic injury with bupivacaine had no effect on body weight or heart mass, but it had a significant effect on the EDL muscle. At 2 days postinjury, there was a marked decrease in absolute protein content (P ⬍ 0.05), which did not return to control values until 21 days postinjury (Table 1). It is well established that 2 days after intramuscular injection with bupivacaine, there is an extensive inflammatory response that is associated with maximal swelling and edema (36). Therefore, the initial decrease in absolute protein was offset presumably by this swelling and edema, such that injured muscles were heavier than uninjured controls at 2 days postinjury (P ⬍ 0.05). However, this was followed by a loss of muscle mass (day 7; P ⬍ 0.05), recovery to normal mass (day 14), and then compensatory hypertrophy (day 21; P ⬍ 0.05), as illustrated in Fig. 1.

Fig. 1. Uninjured and injured extensor digitorum longus muscle mass from rats treated with saline or fenoterol for 2, 7, 14, or 21 days. Note that the increase in mass at 2 days postinjury was lower in the fenoterol-treated rats. The mass of uninjured muscles was greater in fenoterol-treated compared with saline-treated rats after 7 days of administration. The mass of injured muscles from fenoterol-treated rats was greater than saline-treated rats at 14 days postinjury. Of all muscle groups tested, injured muscles from rats treated with fenoterol for 21 days exhibited the greatest mass. Values are means ⫾ SE. *Significantly different from time-matched saline-uninjured (control) muscles, P ⬍ 0.05. #Significant difference between time-matched saline-injured and fenoterol-injured muscles, P ⬍ 0.05.

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2 Days n Absolute protein, mg [Protein], mg/g %Contractile protein 7 Days n Absolute protein, mg [Protein], mg/g %Contractile protein 14 Days n Absolute protein, mg [Protein], mg/g %Contractile protein 21 Days n Absolute protein, mg [Protein], mg/g %Contractile protein

Uninjured

Fenoterol

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tration (41). Injured muscle mass in fenoterol-treated rats was 9 and 12% greater than in saline-treated rats at 14 and 21 days postinjury, respectively (P ⬍ 0.05; Fig. 1). Fenoterol decreased fiber CSA in uninjured muscles after 2 days of treatment (P ⬍ 0.05), but increased fiber CSA thereafter, so that after 21 days of treatment, fiber CSA was 26% greater than in control rats (P ⬍ 0.05). A similar effect on fiber CSA was apparent in injured tissue, with significantly increased CSA at 14 and 21 days postinjury, being 30 and 26% greater than in injured tissue without fenoterol treatment, and similar in magnitude to the fiber CSA in uninjured tissue (Figs. 2 and 3). Muscle protein content was measured to confirm that increases in muscle mass and fiber CSA were due to an increase in functional material. Fenoterol had no effect on protein concentration in uninjured muscles at any time point, but it increased absolute protein content by 31% after 14 days (P ⬍ 0.05; Table 1). Absolute protein content in injured muscles from fenoterol-treated rats was also 33% greater than in salinetreated rats by 21 days postinjury (P ⬍ 0.05; Table 1). In fenoterol-treated rats, absolute protein content had returned to control levels by 14 days postinjury, and by 21 days postinjury it was 21% greater than in control muscles (P ⬍ 0.05, Table 1). In saline-treated rats, absolute protein content in injured muscles had returned to control levels by 21 days postinjury (Table 1). Although the protein concentration of injured muscles from fenoterol-treated rats had returned to control levels by 21 days postinjury, in saline-treated rats, protein concentration was still significantly lower than in controls at this time point. At no time was the proportion of contractile protein as a percentage of total protein affected by drug treatment or injury (Table 1). Fenoterol increased Po in both uninjured tissue by 12, 17, and 13% with 7, 14, and 21 days of treatment, respectively (P ⬍ 0.05), and in injured tissue by 19 and 18% at 14 and 21 days postinjury, respectively (P ⬍ 0.05; Fig. 4). The rate of restoration of force producing capacity was greater with fenoterol treatment such that, at 21 days postinjury, Po was restored to 90% of control values compared with 76% in untreated rats (Fig. 4). When Po was normalized for muscle size, the sPo was not different with fenoterol treatment of uninjured muscles or for injured muscles (Table 2). After 14 and 21 days of fenoterol treatment, Pt of uninjured muscles was 22 and 15% greater than muscles of untreated rats. For injured muscles, Pt was 52, 19, and 17% greater in fenoterol-treated rats (Table 2). Fenoterol treatment did not affect TPT or RT1/2 for either uninjured or injured muscles at any time point (Table 2). In uninjured muscle, fenoterol did not affect the ␤2-adrenoceptor density until 21 days postinjury, when a marked downregulation was observed (P ⬍ 0.05; Fig. 5). In injured tissue, fenoterol attenuated the upregulation observed in saline-treated rats after 7 days of treatment, with a gradual decline in ␤2-adrenoceptor levels thereafter (Fig. 5).

