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CARDIOVASCULAR
2-Adrenoceptor Agonist Prodrug BRL-47672 Impairs Rat Skeletal Muscle Function by Inducing a Comprehensive Shift to a Faster Muscle Phenotype
Centre for Integrated Systems Biology and Medicine, School of Biomedical Science, University of Nottingham, Nottingham, United Kingdom (D.J.B., D.C.-T., S.W.J., P.L.G.); and Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden (J.A.T.)
Received April 27, 2006; accepted July 13, 2006.
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Abstract |
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2-Agonist treatment increases muscle mass, yet it alters fiber proportions such that the net consequences on muscle function remain unclear. In the present study, we focus on the contractile and metabolic consequences of chronic treatment with the 2-agonist prodrug BRL-47672 (BRL). Gastrocnemius-plantaris-soleus (GPS) muscles were harvested at rest and studied for fatigue characteristics during 4 and 20 s of isometric stimulation (30 Hz; 10 V; 200 ms) using the perfused hind limb model. BRL treatment increased GPS mass by 21% (P < 0.05), whereas greater fatigue occurred during 20 s of contraction (45% less work; P < 0.05). Phenotypically, BRL resulted in 17% more type IIb myosin heavy chain protein expression (P < 0.001) and greater adenine nucleotide catabolism during 20 s of contraction (P < 0.05). Chronic BRL treatment impaired maximal lipid oxidation capacity by 30% (P < 0.05) and reduced glutamate dehydrogenase activity by 15% (P < 0.05). We conclude that 2-agonist induced muscle hypertrophy may be clinically limited as impaired energy metabolism and function occur, presumably as a consequence of the shift in muscle phenotype
; Ryall et al., 2004) have a negative impact on quality of life. The maintenance, or ideally, improvement of muscle strength and function (fatigue resistance) is therefore of clinical importance. Chronic administration of a 2-agonist, such as clenbuterol or fenoterol, undoubtedly induces muscle fiber hypertrophy in animal models (Emery et al., 1984; Kim et al., 1987; MacLennan and Edwards, 1989; Yang and McElligott, 1989; Cartana et al., 1994), highlighting its potential for use in a clinical setting. However, chronic 2-agonist treatment also results in impaired cardiac function (Sleeper et al., 2002) and a change in skeletal muscle fiber composition toward a faster phenotype (Zeman et al., 1988; Morton et al., 1995; Dodd et al., 1996; Rajab et al., 2000; Ryall et al., 2002, 2004; Timmons, 2002; Jones et al., 2004). For example, in young animals, the maturation-related increase in slow myosin heavy chain (MyHC) expression is attenuated following chronic 2-agonist treatment, resulting in a greater fast muscle fiber population (Timmons, 2002; Jones et al., 2004). Furthermore, it seems that this response is at least partly the result of a rapid 2-agonist-mediated increase in the expression of the "fast muscle" transcription factor MyoD (Jones et al., 2004). This is in concordance with other studies demonstrating the development of fast muscle twitch characteristics in vitro (Ryall et al., 2002, 2004) and an increase in muscle ATP content and cellular energy charge (EC) (Rajab et al., 2000) as a result of chronic 2-agonist treatment. Collectively, these findings point toward a global shift in muscle phenotype, not just a shift in contractile protein expression. Interestingly, the shorter muscle relaxation rates and greater muscle peak twitch forces observed following chronic administration of clenbuterol (Zeman et al., 1988) can be reversed with concurrent administration of 2-antagonists (Zeman et al., 1988; Morton et al., 1995), demonstrating that the underlying control mechanism(s) relates directly to 2 agonism.
