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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Extracellular signal-regulated kinase pathway is differentially involved in -agonist-induced hypertrophy in slow and fast muscles

¹Department of Animal Sciences and ²Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana

Submitted 30 August 2006 ; accepted in final form 24 November 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The molecular mechanisms controlling -adrenergic receptor agonist (BA)-induced skeletal muscle hypertrophy are not well known. We presently report that BA exerts a distinct muscle- and muscle fiber type-specific hypertrophy. Moreover, we have shown that pharmacologically or genetically attenuating extracellular signal-regulated kinase (ERK) signaling in muscle fibers resulted in decreases (P < 0.05) in fast but not slow fiber type-specific reporter gene expressions in response to BA exposure in vitro and in vivo. Consistent with these data, forced expression of MAPK phosphatase 1, a nuclear protein that dephosphorylates ERK1/2, in fast-twitch skeletal muscle ablated (P < 0.05) the hypertrophic effects of BA feeding (clenbuterol, 20 parts per million in water) in vivo. Further analysis has shown that BA-induced phosphorylation and activation of ERK occurred to a greater (P < 0.05) extent in fast myofibers than in slow myofibers. Analysis of the basal level of ERK activity in slow and fast muscles revealed that ERK1/2 is activated to a greater extent in fast- than in slow-twitch muscles. These data indicate that ERK signaling is differentially involved in BA-induced hypertrophy in slow and fast skeletal muscles, suggesting that the increased abundance of phospho-ERK1/2 and ERK activity found in fast-twitch myofibers, compared with their slow-twitch counterparts, may account, at least in part, for the fiber type-specific hypertrophy induced by BA stimulation. These data suggest that fast myofibers are pivotal in the adaptation of muscle to environmental cues and that the mechanism underlying this change is partially mediated by the MAPK signaling cascade.

muscle fiber type; mitogen-activated protein kinase signaling pathways; mechanism

Skeletal muscle is largely composed of heterogeneous populations of muscle fibers, which differ markedly in their metabolism and contractile function (5, 9). On the basis of metabolic and contractile properties and myosin heavy chain (MyHC) isoforms, muscle fibers can be broadly classified as type I, oxidative, slow-twitch fibers and type II, fast-twitch fibers. Type II muscle fibers can be further classified into type IIa fibers, which possess a more oxidative type of energy metabolism, or type IIx and IIb fibers, which are more glycolytic in nature (5). When cued by a myriad of potential stimuli such as exercise, aging, various atrophy models (30), microgravity (8, 14), obesity, and diabetes (20, 25, 42), muscle responds by altering its metabolism and contractile function to adapt to such needs. The link between or among fiber type, hypertrophy, and BA stimulation is well-supported in the literature. Oishi et al. (28) reported a disproportional increase in the size of fast fibers of rats fed clenbuterol. Furthermore, Depreux et al. (12) clearly demonstrated that pigs fed the muscle growth promotant BA ractopamine possessed muscle with greater amounts of type IIb MyHC at the expense of slower contracting, more oxidative types of muscle fibers. The molecular mechanisms underlying this differential response of muscle fibers to various stimuli, however, are largely unknown. Therefore, the objective of this study was to use an in vivo mouse model and cell culture studies to elucidate the biochemical basis that accounts for the differential response of slow and fast muscles and fibers to BA stimulation. We postulate that the differential responsiveness of slow and fast muscles and muscle fibers to BA stimulation is governed by changes in cell signaling pathways.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Experimental design. A mouse model was used to study the muscle fiber type-specific effects that occur with BA feeding. Mice were fed clenbuterol, and the cross-sectional areas of individual fibers within select muscles were determined. Using C₂C₁₂ cell cultures and muscle fiber type-specific reporter gene constructs as an in vitro model, we studied the signaling events that convey this response. Finally, we validated our in vitro results with studies using rodent models.

Plasmids. MyHC I-luciferase reporter gene construct containing MyHC I sequences from –3542 to +89 bp was kindly provided by Dr. K. Hasegawa (18). Reporter genes MyHC IIB-luciferase, containing MyHC IIB sequences from –2560 to +13 bp, and sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)-1-luciferase, containing SERCA-1 sequences from –1373 to +172 bp, were secured from Dr. S. J. Swoap (40, 41), and enhanced green fluorescent protein (EGFP)-tagged MAPK phosphatase-1 (MKP-1) was supplied by Dr. A. M. Bennett (4). Plasmid pRL-SV40 expressing Renilla luciferase was purchased from Promega (Madison, WI).

