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CELLULAR AND MOLECULAR

The Effect of the ₂-Adrenoceptor Agonist Prodrug BRL-47672 on Cardiovascular Function, Skeletal Muscle Myosin Heavy Chain, and MyoD Expression in the Rat

Centre for Integrated Systems Biology and Medicine, Queens Medical Centre, University of Nottingham, Nottingham, United Kingdom (S.W.J., D.J.B., S.M.G., T.B., P.L.G.); and Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (J.A.T.)

Received for publication May 17, 2004
Accepted July 7, 2004.

	Abstract

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The intracellular mechanisms that regulate changes in postnatal myosin heavy chain (MHC) expression are not well established. The major objective of this study was to examine the acute and chronic effects of administration of BRL-47672, the prodrug of the ₂-adrenoceptor agonist clenbuterol on MHC and MyoD transcription factor expression to determine whether or not changes in MHC composition are preceded by changes in MyoD protein expression. To assess to what extent the use of BRL-47672 minimized cardiovascular effects, its hemodynamic actions were compared with those of clenbuterol. The effect of BRL-47672 on heart rate, mean arterial blood pressure, and hindquarters vascular conductance was significantly less than that of clenbuterol after a single i.p. injection (250 µg kg^-1 body mass). In the main study, 4-week old rats were given BRL-47672 (900 µg kg^-1 body mass) or an equivalent volume of saline (control) daily for 1, 28, or 56 days. Soleus muscle (SOL) was excised and MHC and MyoD expression analyzed. After 4 weeks, SOL from the BRL-47672-treated animals had significantly faster MHC composition (49 ± 2% MHCIIA) compared with those from the control animal (39 ± 3% MHCIIA, P < 0.05). MyoD expression increased by 40% after 1 day of BRL-47672 administration (P < 0.05) before a change in MHC composition. In conclusion, these data suggest that increased expression of fast-type MHCIIA expression in rat SOL induced by BRL-47672 administration is preceded by changes in the level of MyoD transcription factor expression.

Understanding of the determination and differentiation of muscle fiber type has been advanced with the discovery of a family of myogenic regulatory factors (MRFs), namely, MyoD, myogenin, myf5, and MRF4. These transcription factors share 80% homology within a basic helix-loop-helix motif (Murre et al., 1989) that mediates dimerization and DNA binding to the E-box consensus sequence (CANNTG) found in the control regions of muscle-specific genes, including the MHCIIB gene (Wheeler et al., 1999). It has been proposed that these myogenic factors are essential for development and differentiation of skeletal muscle from myoblast cells (Weintraub, 1993; Olson and Klein, 1994). Knockout studies in mice have shown that during murine embryogenesis, MyoD, myf5, myogenin, and MRF4 are expressed in overlapping but distinct patterns, with Myf5 and MyoD acting early to establish myoblast lineage and myogenin acting later to control differentiation (Rawls et al., 1998). After birth, myogenic transcription factor expression decreases, but low levels of expression do persist in adult tissue (Hughes et al., 1993), indicating a possible role in maintaining MHC phenotype and muscle remodeling in adult tissue. Indeed, it has been shown that during development in rat skeletal muscle, MyoD and myogenin accumulate differentially in fast and slow muscles (Hughes et al., 1997), suggesting that MyoD and myogenin may regulate fast and slow myosin expression, respectively. However, it has been shown previously that overexpression of myogenin in transgenic mice resulted in an increase in oxidative enzymes but no change in MHC composition (Hughes et al., 1999). Therefore, it would appear that MyoD expression is a more likely determinant of MHC phenotype than myogenin.