Fig. 2. Muscle fiber cross-sectional area (CSA) of uninjured and injured extensor digitorum longus muscles from rats treated with saline or fenoterol for 2, 7, 14, or 21 days. At 2 days postinjury, no measurements were possible because degeneration is maximal and fibers cannot be identified. Note the significant increase in fiber size with fenoterol treatment in both uninjured and injured muscles by 7 and 14 days, respectively. Fiber CSA of injured muscles from fenoterol-treated rats had returned to control levels by 14 days postinjury. Values are means ⫾ SE. *Significantly different from time-matched salineuninjured (control) muscles, P ⬍ 0.05 #Significant difference between timematched saline-injured and fenoterol-injured muscles, P ⬍ 0.05.

DISCUSSION


This is the first report to identify and characterize the response of ␤2-adrenoceptors during the process of functional muscle regeneration after injury. We showed that ␤2-adrenoceptors undergo a marked increase in number from the early stages of muscle regeneration and that stimulating these receptors with fenoterol enhanced the rate of functional repair after injury. By 21 days postinjury, injured muscles from fenoterol-

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The decrease in muscle protein was reflected by a decrease in fiber CSA to the extent that 2 days after injury fibers could not even be identified and CSA could not be measured. Fiber CSA recovered gradually, to 36% of control values by day 7 (P ⬍ 0.05), and 71% by day 14 (P ⬍ 0.05), but it was still significantly decreased (76% of uninjured control values) at 21 days postinjury (P ⬍ 0.05; Figs. 2 and 3). As expected, the decrease in fiber CSA was accompanied by a marked decrease in Po, which was 34% of control values by 7 days postinjury (P ⬍ 0.05). Without treatment, the recovery of Po was steady, but slow, such that Po was still only 76% of control values by 21 days postinjury (P ⬍ 0.05; Fig. 4). When Po was normalized for muscle size, the sPo of injured muscles was 48, 66, and 71% of values for uninjured muscles, at 7, 14, and 21 days postinjury, respectively (Table 2). Peak twitch tension (Pt) of injured muscles was 32, 67, and 72% of values for uninjured controls at 7, 14, and 21 days postinjury, respectively (Table 2). Time to peak twitch tension (TPT) and one-half relaxation time (RT1/2) were prolonged in injured muscles at 7 days postinjury (P ⬍ 0.05) but not at any other time point (Table 2). Injury resulted in a deficit in sPo, which had not recovered by 21 days postinjury. Myotoxic injury caused a large increase (⬃3.5 fold) in ␤2-adrenoceptor density, which persisted throughout the 21day recovery period (Fig. 5), clearly identifying that ␤2adrenoceptors are present in regenerating muscle. Effects of fenoterol. Fenoterol treatment had no effect on body mass, but it increased the mass of the heart (by 12%) after 14 days, although heart mass relative to body mass was unaffected. After 21 days of treatment, absolute and relative heart mass was 11% greater than in controls (Table 3; P ⬍ 0.05). The ␤2-agonist caused anabolic effects in healthy muscle, and accelerated recovery of injured muscle. In uninjured muscle, mass was increased 12, 19, and 13% after 7, 14, and 21 days of treatment, respectively (P ⬍ 0.05; Fig. 1), clearly demonstrating the previously observed hypertrophy of skeletal muscle after 28 days of fenoterol adminis-


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treated rats exhibited greater restoration of force-producing capacity than saline-treated rats, and Po was 90% of uninjured values (control muscles), compared with 76% for saline-treated rats. These findings strongly support the hypothesis that fenoterol treatment hastens regeneration of skeletal muscle following myotoxic injury. The potent anabolic effect of fenoterol treatment on skeletal muscle was clearly evident: mass, fiber CSA, protein content, and force production of uninjured muscles were greater in fenoterol-treated than saline-treated rats. These results support previous findings of enhanced force production, muscle mass, protein content, and fiber CSA after administration of various ␤2-agonists (31, 43) and confirm that fenoterol has powerful anabolic effects on skeletal muscle (10, 37). Synthetic ␤2J Appl Physiol • VOL

agonists promote muscle hypertrophy via activation of cAMPdependent mechanisms that increase protein synthesis and inhibit protein degradation pathways (8, 28, 34). The high efficacy of fenoterol (relative to clenbuterol) has previously been attributed to its full-agonist action to mediate greater cellular cAMP production (5). The potency of fenoterol to promote muscle hypertrophy may be influenced by the concentration of ␤2-adrenoceptors available for stimulation, which remained at control levels after 14 days but were downregulated with 21 days of administration of fenoterol. The timing of the downregulation of ␤2-adrenoceptors observed in the uninjured muscles from fenoterol-treated rats may be an important factor, contributing to the recognized potency of fenoterol compared with other more extensively studied ␤2-