Pharmacological reagents and antibodies. PD-98059 and U0126 were purchased from Cell Signaling Technology (Beverly, MA); SB203580, SP600125, H89, and c-Raf inhibitors were obtained from EMD Biosciences (La Jolla, CA). Anti-ERK and anti-phospho-ERK antibodies were purchased from Cell Signaling Technology; anti-GFP antibody was purchased from Molecular Probes (Eugene, OR); anti-dystrophin antibody was obtained from Chemicon International (Temecula, CA). Antibodies against MyHC are as follows: type I (A4.840; Ref. 22), type IIa (6B8; Ref. 12), and type IIb (BF-F3; Ref. 35).

Animal model for muscle hypertrophy. Four-week-old male C57BL/6 mice (Harlan, Indianapolis, IN) were randomly divided into two groups, six mice in each. The mice were then fed 20 parts per million (ppm) clenbuterol in drinking water without any restriction on food intake. Two weeks later, mice were weighed to obtain the final body weight. Soleus, extensor digitorum longus (EDL), tibialis anterior (TA), and gastrocnemius muscles from the left leg were dissected, weighed, and snap frozen in liquid nitrogen, whereas soleus and gastrocnemius muscles from the right leg were mounted in OCT and immediately frozen in liquid nitrogen-cooled isopentene. Where indicated, 4-wk-old Sprague-Dawley male rats were used in the experiment and treated the same as the mice. All procedures were approved by the Purdue Animal Care and Use Committee.

Adult skeletal muscle surgery and electroporation. Adult male Sprague-Dawley rats (4 wk old) were subjected to minor surgery to expose the soleus and gastrocnemius, and plasmids purified with an EndoFree plasmid mega kit (Qiagen, Valencia, CA) with DNA at a final concentration of 0.5 µg/µl in 0.9% NaCl were injected into the muscles along the muscle length. About 10 or 50 µl of reporter gene constructs or expression plasmid solution were injected into soleus and gastrocnemius muscles, respectively. pRL-SV40 plasmid was coinjected to normalize transfection efficiency. Electroporation was performed as described previously (32). Briefly, two spatula electrodes were placed on each side of the muscle belly, and eight pulses (1 Hz, 200 V/cm) at 20-ms intervals were applied using a BTX ECM 830 electroporator (Genetronics, San Diego, CA). One week later, muscle samples were collected and homogenized in passive lysis buffer (Promega), and luciferase activity was assayed using a Dual-Luciferase assay kit (Promega). The gastrocnemius and soleus muscles of C57BL/6 mice (4 wk old) also were electroporated with EGFP-tagged MKP-1 expression plasmid. Ten days after electroporation, soleus and gastrocnemius muscles were frozen in liquid nitrogen-cooled isopentene for fluorescent immunohistochemistry.

Immunohistochemical staining and fiber size measurement. Serial sections of skeletal muscles were cut to 8 µm and stored at –80°C until use. Sections were air-dried for 30 min, followed by 10 min of fixation in 1% paraformaldehyde. Sections were blocked in 5% goat serum for 30 min at room temperature, followed by incubation with the primary antibody diluted in 5% goat serum for 1 h. After three 5-min washes in PBS, Cy3-goat anti-rabbit or biotinylated goat anti-mouse IgG or IgM were applied for 1 h. Cy2-streptavidin was used to detect biotinylated second antibody. The ABC/DAB kit (Vector Laboratories, Burlingame, CA) was used for the colorimetric detection of the MyHC. For assigning fiber type and size, fibers were stained with MyHC I, IIa, and IIb antibodies on serial muscle sections, and images of cross sections were captured and digitized using the commercial software Photoshop (Adobe Systems, San Jose, CA). Ten randomly selected fields were analyzed for muscle fiber-type frequency and size. Muscle fibers staining positive for MyHC type I, IIa, or IIb were identified and demarcated, and muscle fiber cross-sectional area was determined. Fibers not reacting with the aforementioned MyHC antibodies were classified as type IIx fibers. The area and frequency of each muscle fiber type in each muscle of each mouse analyzed were determined by assessing at least 500 fibers.