One tool that has been used to study the regulation of skeletal muscle fiber type is the administration of ₂-adrenoceptor agonists (₂-agonists) such as clenbuterol that, when given chronically, have been reported to induce slow-type I to fast-type II fiber transitions (Zeman et al., 1988), possibly by signaling through MyoD since changes in muscle fiber type have been associated with altered levels of MyoD expression (Hughes et al., 1993; Goblet and Whalen, 1995; Kraus and Pette, 1997). Since MyoD expression has been reported to be higher in fast muscles than in slow muscles (Hughes et al., 1997; Sakuma et al., 1999), it is not known whether these changes in expression of MyoD following fiber type transitions are a consequence or cause of change in MHC phenotype. Furthermore, although administration of clenbuterol is known to bring about slow to fast fiber type transitions, it also causes substantial hemodynamic changes (Rothwell et al., 1987), which might preclude its use in a clinical setting. Theoretically, one way of reducing these undesirable effects would be to administer a prodrug that is metabolized to a ₂-agonist in vivo (Sillence et al., 1995), but it is not known if such a strategy would also reduce the phenotypic effects. BRL-47672 has a chemical structure similar to the ₂-agonist clenbuterol (Fig. 1) but has little direct action on ₂-adrenoceptors. The work of Sillence et al. (1995) demonstrated that, in vitro, BRL-47672 had a low affinity for rat ₂-adrenoceptors compared with clenbuterol and was a poor activator of rat adenylyl cyclase activity in rat skeletal muscle. Conversely, acute administration in vivo resulted in increased adenylyl cyclase activation and, over 6 days of administration, an increase in skeletal muscle mass. Furthermore, these anabolic effects of BRL-47672 were not blocked by daily injection of the ₂-adrenoceptor-selective antagonist ICI-118551 but were blocked when ICI-118551 was administered in the diet. The addition of the ₁-adrenoceptor-selective antagonist CGP-20712A to the diet failed to dampen the anabolic effects of BRL-47672. This led us to conclude that BRL-47672 has little direct action on ₂-adrenoceptors per se but is metabolized rapidly in vivo to a potent ₂-agonist. Finally, Sillence et al. (1995) claimed that BRL-47672 was a less potent stimulator of heart rate than clenbuterol, which seems logical given its reported lack of direct effect on ₂-adrenoceptors. On the basis of the observations of Sillence et al. (1995), it is not unreasonable to infer that chronic administration of BRL-47672 would influence cell signaling pathways associated with skeletal muscle mass and fiber type in the rat in a manner similar to clenbuterol while having a comparatively attenuated hemodynamic effect.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Structural properties of clenbuterol and BRL-47672 (Sillence et al., 1995).

The first objective, therefore, of the present work was to compare the in vivo hemodynamic effects of clenbuterol and BRL-47672 in conscious rats. The second objective was to examine the acute and chronic effects of BRL-47672 administration on MHC and MyoD transcription factor expression to determine whether or not ₂-agonist-induced changes in MHC composition are preceded by changes in MyoD protein expression. Knowledge of the intracellular regulatory factors that underlie ₂-agonist-induced changes in postnatal MHC expression would aid an understanding of how skeletal muscle adaptations occur, not only during normal development but also in disease states that lead to muscle dysfunction. This information could be of considerable clinical importance if, for example, selective fast fiber type atrophy causing debilitating muscular dysfunction could be halted or even reversed by administration of a ₂-agonist compound that promotes fast-type myosin expression.

	Materials and Methods

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Cardiovascular Studies
Cardiovascular measurements were performed on male Sprague-Dawley rats (n = 8) weighing between 420 and 470 g. Surgery was performed under general anesthesia (fentanyl; Janssen-Cilag Ltd., High Wycombe, UK) and medetomidine (Pfizer Limited, Sandwich, UK), 300 µg kg^-1 of each, i.p., reversed with nalbuphine (Bristol-Myers Squibb, Hounslow, UK) and atipamezole (Pfizer), 1 mg kg^-1 of each, s.c., as described previously (Gardiner et al., 2002). Catheters were placed in the distal abdominal aorta (via the caudal artery) to monitor arterial blood pressure and heart rate and also in the peritoneum for drug administration. Hindquarters blood flow was monitored by a pulsed Doppler probe implanted around the distal abdominal aorta.