We have previously demonstrated that administration of the 2-agonists prodrug BRL-47672 (BRL), which is structurally similar to clenbuterol (Sillence et al., 1995) and is metabolized in vivo to its active component, results in an attenuation of unwanted cardiovascular responses, which is a more favorable profile for use in a clinical setting compared with direct agonists (Jones et al., 2004). The impact that BRL has on contractile protein expression, metabolic phenotype, and function has not been the subject of scrutiny. Any deleterious alterations, even in the face of an improved cardiovascular side effect profile, would have major implications for muscle function and therefore require investigation before proceeding with such ideas beyond the preclinical laboratory. In rodent models, 2-agonist treatment has been reported to improve muscle function following hind limb unloading (Dodd and Koesterer, 2002), and more recently, to reverse age associated decline in muscle mass (Ryall et al., 2002). These positive effects were noted because function reflected the increase in total muscle mass, a parameter that may be most relevant for patients with extreme muscle wasting. We must also consider that the 2-agonist-induced increase in fast MyHC expression may promote fatigue development during sustained contraction, because of a limited inherent capacity for fast muscle fibers to maintain ATP turnover (Hultman et al., 1991). The issue is further complicated by the observation that an increase in mitochondrial enzyme expression has been noted following chronic 2-agonist treatment (Dodd et al., 1996), which could be beneficial to function, during sustained contraction. In light of these considerations, and the potential clinical benefits from chronic 2-agonist treatment, the current study investigated the effects of chronic treatment with BRL on muscle twitch characteristics and isometric tension development during sustained contraction. We determined muscle adenine nucleotide breakdown during contraction, as a measure of the ability of muscle to maintain ATP turnover during contraction. Finally, mitochondrial enzyme activities and the maximal in vitro rate of mitochondrial ATP production were determined to inform us about changes in mitochondrial density and function, respectively.
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Materials and Methods |
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2-Agonist Prodrug Treatment. Sixty male Wistar rats (150-160 g; Charles River, Margate, Kent, UK) were divided into two treatment groups. One group (n = 30) received daily subcutaneous injections of 2-agonist prodrug BRL at 900 µg kg-1 body mass for 1 day (n = 10), 4 weeks (n = 10), or 8 weeks (n = 10). The second group (n = 30) received daily subcutaneous injections of an isovolume of saline for 1 day (n = 10), 4 weeks (n = 10), or 8 weeks (n = 10). The day following the final injection of either 2-agonist or saline, the rats were anesthetized (inactin; 120 mg kg-1 body mass i.p.), and the gastrocnemius muscles were excised, frozen in liquid nitrogen, and then weighed before subsequent separation and quantification of MyHC expression as described below. All experimental use of animals was approved by the UK Home Office and conducted in accordance with the laws governing the use of animals in research Act of 1986.
Myosin Heavy Chain Analysis. Crude myosin was prepared as described previously (Jones et al., 2004). Protein extract (0.1-0.5 µg) was electrophoretically separated using SDS-polyacrylamide gel electrophoresis (6%) gels containing 40% glycerol and then silver-stained using Silver Stain Plus kit (Bio-Rad, Hemel, Hempstead, UK), as done previously (Jones et al., 2004). Gels were then scanned, and computerized densitometry was used to quantify band intensity for each MyHC isoform, which was expressed as a percentage of the total MyHC content, and thus is representative as MyHC content as a proportion of the total expression.
Experiment to Determine the Effect of Chronic 2-Agonist Prodrug Treatment on Muscle Function and Metabolism. Forty-one male Wistar rats (250-350 g; Charles River) received daily subcutaneous injections of BRL (900 µg kg-1 body mass) or an isovolume of saline for 8 wk. The day following the final injection, rats were terminally anesthetized to allow vascular isolation of the gastrocnemius-plantaris-soleus (GPS) muscle group. Animals then underwent 60 min of perfusion (resting muscle: saline, n = 8; BRL, n = 6) or 60 min of perfusion followed immediately by isometric contraction at maximal intensity for either 4 s (saline, n = 8; BRL, n = 6) or 20 s (saline, n = 7; BRL, n = 6).
Hind Limb Perfusion Model. We used our novel version (Baker et al., 2005, 2006) of the standard perfused rat hind limb preparation. Male Wistar rats were anesthetized (inactin; 120 mg kg-1 body mass i.p.), and the musculature of the left hind limb was exposed by removal of the skin from this region. The branches of the femoral artery and vein were ligated using silk ligatures or thermocautery up to the point where these vessels entered the GPS muscle complex. This ensured that blood flow to and from this muscle group was solely via the intact femoral artery and vein. The bicep femoris was then removed, and a length of thread was tied around the Achilles tendon and cut distal to the ligature. This resulted in the GPS muscle group remaining fixed to the limb on the dorsal side of the knee joint. The femoral artery and vein were then cannulated, and heparinized saline (10 U ml-1) was slowly flushed through the vasculature of the GPS muscle group. An arterial cannula was then attached to a primed perfusion system that was contained in an enclosed chamber that maintained an ambient temperature of 37°C, and the muscle group was perfused with previously prepared perfusion media (see below for details). The animal was then killed humanely (according to UK Home Office Guidelines) and placed ventral surface down to enable the tibia to be secured using a clamp fixed to a stereological frame, after which the thread from the Achilles tendon was attached to an isometric force transducer (Grass Instruments, Warwick, RI). Clamping the tibia facilitated the measurement of muscle force production during contraction by minimizing inertia generated by movement of the animal.