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Fig. 3. Hematoxylin and eosin-stained sections of injured extensor digitorum longus muscles from rats treated with saline or fenoterol for a period or 2, 7, 14, or 21 days. Degeneration is maximal 2 days after the bupivacaine injection. Arrows show central nuclei, which indicate regenerating fibers. Note the greater muscle fiber CSA after 14 days of fenoterol treatment. (Scale applies to all panels.)

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agonists, such as clenbuterol (41), which causes marked downregulation within 10–12 days in rats (18, 40). This effect is associated with an attenuation of the muscle anabolic response (17, 40). Therefore, the absence of a fenoterol-mediated increase in protein content in the uninjured muscles at 21 days of treatment may be associated with the downregulation of adrenoceptors. Importantly, this study has identified that ␤2-adrenoceptors are actually present on regenerating skeletal muscle fibers. Although evidence for the existence of ␤2-adrenoceptors on Table 2. Contractile properties of uninjured and injured EDL muscles from rats treated with saline or fenoterol for 7, 14, or 21 days Saline Treatment Duration

7 Days n sPo, kN/m2 Pt, mN TPT, ms RT1/2, ms 14 Days n sPo, kN/m2 Pt, mN TPT, ms RT1/2, ms 21 Days n sPo, kN/m2 Pt, mN TPT, ms RT1/2, ms

Uninjured

Uninjured

myoblasts and myotubes has been conflicting, it has been postulated that adrenergic stimulation plays an important role in muscle development through increases in adenylate cyclase activity (24, 30, 38, 42). The marked increase in ␤2-adrenoceptor concentration observed in injured muscles from both saline-treated and fenoterol-treated rats at 2 days postinjury indicates that ␤2-adrenoceptors may play an important physiological role in the process of skeletal muscle regeneration. We believe that the upregulation of ␤2-adrenoceptors after injury would maximize endogenous catecholamine stimulation and would promote protein accretion and growth in regenerating muscles. Additional studies are required to characterize the downstream signaling and cellular response after activation of these ␤2-adrenoceptors in regenerating muscle and to determine whether recovery from muscle injury is impaired by ␤2-adrenoceptor antagonists. Table 3. Morphometric properties of rats treated with saline or fenoterol for 2, 7, 14, or 21 days

Fenoterol Injured

Fig. 5. ␤2-Adrenoceptor concentration of uninjured and injured EDL muscles from rats treated with saline or fenoterol for 2, 7, 14, or 21 days. It is important to note the marked increase in ␤2-adrenoceptor concentration at 2 days postinjury for both treatment groups. Downregulation of adrenoceptors occurred in both uninjured and injured muscle groups with fenoterol treatment. Values are means ⫾ SE. *Significantly different from time-matched salineuninjured (control) muscles, P ⬍ 0.05. #Significant difference between timematched saline-injured and fenoterol-injured muscles, P ⬍ 0.05.

Injured Treatment Duration

10 258.8⫾9.4 888⫾55 24.4⫾0.6 22.6⫾1.0

10 123.7⫾10.9* 281⫾24* 31.6⫾1.4* 35.3⫾1.3*

9 253.5⫾8.0 1002⫾52 26.9⫾1.2 25.4⫾1.1

9 128.3⫾11.6* 427⫾48*† 33.0⫾1.0* 38.0⫾2.3*

10 269.3⫾8.4 975⫾41 27.0⫾2.0 26.4⫾2.3

10 177.5⫾5.0* 640⫾28* 27.1⫾1.1 28.7⫾1.3

9 260.3⫾7.2 1189⫾34* 26.1⫾0.8 23.0⫾0.6

9 190.1⫾10.4* 761⫾64*† 25.7⫾0.7 26.8⫾0.9

10 277.2⫾4.5 1121⫾28 25.4⫾0.5 21.8⫾0.6

10 197.6⫾5.8* 811⫾38* 25.2⫾0.3 24.2⫾0.9

9 273.6⫾6.2 1293⫾32* 26.2⫾0.7 21.9⫾0.9

9 205.2⫾3.9* 952⫾40*† 24.7⫾0.7 23.2⫾1.7

Values are means ⫾ SE; n, no. of animals in each group. sPo, specific or normalized force; Pt, maximal isometric twitch force; TPT, time to peak twitch tension; RT1/2, one-half relaxation time. *Significantly different from timematched saline-uninjured (control) muscles, P ⬍ 0.05. †Significant difference between time-matched saline-injured and fenoterol-injured muscles, P ⬍ 0.05. J Appl Physiol • VOL