Animals were administered either clenbuterol (n = 8) or the BRL-47672 (n = 8) at 250 µg kg^-1 i.p., which is equivalent to molar concentrations of 0.80 and 0.82 µM, respectively. Continuous recordings of cardiovascular variables were made using a customized computer-based system [Hemodynamics Data Acquisition System (HDAS), University of Limberg, Maastricht, The Netherlands] connected to the Gould transducer amplifier (model 13-4615-50; Gould Instrument Systems Inc., Cleveland, OH). Raw data were collected every 2 min and stored to disc at 5-s intervals for up to 40 min after drug administration. Within-group analysis was conducted using Friedman's test (Theodorsson-Norheim, 1987), and between-group comparisons were made with the Mann-Whitney U test applied to areas under curves. A P value of <0.05 was taken as significant.

Muscle Studies
Dose-Dependent Effects of BRL-47672. Six-week-old male Wistar rats were given daily subcutaneous injections of either 250, 450, or 900 µgkg^-1 body mass of BRL-47672 (n = 4) or an equivalent volume of saline (n = 4) for 8 weeks. On the day after the final injection, rats were anesthetized with thiobutabarbital sodium (Inactin, 120 mg kg^-1, i.p.) and SOL of the hind limb excised, snap frozen in liquid nitrogen, and then stored at -80°C for subsequent analysis. Given that the SOL muscle is known to contain only type I and type IIA myosin, the gastrocnemius muscle of the saline-treated animals was also excised to best illustrate the separation of MHCs using SDS-PAGE (6%) gels.

Time-Dependent Effects of BRL-47672. Four-week-old male Wistar rats were given daily subcutaneous injections of 900 µg kg^-1 body mass of BRL-47672 or saline (control) for 1 day (n = 10), 4 weeks (n = 10), or 8 weeks (n = 10). The day after the final injection, rats were anesthetized (Inactin, 120 mg kg^-1, i.p.), and the SOL was excised and frozen as described above.

All animals were kept under 12-h light/dark cycles and food and water was provided ad libitum. All work was conducted in accordance with the Home Office Scientific Animals (Scientific Procedures) Act 1986, and the animals were killed humanely at the end of the experiment.

Myosin Heavy Chain Analysis. Crude myosin was prepared as described previously (Termin et al., 1989). In brief, SOL (approximately 50 mg) was homogenized in 10 vol of buffer (0.3 M KCl, 0.1 M KH₂PO₄, 50 mM K₂HPO₄, 10 mM EDTA, and 5 mM DTT, pH 6.5) containing 10 µg ml^-1 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 0.01 µg ml^-1 pepstatin A, and 0.1 µg ml^-1 leupeptin. Muscle homogenate was then left on ice for 15 min with periodic vortexing to facilitate protein extraction. Samples were then centrifuged at 6000g for 15 min, and the supernatant was mixed 1:1 with glycerol before being stored at -20°C. Supernatant total protein was determined (Bradford, 1976), and MHCs (MHCI and MHCIIA) were electrophoretically separated by applying between 0.1 and 0.5 µg of protein extract to 0.75-mm thick SDS-PAGE (6%) gels containing 40% glycerol. Electrophoresis was run at 70 V for 17 to 18 h, and the gels were then silver-stained using Bio-Rad Silver-Stain Plus kit (Bio-Rad, Hemel Hempstead, UK).

Histochemistry. To determine whether SDS-PAGE analysis of the MHCs was a reflection of fiber type composition rather than fiber hypertrophy, SOL from 8-week BRL-47672-treated and control animals (n = 5 in each) was subjected to histochemical analysis. Serial cross-sections (20 µm thick) were cut on a cryostat at -20°C and stained for myofibrillar ATPase after prior preincubation of sections at pH 4.2 and 10.3 (Brooke and Kaiser, 1970). Acid preincubation consisted of 5 min in 142 mM barbital acetate buffer, whereas alkaline preincubation consisted of 20 min in 36 mM CaCl₂, buffered with 100 mM Tris. Sections were then incubated at room temperature for 45 min in 20 mM sodium barbital, 18 mM CaCl₂, and 4.5 mM ATP (pH 9.4).