Cell culture and transient transfection. C₂C₁₂ myoblasts were grown in DMEM containing 10% FBS until 70–80% confluence. Plasmids were transfected into the cells using FuGene 6 (Roche Applied Science, Indianapolis, IN) according to the manufacturer's recommendation. Eight hours later, the growth medium was replaced by the differentiation medium containing 2% horse serum. Thirty-six hours after transfection, cells were lysed with passive lysis buffer and luciferase activity was assayed as described above. pRL-SV40 plasmid was cotransfected for transfection efficiency.

Immunoblotting. Muscles were quickly frozen in liquid nitrogen and stored at –80°C until homogenization in ice-cold RIPA lysis buffer (1% Nonidet P-40, 0.1% SDS in 50 mM NaCl, and 20 mM Tris, pH 7.6) in the presence of 1 mM PMSF, aprotinin (10 µg/ml), leupeptin (10 µg/ml), 50 mM NaF, and 1 mM Na₃VO₄. The homogenates were centrifuged at 13,000 g at 4°C for 10 min, and supernatants were analyzed for protein concentration with a protein assay kit (Bio-Rad, Hercules, CA) and stored at –80°C. An aliquot (100 µg) was separated using SDS-PAGE (10%). Proteins were transferred to polyvinylidene difluoride membranes, which were blocked and blotted with various antibodies. Antibody-antigen complexes were visualized using ECL (Amersham, Piscataway, NJ).

Immunoprecipitation kinase assay. ERK kinase activity was measured with a nonradioactive MAPK (ERK1/2) activity assay kit (Chemicon) per the manufacturer's instructions. Briefly, muscles were homogenized in RIPA lysis buffer, and 200 µg of total protein in 500 µl of solution were incubated with 2 µg of rabbit polyclonal anti-ERK antibody overnight with end-over-end rotation at 4°C. The immune complexes were immunoprecipitated with 20 µl of protein A-agarose beads for 8 h with end-over-end rotation at 4°C. The immune complex-beads were washed twice with RIPA buffer and once with the kinase assay buffer included in the kit. ERK kinase was assayed with biotinylated myelin basic protein (MBP) as the substrate. The reaction mix was then transferred to a streptavidin-coated 96-well plate. Mouse anti-phospho-MBP antibody was then used to detect the phosphorylated MBP. A peroxidase-conjugated secondary antibody and 3,3',5,5'-tetramethyl benzidine were used to detect the signal. Data are expressed as relative kinase activity compared with the soleus control.

Data analysis. All data are expressed as means ± SE (represented as error bars). Statistical analysis was performed using Student's t-test or ANOVA, and the significance level was set as P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Clenbuterol induces skeletal muscle growth and shifts muscle fiber toward the fast-twitch phenotype. To investigate the effect of BA stimulation on muscle fiber type distribution, we fed mice 20 ppm clenbuterol for 2 wk. Absolute muscle weights of soleus (slow), TA (fast), and EDL (fast) muscle increased (P < 0.05) 25, 49, and 50%, respectively, in response to clenbuterol stimulation (Fig. 1A, left). When normalized for final body weight, the increase was 18, 32, and 32%, respectively (Fig. 1A, right), and no significant increase in soleus was observed. In addition to stimulating skeletal muscle growth, clenbuterol feeding induced skeletal muscle fiber type transition. Most striking was the fact that MyHC type IIb, which is normally not expressed in the soleus (Fig. 1B, left), was expressed in the presence of clenbuterol (Fig. 1B, right). This phenomenon was captured in relative frequency distributions of type I, type I/IIa hybrid, type IIa or IIx, and type IIb fibers, where the solei of untreated mice possessed 37, 3, 60, and 0% fiber, respectively, and clenbuterol treated mice had 25, 10, 60, and 5% fiber, respectively (Fig. 1C). Together, these data show that clenbuterol induces skeletal muscle growth and muscle fiber transition more markedly in fast muscles.