The perfusion media contained isolated porcine red blood cells suspended in a modified Krebs' buffer containing 5% bovine serum albumin, 100 µU ml-1 insulin, and 0.15 mM pyruvate (adjusted to pH 7.4; 47% hematocrit and 6 mM glucose). The GPS was perfused with the cell suspension for 60 min (15 ml min-1 100 g-1 wet muscle) before undergoing assessment of single twitch characteristics, and then in a randomized order one of the following steps was conducted: 1) excision of the GPS muscle group, which was then immediately snap-frozen in liquid nitrogen (rested baseline muscle); or 2) 4 s or 3) 20 s of maximal intensity tetanic isometric contraction (30 Hz; 200 ms; 10 V), achieved via direct electrical stimulation of the sciatic nerve using a hook electrode (Harvard Apparatus, Holliston, MA). Isometric tension was recorded throughout (MacLab 400; ADInstruments Pty Ltd., Castle Hill, Australia). Immediately following contraction, the GPS muscle group was rapidly excised and snap-frozen in liquid nitrogen. The muscle group was subsequently weighed frozen and then stored in liquid nitrogen until further analysis was performed.
Muscle Metabolite Analysis. The GPS muscle group was crushed under liquid nitrogen and thoroughly mixed to create a homogenous representation of the whole GPS muscle group. An aliquot of this pool of crushed, frozen tissue was then freeze-dried overnight and stored at -80°C. At a later date, this freeze-dried muscle was powdered and extracted (Harris et al., 1974), and then it was used for the determination of muscle ATP, ADP, AMP, IMP, inosine, hypoxanthine, xanthine, and uric acid concentrations by high-performance liquid chromatography (Idstrom et al., 1990), and for determination of muscle glycogen concentration using spectrophotometry (Harris et al., 1974).
Mitochondrial ATP Production Rates. Approximately 50 mg of muscle was excised from the middle part of each soleus muscle undergoing perfusion only (i.e., resting), and, following rapid weighing, was used immediately for the bioluminometric determination of ATP production (mitochondrial ATP production rate) rates in isolated mitochondria using a variety of substrates (Wibom et al., 1990). Mitochondria were prepared by differential centrifugation following muscle homogenization in a Potter-Elvehjem glass homogenizer fitted with a Teflon pestle. This method is based on the reaction of ATP with firefly luciferase, providing a light signal proportional to the concentration of ATP in the solution. The following substrate concentrations (in the cuvette) were used to measure mitochondrial activity: 1) 1 mM pyruvate + 5 µM palmitoyl L-carnitine + 10 mM 
-ketoglutarate + 1 mM malate 1, 2) 10 mM glutamate, 3) 10 mM -ketoglutarate, 4) 5 µM palmitoyl L-carnitine + 1 mM malate, 5) 1 mM pyruvate 1 + 1 mM malate, 6) 20 mM succinate and 0.1 mM rotenone, and 7) 20 mM succinate. The same mitochondrial suspension was used at a later date for the measurement of glutamate dehydrogenase activity. The yield for mitochondria for each mitochondrial preparation was determined from the ratio between glutamate dehydrogenase activity determined in the whole muscle homogenate and that determined in the mitochondrial suspension.
Enzyme Activities. The activities of muscle glutamate dehydrogenase (GDH) and citrate synthase (CS) were determined as markers for mitochondrial matrix, -hydroxy-acyl-CoA dehydrogenase (HAD) was determined as an index of mitochondrial oxidation, and glyceraldehyde-3-phosphate dehydrogenase (Gly3PDH) was determined as a measure of cytosolic-based glycolysis. Frozen muscle (5-10 mg) was homogenized on ice in a solution containing 50 mM KH2PO4, 1 mM EDTA, and 0.05% Triton X-100. The muscle homogenates were then analyzed for the enzymes activities outlined above as described by Opie and Newsholme (1967) and Zammit and Newsholme (1976). Protein content in the mitochondrial suspension was determined by the Bradford method (Bradford, 1976).
Statistics. Comparison of means was performed using two-way analysis of variance with an least significant difference post hoc test or Student's unpaired t test where appropriate. Pearson's correlation coefficient was used to determine any significant relationships. Values of P < 0.05 were deemed to be significant. Values in text, table, and figures represent mean ± S.E.M.