MyoD Analysis. Frozen samples of SOL were homogenized in buffer (20 mM Tris, 5 mM EDTA, and 5 mM DTT, pH 7.5) containing 300 mM NaCl to extract nuclear protein. Protease inhibitors were added as for MHC analysis. Homogenates were left on ice for 30 min with periodic vortexing to facilitate nuclear protein extraction before being centrifuged at 10,000g for 20 min. Supernatant protein content was measured (Bradford, 1976), and an aliquot of supernatant was then mixed 1:1 with 2x SDS mix. Protein extracts (100 µg) were analyzed by 10% SDS-PAGE gels. Separated protein was electrotransferred onto Hybond ECL membrane (Amersham Biosciences UK, Ltd., Little Chalfont, UK) at 200 mA for 2 h (Towbin et al., 1979) and then blots were probed with a commercially available MyoD (M-318; 1:1000) polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and visualized using enhanced chemiluminescence (ECL; Amersham Biosciences UK, Ltd.). To confirm equal protein loading, immunoblots were re-probed with a monoclonal sarcomeric -actinin antibody (Sigma-Aldrich, St. Louis, MO) at a dilution of 1:1000.

Preparation of Nuclei from Skeletal Muscle. To confirm that the MyoD antibody was detecting a nuclear protein on the immunoblots of the supernatant protein extracts, nuclei from skeletal muscle were prepared by centrifugation through high sucrose gradients (Koffer and Brownson, 1982). Fresh SOL (100 mg) was diced and gently homogenized using a Kontes glass homogenizer in 10 volumes of ice-cold TM buffer (10 mM Tris, 5 mM MgCl₂, pH 7.6) containing 0.25 M sucrose, 2 mM DTT, and a cocktail of protease inhibitors (Sigma-Aldrich). The homogenate was then filtered through gauze, and the filtrate was centrifuged at 2500g for 15 min at 4°C. The sucrose supernatant was then discarded, and the pellet was homogenized in 1 ml of TTM buffer (TM buffer containing 0.5% Triton) with 0.25 M sucrose for 5 min before being centrifuged at 2500g for 15 min at 4°C. The sucrose supernatant was discarded, and the pellet was homogenized in 0.5 ml of 2.4 M sucrose in TM buffer and centrifuged at 35,000g for 1 h at 4°C to pellet the nuclei. The nuclear pellet was then resuspended in 70 µl of TM buffer and stored at -80°C. Twenty microliters were then mixed with an equal volume of 2x SDS mix and run on 10% SDS-PAGE and MyoD protein detected, as described for whole muscle analysis.

Quantification and Statistical Analysis. Silver-stained gels and immunoblots were scanned, and the densitometric intensity of the bands was analyzed using the OPTIMAS software package (Media Cybernetics, Inc., Silver Spring, MD). The ECL signal was in the linear range, and a standard whole cell lysate, prepared from skeletal muscle, was used to enable blot-to-blot comparisons to be made. Values in text are represented as mean ± S.E.M. ANOVA (one-way, plus least significant difference post hoc test) was used to examine dose-dependent effects of BRL-47672 on MHC expression. ANOVA (two-way) was used to examine time-dependent effects of BRL-47672 on MHC and MyoD expression. Where time and treatment effects were observed, unpaired t-tests were used to examine differences between treatment groups and time points. Statistical significance was accepted at the P < 0.05 level. The Pearson correlation coefficient was used to describe the linear relationship between the percentage of MHC and percentage of fiber type.

	Results

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Cardiovascular Studies
Clenbuterol produced significant cardiovascular effects within 5 min of administration, with increased heart rate, reduced mean arterial pressure, and raised vascular conductance in the hindquarters. These effects were sustained over the 40-min recording period. In contrast, the rates of onset and magnitude of the cardiovascular effects following administration of BRL-47672 were significantly lower compared with those observed following clenbuterol administration (Fig. 2).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Cardiovascular responses to clenbuterol or BRL-47672. Cardiovascular responses to clenbuterol or BRL-47672 (250 µg kg-1) in conscious Sprague-Dawley rats (n = 8). Values represent mean, and the vertical bars show S.E.M. *, P < 0.05 versus baseline within group (Friedman's test). #, P < 0.05 BRL-47672 versus clenbuterol (Mann-Whitney U test applied to areas under curves).