2 Days n Body mass, g Heart mass, g Heart mass/body 7 Days n Body mass, g Heart mass, g Heart mass/body 14 Days n Body mass, g Heart mass, g Heart mass/body 21 Days n Body mass, g Heart mass, g Heart mass/body

Saline

Fenoterol

mass, mg/g

8 348⫾10 1.02⫾0.04 0.29⫾0.01

8 348⫾6 1.09⫾0.04 0.31⫾0.01

mass, mg/g

10 375⫾11 1.10⫾0.04 0.29⫾0.01

9 376⫾12 1.19⫾0.02 0.32⫾0.01

mass, mg/g

10 415⫾9 1.14⫾0.04 0.28⫾0.01

9 428⫾11 1.28⫾0.03* 0.30⫾0.01

mass, mg/g

10 446⫾9 1.18⫾0.04 0.27⫾0.01

9 439⫾9 1.31⫾0.04* 0.30⫾0.01*

Values are means ⫾ SE; n, no. of animals in each group. *Significantly different from saline-treated rats, P ⬍ 0.05.

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Fig. 4. Maximal force production (Po) of uninjured and injured extensor digitorum longus muscles from rats treated with saline or fenoterol for 7, 14, or 21 days. Compared with control muscles, Po of uninjured muscles was greater after 7 days of fenoterol treatment. Injured muscles from fenoteroltreated rats exhibited a greater force-producing capacity than injured muscles from saline-treated rats at 14 days postinjury, indicating an improvement in contractile function of the regenerating muscles with fenoterol treatment. Values are means ⫾ SE. *Significantly different from time-matched salineuninjured (control) muscles, P ⬍ 0.05. #Significant difference between timematched saline-injured and fenoterol-injured muscles, P ⬍ 0.05.



at 21 days postinjury. Previous studies have also noted full recovery of morphometric and histochemical properties within 3–4 wk after intramuscular bupivacaine injection (3, 13). However, the evidence regarding the recovery of muscle function after injury has been conflicting. Rosenblatt (35) reported that isometric twitch and tetanic tension were at control levels within 21 days, whereas other studies suggest that at least 60 days are required for restoration of function after bupivacaine injection (7). Regardless of the duration for complete recovery, fenoterol treatment could potentially reduce the time required for full restoration of muscle function. This investigation has clearly identified the benefits of fenoterol for hastening restoration of muscle function after myotoxic injury. Future studies should determine whether such treatment can promote recovery of muscle function in conditions where normal muscle regeneration is impaired. Treatment with powerful ␤2-agonists like fenoterol may prove useful in promoting functional recovery of muscle after severe trauma or after plastic and reconstructive surgery, and for combating muscle wasting and weakness associated with ageing, neuromuscular disorders, prolonged sepsis, acquired immunodeficiency syndrome, burn injury, and cancer cachexia (20, 21). GRANTS This work was supported by research grants from the Muscular Dystrophy Association (USA), the Rebecca L. Cooper Medical Research Foundation, and The University of Melbourne. REFERENCES 1. Auman JT, Seidler FJ, Tate CA, and Slotkin TA. Are developing ␤-adrenoceptors able to desensitize? Acute and chronic effects of ␤-agonists in neonatal heart and liver. Am J Physiol Regul Integr Comp Physiol 283: R205–R217, 2002. 2. Awede BL, Thissen JP, and Lebacq J. Role of IGF-I and IGFBPs in the changes of mass and phenotype induced in rat soleus muscle by clenbuterol. Am J Physiol Endocrinol Metab 282: E31–E37, 2002. 3. Benoit PW and Belt WD. Destruction and regeneration of skeletal muscle after treatment with a local anaesthetic, bupivacaine (Marcaine). J Anat 107: 547–556, 1970. 4. Bischoff R. Analysis of muscle regeneration using single myofibers in culture. Med Sci Sports Exerc 21: S164–S172, 1989. 5. Bremner P, Siebers R, Crane J, Beasley R, and Burgess C. Partial vs full receptor agonism—a clinical study of inhaled albuterol and fenoterol. Chest 109: 957–962, 1996. 6. Brooks SV and Faulkner JA. Contractile properties of skeletal muscles from young, adult and aged mice. J Physiol 404: 71–82, 1988. 7. Carlson BM and Faulkner JA. The regeneration of noninnervated muscle grafts and marcaine-treated muscles in young and old rats. J Geronotol 51A: B43–B49, 1996. 8. Choo JJ, Horan MA, Little RA, and Rothwell NJ. Anabolic effects of clenbuterol on skeletal muscle are mediated by ␤2-adrenoceptor activation. Am J Physiol Endocrinol Metab 263: E50–E56, 1992. 9. Emery PW, Rothwell NJ, Stock MJ, and Winter PD. Chronic effects of ␤2-adrenergic agonists on body composition and protein synthesis in the rat. Biosci Rep 4: 83–91, 1984. 10. Garrett RP, Britain KG, Savell JW, Edwards JW, and Smith SB. Body composition of lambs receiving 30 or 60 days of exercise training and (or) fenoterol treatment. Meat Sci 52: 235–246, 1999. 11. Gregorevic P, Williams DA, and Lynch GS. Hyperbaric oxygen increases the contractile function of regenerating rat slow muscles. Med Sci Sports Exerc 34: 630–636, 2002. 12. Hall-Craggs ECB. Rapid degeneration and regeneration of a whole skeletal muscle following treatment with bupivacaine (Marcain). Exp Neurol 43: 349–358, 1974. 13. Hall-Craggs ECB and Seyan HS. Histochemical changes in innervated and denervated skeletal muscle fibres following treatment with bupivacaine (Marcain). Exp Neurol 46: 345–354, 1975.