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Results |
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Saline administration did not alter type I MyHC protein expression over the course of the experiment. No difference in type I MyHC protein expression existed between saline- and BRL-treated groups after 1 day of administration. However, following 4 weeks of BRL administration, the expression of type I MyHC protein had decreased by 50% compared with 1 day (P < 0.01) such that a clear difference was evident between treatment groups at this time point (P < 0.001), which was maintained at the 8-week time point (P < 0.01).
Type IIa MyHC protein expression did not differ between treatments after 1 day of administration (8.7 ± 0.7 versus 9.5 ± 0.3%). After 4 weeks of saline treatment, an increase in IIa protein expression was observed (11.0 ± 0.9%; P < 0.05 compared with 1 day), which remained unchanged for the rest of the study. BRL treatment decreased type IIa protein expression to 7.8 ± 0.7% after 4 weeks (P < 0.05 compared with 1 day and saline treatment), which was also maintained for the remainder of the study (significantly lower than saline-treated group; P < 0.05).
Type IIx MyHC protein expression was no different between treatment groups at day 1. Expression was unchanged in the saline-treated group after 4 weeks, but a significant increase was observed after 8 weeks (P < 0.05 compared with 4 weeks; P < 0.001 compared with 1 day), reflecting maturation-related changes. Type IIx MyHC protein expression remained unchanged over the course of the study in the BRL-treated group, such that expression was 25% less in this group compared with saline after 8 weeks (P < 0.001).
Type IIb MyHC protein expression decreased in the saline-treated group by 11% over the course of the study, the decline of which was similar in magnitude to the increase seen in type IIx MyHC protein expression (9%) in the same group. There was no difference in the type IIb MyHC protein expression between saline- and BRL-treated groups at 1 day; however, after 4 weeks, expression in the BRL-treated group was 
10% greater compared with the saline-treated group (P < 0.01) and increased further to 17% after 8 weeks of administration (P < 0.001).
Muscle Function. There was no difference in twitch peak tension between treatments. Time to peak tension was 14% shorter following 8 weeks of BRL administration compared with saline (P < 0.001; data not shown) Likewise, relaxation time, calculated as the time from peak tension to resting tension, was 20% lower following BRL administration compared with saline (Fig. 2A). Figure 2B shows muscle tension development (expressed as a percentage of initial peak tension) after 4 and 20 s of contraction following 8 weeks of saline or BRL administration. There was no difference in the percentage of decline of force from peak tension between saline and BRL treatment groups after 4 s of contraction (39.5 ± 5.1 versus 36.7 ± 6.0%, respectively). Muscle tension in the saline-treated group was unchanged between 4 and 20 s. BRL treatment, however, caused a further 13% reduction in force from 4 to 20 s of contraction compared with saline treatment (P < 0.05; 37.0 ± 4.7 versus 50.4 ± 2.4%, respectively). Figure 2C shows the area under the tension x time curve in both treatment groups following 4 and 20 s of contraction, which represents the total amount of work performed. In line with Fig. 2A, no difference in total work output was observed after 4 s of contraction when comparing groups. However, by 20-s BRL treatment resulted in 45% less work being performed compared with the saline-treated group (P < 0.05).
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Muscle Metabolites, Energy Charge, and Glycogen Content. Table 1 shows muscle metabolites, energy charge, and glycogen content at rest and after 4 and 20 s of maximal intensity contraction following 8 weeks of saline or BRL treatment. Resting muscle ATP concentration was greater in the BRL-treated group compared with saline (P < 0.05), and resting AMP concentration was lower (P < 0.05). These differences were reflected by a higher cellular energy charge (ATP + 0.5 ADP/ATP + ADP + AMP) at rest in the BRL-treated group compared with the saline-treated group.
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Muscle ATP concentration did not change from its resting value in the saline-treated group during contraction (Table 1). However, muscle ATP was significantly lower (27.8 ± 0.5 mmol · kg-1 dry mass) by 20 s of contraction in the BRL-treated group (P < 0.05). Muscle IMP concentration increased from rest in both treatment groups by 20 s of contraction; however, this increase was 2-fold greater in the BRL-treated group compared with saline. No difference was observed between or within treated groups for muscle ADP, inosine, hypoxanthine, and xanthine concentrations (Table 1). Figure 3A presents the sum of the products of muscle adenine nucleotide degradation at rest and following 4 and 20 s of contraction in each treatment group. There was no difference between treatment groups at rest. Furthermore, contraction did not result in any change from rest in the saline-treated group. However, BRL treatment was associated with a cumulative increase in adenine nucleotide breakdown products after 4 s (P < 0.05) and 20 s (P < 0.01) of contraction (Fig. 3A). Figure 3B shows that a significant correlation existed between the magnitude of muscle fatigue development (percentage of decline from peak tension) and accumulation of muscle adenine nucleotide breakdown products (R2 = 0.656; P < 0.05) over the course of 20 s of contraction. Resting muscle glycogen content was 17% greater in the saline-treated group (Table 1; P < 0.05). However, the magnitude of glycogen degradation was no different between treatments, being 31 mmol · kg-1 dry mass over the course of 20 s of contraction.