Muscle Studies
Dose-Dependent Effects of BRL-47672 on MHC Expression. SDS-PAGE resolved two MHCs in SOL, type I and II, with no expression of fast type IIB or IIX myosin in either treatment group (Fig. 3). Densitometric analysis of the MHC silver-stained gels showed that after 8 weeks, SOL from control animals was composed of 83 ± 3% MHCI and 17 ± 3% MHCIIA (Fig. 4, a and b). Administration of BRL-47672 at 250 µg kg^-1 increased the proportion of MHCIIA by 15% compared with control, whereas the 450 and 900 µg kg^-1 doses increased the proportion of MHCIIA by 18 and 23%, respectively (Fig. 4, a and b).

View larger version (64K): [in this window] [in a new window]   Fig. 3. SDS-PAGE analysis of myosin heavy chains. Myosin heavy chain expression in SOL after 8 weeks of administration of saline control (C) or BRL-47672 (T) compared with rat gastrocnemius (GAS) myosin, analyzed on 6% SDS-PAGE gels. Approximately 0.1 µg of protein was loaded in each lane and the gel run at 70 V for 17 h.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Dose-dependent effects of BRL-47672 on myosin heavy chain expression. a, representative 6% SDS-PAGE gel of myosin heavy chain expression after 8 weeks of saline control (C) or after 250, 450, or 900 µg kg-1 body mass of BRL-47672. Approximately 0.5 µg of protein were loaded in each lane, and the gel was run at 40 V for 17 h. b, dose-response curve of the effects of BRL-47672 on the percentage of type I and type IIA myosin. Values are mean (n = 4) with S.E.M. shown by vertical bars. *, P < 0.01 versus control (one-way ANOVA plus least significant difference post hoc test).

Time-Dependent Effects of BRL-47672 on MHC Expression. Over the 8-week study, SOL shifted toward a slower more oxidative phenotype. This was seen as an increase in MHCI expression from 58 ± 2% to 72 ± 3% and a corresponding reduction in fast-type MHCIIA expression from 42 ± 2% to 28 ± 3% (P < 0.01; Fig. 5). There was no significant difference in MHC expression between the BRL-47672-treated and control group after 1 day of dosing (Fig. 5); however, after 4 weeks of BRL-47672 administration, the proportion of MHCIIA (49 ± 2%) was significantly higher than at the 1-day time point (41 ± 2%; P < 0.05) and significantly higher than in the control group at that time (39 ± 3% P < 0.05) (Fig. 5). At 8 weeks, the level of MHCIIA expression in the BRL-47672-treated group was not different from the 1-day time point, although compared with the control group, MHCIIA expression was significantly higher (P < 0.01) and MHCI significantly lower (P < 0.01).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Time-dependent effects of BRL-47672 on myosin heavy chain expression. Changes in MHC expression after BRL-47672 administration (900 µg kg-1 body mass) or saline for 8 weeks. Values are expressed as mean ± S.E.M. (n = 10). *, P < 0.05 and **, P < 0.01, different from 1-day value within group. , P < 0.05 and , P < 0.01, different from corresponding control value (two-way ANOVA).

Histochemistry. To determine whether the measurements of MHC reflected fiber type composition, SOL from treated and control animals was subjected to myofibrillar ATPase staining. Histochemical analysis (Fig. 6a) showed that the percentage of type IIA fiber type was significantly higher (P < 0.01) in the BRL-47672-treated animals (38 ± 4%, n = 5) compared with control (16 ± 3%, n = 5). Furthermore, plotting the percentage of fiber type as determined by ATPase staining against the percentage of MHC as determined by SDS-PAGE revealed a strong linear correlation (r = 0.99) between fiber type and MHC expression (Fig. 6b).

View larger version (73K): [in this window] [in a new window]   Fig. 6. Correlation of myofibrillar ATPase staining and MHC analysis by SDS-PAGE. a, myofibrillar ATPase staining of SOL cross-sections (20 µm thick) after preincubation at pH 10.3 and 4.2 from 8-week BRL-47672 treatment and from control animals (n = 5). b, correlation of percentage of MHCIIA (as determined by SDS-PAGE) against the percentage of type IIA fibers (as determined by histochemical staining). Open boxes, control samples; closed diamonds, BRL-47672-treated samples.