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Whereas the receptor density in the regenerating muscles of saline-treated rats was strikingly higher than in uninjured muscles at all time points, the receptor density in regenerating muscles from fenoterol treated rats was only greater than controls at 2 days postinjury, with significant reduction in adrenoceptor density apparent from 7 days onward. The findings indicate that there may be a potentially diminishing influence of fenoterol as regeneration proceeds due to the reduction in ␤2-adrenoceptor density with chronic treatment. Downregulation of ␤2-adrenoceptors with fenoterol treatment occurred more rapidly in injured compared with uninjured muscles. It is possible that during regeneration, the ␤2-adrenoceptors may be more susceptible to characteristic phosphorylation and subsequent sequestration and downregulation processes that occur during chronic exposure to ␤2-agonists (2, 16). In future experiments, this issue could be resolved by examination of adrenoceptor activity in regenerating muscles after chronic fenoterol administration. Fenoterol may influence the early stages of regeneration before the formation of new muscle fibers is complete. Possible mechanisms of enhanced muscle repair after treatment include a ␤-agonist-mediated increase in vascularization, increased satellite cell proliferation and differentiation (32, 33), an increased expression of local growth factors such as IGF-I (2), inhibition of proteolysis, and promotion of protein synthesis (27). These proposed mechanisms are consistent with fenoterol binding directly to ␤2-adrenoceptors to stimulate downstream signaling pathways, promoting protein accretion and causing hypertrophy of regenerating fibers. Thus the increase in fiber CSA and mass of injured muscles with treatment resulted directly from the fenoterol-induced increase in muscle protein content. Similar to fiber CSA, absolute protein content of injured muscles from fenoterol-treated rats had returned to control levels by 14 days postinjury. By 21 days postinjury, absolute protein content of injured muscles from fenoteroltreated rats not only was greater than that in injured muscle from saline-treated rats but also was the highest of all groups. This is in contrast to muscles from saline-treated rats, in which the deficit in fiber CSA and protein concentration between injured and control muscles was still evident at 21 days postinjury, although the mass of injured muscles had exceeded control levels. This suggests a higher muscle water content in injured muscles from saline-treated rats. These findings show that the beneficial effects of fenoterol in regenerating skeletal muscle were mediated by enhanced protein accretion. Therefore, the enhanced functional capacity of regenerating muscles after fenoterol treatment occurred because of direct increases in muscle protein content, fiber CSA, and muscle mass. However, it should be noted that the Po and sPo of injured muscles in rats from both treatment groups did not return to control levels by 21 days postinjury, despite the fact that muscle protein content, fiber CSA, and mass had recovered to, or exceeded, control levels at this time point. The disparity between the rate of recovery of muscle mass and muscle force-producing capacity could be attributed to the expression of developmental isoforms of contractile and regulatory proteins during muscle regeneration, which have reduced functionality compared with mature isoforms of these proteins (11, 36). These findings indicate that the internal structure of the muscle was undergoing remodeling and maturation processes

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