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-ketoglutarate, and malate and with each of the following: glutamate, -ketoglutarate, palmitoyl L-carnitine and malate, pyruvate and malate, succinate and rotenone, and succinate (Succ). The rate of mitochondrial ATP production in the presence of palmitoyl L-carnitine and malate was 30% lower following BRL treatment compared with saline (9.39 ± 1.31 and 6.74 ± 0.47 mmol ATP min-1 kg-1 wet muscle, respectively; P < 0.05). None of the remaining measurements of mitochondrial function were different between treatment groups.
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Enzyme Activities. BRL treatment did not alter the activity of CS, HAD, or Gly3PDH from that observed in the saline-treated group. However, there was a significant reduction in the activity of GDH following 8 week of BRL treatment compared with saline (Fig. 4B; P < 0.05).
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Discussion |
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2-agonist administration increases muscle mass (Morton et al., 1995). Furthermore, administration of a single i.p. bolus dose of BRL avoids much of the severe hemodynamic responses observed with clenbuterol (Jones et al., 2004), presumably because BRL is a prodrug of clenbuterol itself, thus rendering it a more favorable option for use in a clinical setting. However, BRL treatment is also associated with a shift in muscle fiber composition (greater faster contractile protein expression), giving it the potential to promote muscle fatigue development during sustained contraction and thereby it is of limited clinical use. In addition, it has been postulated that the altered muscle phenotype is secondary to the severe cardiovascular response (Rothwell et al., 1987) such that the net impact of BRL on muscle function was unclear. The present study comprehensively determined the impact of chronic BRL treatment on muscle phenotype and contractile function in the rat. Collectively, these observations provide insight into the long-term viability of this therapeutic strategy.
The major findings of the present study were that although the 21% increase in muscle mass observed following 8 weeks of treatment with BRL was within the typical range of increase seen in rodents (Zeman et al., 1988; Jones et al., 2004), this was also accompanied by 17% greater type IIb MyHC protein expression, a shorter rate of muscle twitch relaxation, and almost 50% greater fatigue development during 20 s of maximal isometric contraction. In accordance with these observations, muscle adenine nucleotide breakdown was greater during contraction following BRL. In addition, the maximal rate of mitochondrial ATP production from fat oxidation was reduced by 30%, as was muscle GDH activity (a marker of mitochondrial density), collectively indicating a transition in the "energy metabolism" machinery. These observations indicate that BRL induced shift in contractile protein expression, but also a myriad of physiological and metabolic adaptations, not least changes in rates of ATP turnover during contraction (Hultman et al., 1991) and alterations in calcium reuptake kinetics (Schertzer et al., 2005). It would also seem clear that muscle phenotype alterations can occur in the face of blunted cardiovascular activation by the 2-agonist, dissociating these two phenomena. It is logical to conclude, therefore, that although chronic 2-agonist treatment can increase muscle mass, the greater fatigability that accompanies this response may limit the clinical usefulness of this strategy.