MyoD Analysis. The cDNA sequence of rat MyoD predicts a protein of 34 kDa molecular weight; however, a number of molecular weights have been reported for MyoD on SDS-PAGE, ranging from 33 to 48 kDa (Hughes et al., 1997; Sakuma et al., 1999; Tamaki et al., 2000), which may, in part, be due to the high number of basic residues in its sequence. Using a commercially available polyclonal MyoD antibody (Santa Cruz Biotechnology, Inc.), a single band of approximately 35 kDa was detected on SDS-PAGE from SOL whole cell salt extracts. A Western blot of nuclei prepared from fresh SOL detected a band of identical mobility (Fig. 7a).

View larger version (31K): [in this window] [in a new window]   Fig. 7. MyoD analysis. a, immunoblot from 10% SDS-PAGE showing detection of MyoD in SOL nuclei prepared by centrifugation through high-sucrose gradients (N) and in SOL whole muscle extract (W). Blot was probed with MyoD (1:1000) antibody and visualized using ECL. Markers, BioRad precision prestained standards. b, representative immunoblot from SDS-PAGE (10%) analysis of SOL extracts after BRL-47672 treatment (900 µg kg-1 body mass; T) or saline control (C) probed with MyoD (1:1000) antibody and visualized using ECL. Exactly 100 µg of total protein was loaded in each lane. Markers as in panel a. Immunoblot was reprobed with -actinin (1:1000) to demonstrate equal protein load. c, mean MyoD expression levels in SOL after 1 day, 4 weeks, and 8 weeks of BRL-47672 or saline (control) administration. Values are means ± S.E.M. (n = 10). *, P < 0.05 (two-way ANOVA), different from corresponding control value.

Densitometric analysis of immunoblots revealed that there was no change in MyoD expression within either treatment group with respect to time; however, MyoD protein expression was significantly higher (P < 0.05) in the BRL-47672-treated group (30 ± 3 units, n = 10) compared with control (21 ± 3 units) after 1 day of administration (Fig. 7, b and c). MyoD expression was maintained at a significantly higher level (P < 0.05) after 4 weeks in the BRL-47672-treated group (26 ± 3 units) compared with control (18 ± 2 units). By 8 weeks, although MyoD expression was on average higher in the BRL-47672-treated group compared with the control group, this was not significantly different (Fig. 7c).

	Discussion

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The objective of the present work was to determine whether there were significant differences between the hemodynamic effects of clenbuterol and its prodrug, BRL-47672, and to determine whether or not changes in MHC expression induced by BRL-47672 administration are preceded by changes in MyoD protein expression in rat SOL to ascertain whether MyoD expression plays a role in regulating postnatal MHC composition.

Although clenbuterol is well known to induce slow to fast skeletal muscle fiber type transitions, its substantial cardiovascular actions render it unacceptable for clinical use in conditions where muscle protein accretion and/or remodeling of muscle fiber type might be desirable. Thus, an agent with the anabolic and fiber type remodeling actions of clenbuterol but with lesser cardiovascular effects would be of clinical interest. Since the ₂-adrenoceptors that mediate the effects of clenbuterol on skeletal muscle are pharmacologically indistinguishable from those responsible for its cardiovascular actions (Rothwell et al., 1987), improving target selectivity does not appear to be a likely option. Therefore, the question arises whether or not a prodrug that is metabolized to clenbuterol in vivo would have lesser cardiovascular effects.

Comparing clenbuterol and BRL-47672, it was clear that the effect of the latter on heart rate, mean arterial pressure, and hindquarters vascular conductance were blunted; however, BRL-47672 was at least as potent as clenbuterol at inducing MHC remodeling. Thus, administration of BRL-47672 at a dose of 250 µg kg^-1 for 8 weeks induced a 15% increase in MHCIIA expression, although at the higher dose (900 µg kg^-1), there was a 23% increase in MHCIIA expression. These changes are comparable with results we have previously published showing that clenbuterol administration (250 µg kg^-1 for 8 weeks) increased MHCIIA expression by 13 to 23% (Rajab et al., 2000). Furthermore, it has been reported that rats fed clenbuterol (1.6 mg kg^-1 for 8 weeks) showed a 23% increase in MHCIIA expression (Zeman et al., 1988), which is similar to the increase in MHCIIA we report here with BRL-47672 but using a considerably lower dose (900 µg kg^-1).

To determine whether or not ₂-agonist-induced changes in MHC expression were preceded by up-regulation of MyoD expression, time-dependent changes in MHC and MyoD protein expression were measured. During the 8 weeks of the study, the MHC composition of SOL in the control animals changed, becoming slower because of a transition from MHCIIA to MHCI (Fig. 5). This pattern of transition, however, did not occur in the SOL of the BRL-47672-treated animals. As a result, the proportion of MHCIIA was significantly higher (P < 0.01), and consequently MHCI was significantly lower in the BRL-47672-treated animals compared with control after 4 and 8 weeks.

The strong correlation between the percentage of fiber type and MHC expression (Fig. 6b) clearly indicates that the changes in MHC expression reported here are likely to have been predominantly due to changes in muscle fiber type composition as opposed to hypertrophy of specific fiber types. A change of fiber type with age in rats has been reported previously (Maltin et al., 1989), where the fiber type profile of SOL changed throughout the first year of life. Since the MHC composition of rat muscles in this period is not static, the current data suggest that the effect of BRL-47672 administration was to induce and maintain fast MHCIIA expression in SOL, as opposed to "switching" per se (Fig. 5). Thus, BRL-47672 administration seems to prevent the normal developmental MHC switch from MHCIIA to MHCI in SOL.

Analysis of MyoD protein expression revealed that MyoD expression was significantly increased after 1 day of BRL-47672 treatment (Fig. 7, b and c). These data support previous observations (Hughes et al., 1993) showing that treatment with clenbuterol and thyroid hormone (T₃) resulted in increased MHCIIA and MyoD expression. Notably, however, we report here for the first time that elevation of MyoD protein expression occurred before any change in MHC composition. This suggests that an up-regulation of MyoD is a prerequisite for induction of MHCIIA expression in SOL following ₂-agonist administration.

In the control animals, despite the significant increase in MHCI and reduction in MHCIIA during the 8 weeks (Fig. 5), there was no change in the expression of MyoD with time (Fig. 7c). This suggests that during normal development of SOL, MyoD expression is kept at basal levels and that the increase in MHCI and parallel loss of MHCIIA may be due to maintenance of these low levels of MyoD. If this were the case, the relatively high abundance of the transcription factor, myogenin, which is associated with slow muscle, might be responsible for the increase in type I myosin as the muscle reaches maturation. Indeed, there is evidence to suggest that the expressions of MyoD and myogenin are regulated through independent mechanisms. Thus, in rat, administration of clenbuterol in the diet for 3 days resulted in an increase in MyoD mRNA expression relative to myogenin (Delday and Maltin, 1997). Interestingly, however, clenbuterol, fed to rats with immobilized hind limbs over the same time period, led to a marked increase in myogenin but no change in MyoD. It is worth noting, therefore, that the expression of myogenic transcription factors in response to the administration of ₂-agonists such as clenbuterol could depend on the metabolic status of the muscle.

In summary, our data indicate that induction and maintenance of MHCIIA in SOL is preceded by up-regulation of the transcription factor, MyoD. The finding that BRL-47672 induced MHC remodeling to the same extent as clenbuterol, but with lesser cardiovascular effects, suggests future therapeutic potential for an ultra-slow release formulation in conditions of debilitating fiber type remodeling and selective fiber type atrophy.

	Footnotes

 
Financial support was provided by Pfizer Global Research and Development, Sandwich, UK.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.071589.

ABBREVIATIONS: MHC, myosin heavy chain; MRF, myogenic regulatory factor; SOL, soleus muscle; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; ECL, enhanced chemiluminescence; ANOVA, analysis of variance; ICI-118551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol; CGP-20712A, ([2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy]-2-propanolmethanesulfonate.

Address correspondence to: Prof. Paul L. Greenhaff, Centre for Integrated Systems Biology and Medicine, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK. E-mail: paul.greenhaff{at}nottingham.ac.uk

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