Changes in contractile characteristics and biochemical properties following 2-agonist treatment have been reported previously (Torgan et al., 1993; Dodd et al., 1996); however, we have combined representative measurements of muscle function, metabolism, and composition for the first time to comprehensively demonstrate that chronic 2-agonist treatment shifts muscle in a global manner toward a faster phenotype. We have previously found that BRL-induced shift in muscle phenotype may be linked to a rapid up-regulation of MyoD, a myogenic transcription factor that predominates in fast muscle (Jones et al., 2004). Ryall et al. (2002) demonstrated that 4-week treatment with the 2-agonist fenoterol increased muscle fiber size but impaired EDL and soleus muscle function during recovery in vitro (Ryall et al., 2002). The present study has been able to demonstrate that this impairment of muscle function persists in a setting, where oxygen supply is adequate and conditions are more similar to the in vivo scenario. Furthermore, because we have systematically characterized muscle metabolism during contraction, we have been able to provide insight as to the potential mechanism(s) by which chronic 2-agonist treatment accelerates fatigue. Specifically, we show that isometric tension development did not differ between treatment groups, during the initial 4 s of contraction. However, after 20 s of contraction, there was a significant increase in fatigue development in the BRL-treated group (Fig. 2B), as evidenced by the 45% reduction in the area under the time-tension curve (Fig. 2C). In accordance with these observations, muscle ATP concentration was maintained throughout contraction in the saline-treated group, but it declined in BRL-treated group and was paralleled by a sequential accumulation of the products of adenine nucleotide pool breakdown (Fig. 3A). The loss of muscle adenine nucleotides during our study with BRL, presenting itself as an increase in muscle IMP, inosine, hypoxanthine, and xanthine, has been closely implicated with fatigue development and is considered to reflect the inability of muscle to maintain ATP turnover during contraction, particularly during high-intensity contraction in fast muscle fibers (Meyer et al., 1980; Dudley and Terjung, 1985; Sewell and Harris, 1992; Harris et al., 1997). When AMP is produced it is broken down to IMP in an effort to stabilize the cellular ATP/ADP ratio under conditions of cellular stress (Atkinson, 1970; Meyer and Terjung, 1980). In addition, because AMP catabolism is regulated by AMP deaminase, which is activated by transient increases in muscle AMP and a decline in muscle pH (Wheeler and Lowenstein, 1979; Dudley and Terjung, 1985), it is plausible that AMP deaminase expression was increased by chronic 2-agonist treatment, particularly because its activity is typically greater in fast muscle fibers. In line with this proposed relationship between muscle adenine nucleotide loss and fatigue development, we observed a significant correlation between the extent of muscle tension loss over 20 s of contraction and the magnitude of accumulation of the products of adenine nucleotide degradation (namely, IMP, inosine, hypoxanthine, xanthine, and uric acid; R2 = 0.656; P < 0.05; Fig. 3B). This suggests that the global transition of skeletal muscle to a faster phenotype following BRL treatment is detrimental to muscle function; however, some evidence suggests that 2-agonist treatment in conjunction with aerobic training can counter these deleterious slow-fast fiber type transformations (Lynch et al., 1996). The clinical usefulness of a 2-agonist and exercise treatment regimen has yet to be elucidated.
In the present study, we determined that muscle glycogen content was reduced in the basal state following 8 weeks of treatment with BRL compared with saline treatment. We think this response could have occurred as a result of chronic 2-adrenreceptor stimulation, resulting in muscle cyclic AMP concentration being chronically elevated, and, in turn, activating glycogen phosphorylase and thereby glycogen degradation. This effect of adrenoreceptor agonism on muscle glycogen degradation has been shown to occur in resting human muscle (Chasiotis et al., 1983) and in the rat (Chasiotis, 1985) following acute administration of adrenaline. Conversely, adrenoreceptor antagonists are known to inhibit muscle glycogen degradation during contraction (Van Baak et al., 1995). However, because the rate of glycogenolysis over the 20-s contraction was identical between treatment groups (1.57 versus 1.56 mmol glucosyl units s-1 kg-1 dry mass), this suggests that the glycogen availability was not limiting to muscle tension development during contraction. In summary, although 8 weeks of treatment with BRL resulted in a 21% increase in muscle mass, this was paralleled by phenotype changes that resulted in greater fatigue during 20 s of maximal isometric contraction. The tight relationship between muscle adenine nucleotide breakdown and muscle function, coupled with a reduction in in vitro mitochondrial ATP production and mitochondrial density, suggests that impaired energy metabolism may be central to the enhanced fatigue profile. Our findings suggest that although a strategy of chronic 2-agonist prodrug administration may be helpful in reversing muscle atrophy models, the greater fatigability that accompanies such treatment complicates its clinical usefulness, particularly because elderly or frail individuals typically work close to the maximal limits of their muscle capacity (Young, 1987).
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Footnotes |
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ABBREVIATIONS: MyHC, myosin heavy chain; EC, energy charge; GPS, gastrocnemius-plantaris-soleus; GDH, glutamate dehydrogenase; CS, citrate synthase; HAD, -hydroxy-acyl-CoA dehydrogenase; Gly3PDH, glyceraldehyde-3-phosphate dehydrogenase; Succ, succinate.
1 These authors contributed equally to the article and should be considered as joint first authors. 
Address correspondence to: Dr. David J. Baker, Faculty of Kinesiology, University of Calgary, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada. E-mail: dbaker{at}kin.ucalgary.ca
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Atkinson DE (1970) Adenine nucleotides as universal stoichiometric metabolic coupling agents. Adv Enzyme Regul 9: 207-219.[Medline]
Baker DJ, Greenhaff PL, MacInnes A, and Timmons JA (2006) The experimental type 2 diabetes therapy glycogen phosphorylase inhibition can impair aerobic muscle function during prolonged contraction. Diabetes 55: 1855-1861.
Baker DJ, Timmons JA, and Greenhaff PL (2005) Glycogen phosphorylase inhibition in type 2 diabetes therapy: a systematic evaluation of metabolic and functional effects in rat skeletal muscle. Diabetes 54: 2453-2459.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.[CrossRef][Medline]
Busquets S, Figueras MT, Fuster G, Almendro V, Moore-Carrasco R, Ametller E, Argiles JM, and Lopez-Soriano FJ (2004) Anticachectic effects of formoterol: a drug for potential treatment of muscle wasting. Cancer Res 64: 6725-6731.
Cartana J, Segues T, Yebras M, Rothwell NJ, and Stock MJ (1994) Anabolic effects of clenbuterol after long-term treatment and withdrawal in t the rat. Metabolism 43: 1086-1092.[CrossRef][Medline]
Chasiotis D (1985) Effects of adrenaline infusion on cAMP and glycogen phosphorylase in fast-twitch and slow-twitch rat muscles. Acta Physiol Scand 125: 537-540.[Medline]
Chasiotis D, Sahlin K, and Hultman E (1983) Regulation of glycogenolysis in human muscle in response to epinephrine infusion. J Appl Physiol 54: 45-50.
Dodd SL and Koesterer TJ (2002) Clenbuterol attenuates muscle atrophy and dysfunction in hindlimb-suspended rats. Aviat Space Environ Med 73: 635-639.[Medline]
Dodd SL, Powers SK, Vrabas IS, Criswell D, Stetson S, and Hussain R (1996) Effects of clenbuterol on contractile and biochemical properties of skeletal muscle. Med Sci Sports Exerc 28: 669-676.
Dudley GA and Terjung RL (1985) Influence of aerobic metabolism on IMP accumulation in fast-twitch muscle. Am J Physiol 248: C37-C42.[Medline]
Emery PW, Rothwell NJ, Stock MJ, and Winter PD (1984) Chronic effects of beta 2-adrenergic agonists on body composition and protein synthesis in the rat. Biosci Rep 4: 83-91.[CrossRef][Medline]
Harris DB, Harris RC, Wilson AM, and Goodship A (1997) ATP loss with exercise in muscle fibres of the gluteus medius of the thoroughbred horse. Res Vet Sci 63: 231-237.[CrossRef][Medline]
Harris RC, Hultman E, and Nordesjo LO (1974) Glycogen, glycolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values. Scand J Clin Lab Investig 33: 109-120.[Medline]
Hultman E, Greenhaff PL, Ren JM, and Soderlund K (1991) Energy metabolism and fatigue during intense muscle contraction. Biochem Soc Trans 19: 347-353.[Medline]
Idstrom JP, Soussi B, Wanag E, and Bylund-Fellenius AC (1990) Analysis of purine nucleotides in muscle tissue by HPLC. Scand J Clin Lab Invest 50: 541-549.[Medline]
Jones SW, Baker DJ, Gardiner SM, Bennett T, Timmons JA, and Greenhaff PL (2004) The effect of the beta2-adrenoceptor agonist prodrug BRL-47672 on cardiovascular function, skeletal muscle myosin heavy chain, and MyoD expression in the rat. J Pharmacol Exp Ther 311: 1225-1231.
Kim YS, Lee YB, and Dalrymple RH (1987) Effect of the repartitioning agent cimaterol on growth, carcass and skeletal muscle characteristics in lambs. J Anim Sci 65: 1392-1399.
Lynch GS, Hayes A, Campbell SP, and Williams DA (1996) Effects of beta 2-agonist administration and exercise on contractile activation of skeletal muscle fibers. J Appl Physiol 81: 1610-1618.
MacLennan PA and Edwards RH (1989) Effects of clenbuterol and propranolol on muscle mass. Evidence that clenbuterol stimulates muscle beta-adrenoceptors to induce hypertrophy. Biochem J 264: 573-579.[Medline]
Meyer RA, Dudley GA, and Terjung RL (1980) Ammonia and IMP in different skeletal muscle fibers after exercise in rats. J Appl Physiol 49: 1037-1041.
Meyer RA and Terjung RL (1980) AMP deamination and IMP reamination in working skeletal muscle. Am J Physiol 239: C32-C38.[Medline]
Morton RH, Agbenyega ET, Hatton PA, and Wareham AC (1995) Effects of clenbuterol and ICI118551, a selective beta 2-antagonist, on the growth of skeletal muscle of suckling rats. Pflueg Arch Eur J Physiol 431: 237-243.[CrossRef][Medline]
Opie LH and Newsholme EA (1967) The activities of fructose 1,6-diphosphatase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle. Biochem J 103: 391-399.[Medline]
Rajab P, Fox J, Riaz S, Tomlinson D, Ball D, and Greenhaff PL (2000) Skeletal muscle myosin heavy chain isoforms and energy metabolism after clenbuterol treatment in the rat. Am J Physiol 279: R1076-R1081.
Rothwell NJ, Stock MJ, and Sudera DK (1987) Changes in tissue blood flow and beta-receptor density of skeletal muscle in rats treated with the beta2-adrenoceptor agonist clenbuterol. Br J Pharmacol 90: 601-607.[Medline]
Ryall JG, Gregorevic P, Plant DR, Sillence MN, and Lynch GS (2002) Beta 2-agonist fenoterol has greater effects on contractile function of rat skeletal muscles than clenbuterol. Am J Physiol 283: R1386-R1394.
Ryall JG, Plant DR, Gregorevic P, Sillence MN, and Lynch GS (2004) Beta 2-agonist administration reverses muscle wasting and improves muscle function in aged rats. J Physiol (Lond) 555: 175-188.
Schertzer JD, Plant DR, Ryall JG, Beitzel F, Stupka N, and Lynch GS (2005) Beta2-agonist administration increases sarcoplasmic reticulum Ca2+-ATPase activity in aged rat skeletal muscle. Am J Physiol 288: E526-E533.
Sewell DA and Harris RC (1992) Adenine nucleotide degradation in the thoroughbred horse with increasing exercise duration. Eur J Appl Physiol Occup Physiol 65: 271-277.[CrossRef][Medline]
Sillence MN, Matthews ML, Moore NG, and Reich MM (1995) Effects of BRL-47672 on growth, beta 2-adrenoceptors, and adenylyl cyclase activation in female rats. Am J Physiol 268: E159-E167.[Medline]
Sleeper MM, Kearns CF, and McKeever KH (2002) Chronic clenbuterol administration negatively alters cardiac function. Med Sci Sports Exerc 34: 643-650.
Timmons JA (2002) Can we "switch" the emphasis please? J Appl Physiol 92: 2221.
Torgan CE, Etgen GJ Jr, Brozinick JT Jr, Wilcox RE, and Ivy JL (1993) Interaction of aerobic exercise training and clenbuterol: effects on insulin-resistant muscle. J Appl Physiol 75: 1471-1476.
Van Baak MA, de HA, Saris WH, van KE, Kuipers H, and van der Vusse GJ (1995) beta-Adrenoceptor blockade and skeletal muscle energy metabolism during endurance exercise. J Appl Physiol 78: 307-313.
Wheeler TJ and Lowenstein JM (1979) Adenylate deaminase from rat muscle. Regulation by purine nucleotides and orthophosphate in the presence of 150 mM KCl. J Biol Chem 254: 8994-8999.
Wibom R, Lundin A, and Hultman E (1990) A sensitive method for measuring ATP-formation in rat muscle mitochondria. Scand J Clin Lab Investig 50: 143-152.[Medline]
Yang YT and McElligott MA (1989) Multiple actions of beta-adrenergic agonists on skeletal muscle and adipose tissue. Biochem J 261: 1-10.[Medline]
Young A (1987) Exercise in chronic disease, in Exercise: Benefits, Limits and Adaptations (Macleod D, Maughan R, Nimmo M, Reilly T and Williams C eds) pp 20-32, E&FN Spon, London.
Zammit VA and Newsholme EA (1976) The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates. Biochem J 160: 447-462.[Medline]
Zeman RJ, Ludemann R, Easton TG, and Etlinger JD (1988) Slow to fast alterations in skeletal muscle fibers caused by clenbuterol, a beta 2-receptor agonist. Am J Physiol 254: E726-E732.[Medline]
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, P < 0.05 signifies different from 4-week value within each treatment group; 
, P < 0.05; 