View larger version (47K): [in this window] [in a new window]   Fig. 1. A: clenbuterol induces skeletal muscle hypertrophy and muscle fiber type transition. C57BL/6 mice (6 in each group) were fed 20 parts per million clenbuterol for 2 wk. Soleus, tibialis anterior (TA), and extensor digitorum longus (EDL) muscles were dissected and weighed. Absolute muscle weight was expressed as %control (left), and relative muscle weight was expressed as %whole body weight (right) of control (open bars) and clenbuterol-treated mice (solid bars). B: clenbuterol stimulated type IIb muscle fiber type formation in the soleus. Muscle cryosections were stained with anti-MyHC IIb antibody. Note portions of gastrocnemius muscle (top left corner of micrograph at left) are positive for type IIb MyHC. Clen, clenbuterol. C: muscle fiber type transition in the soleus is induced by clenbuterol. Serial muscle cryosections were immunostained with MyHC antibodies, and the percentage of each fiber type or their hybrid was determined. Open bars, type I fibers; hatched bars, type I/IIa hybrid fibers; shaded bars, type IIa or IIx fibers; solid bars, type IIb fibers. *P < 0.05 vs. corresponding control.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Clenbuterol induces muscle fiber hypertrophy in a muscle- and fiber type-dependent manner. A: serial cryosections of soleus and gastrocnemius muscles were immunostained with different MyHC antibodies. Clenbuterol-induced relative changes in cross-sectional area (CSA) of different fiber types in the soleus (left) and gastrocnemius muscles (right). Open bars, control; filled bars, clenbuterol. CSA of each muscle fiber type was determined and expressed as %control. *P < 0.05. B and C: frequency histograms of muscle fiber CSA in the soleus (B) and gastrocnemius (C) muscles, respectively.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Isoproterenol (Iso) induces fiber type-specific reporter gene expression in cultured C2C12 myoblasts. A: titration of Iso used for sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-1-luciferase reporter gene assays. L-Ascorbic acid (0.1 mM) was used in all experiments to prevent Iso oxidation. a,b,c,dP < 0.05, different letters indicate significantly different means. B: Iso-induced reporter gene expression is fiber type dependent. Iso (1 nM) was used in the experiment. Open bars, controls; solid bars, Iso. MyHC, myosin heavy chain. *P < 0.05.

View larger version (15K): [in this window] [in a new window]   Fig. 4. ERK signaling pathway is differentially involved with -adrenergic receptor signaling in slow and fast specific reporter gene expression in vitro. A and B: C2C12 myoblasts were transfected with slow and fast fiber type-specific and control reporter luciferase constructs. Cultures were treated with Iso (1 nM) alone or in combination with H89 (10 µM), and PD98059 (50 µM). Open bars, controls; shaded bars, Iso alone; solid bars, Iso + H89 (A) or Iso + PD98059 (B). Values represent data from at least 3 independent experiments. *P <0.05. C: C2C12 myoblasts were transfected with SERCA-1-luciferase reporter and treated with Iso (1 nM) alone or in combination with U0126 (5 µM), SB203580 (5 µM), and SP600125 (5 µM). a,bP < 0.05, different letters indicate significantly different means.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Disruption of ERK signaling antagonizes -agonist-induced fast fiber-specific gene expression and skeletal muscle hypertrophy in vivo. A: MyHC I and IIb reporter genes were electroporated into rat soleus or gastrocnemius muscles in the presence of -Gal (as control) or MAPK phosphatase-1 (MKP-1) expression plasmids. Ten days later, muscle luciferase was assayed and normalized to Renilla luciferase. Mean values represent data from at least 6 mice each. Open bars, controls; shaded bars, 20 ppm clenbuterol; solid bars, clenbuterol with MKP-1. *P < 0.05. B–D: enhanced green fluorescent protein (EGFP)-tagged-MKP-1 plasmid DNA was electroporated into mouse lateral gastrocnemius muscle (fibers are primarily type IIb fibers), and mice were treated with or without clenbuterol for 2 wk. CSA of the various fiber types was calculated and expressed as means (B) and frequency histograms (C). a,bP < 0.05, different letters indicate significantly different means. Note that GFP-positive fibers expressed exogenous MKP-1 (D). Muscle cell sarcolemma was stained for dystrophin (red) to delineate fibers.

ERK signaling pathway is differentially upregulated in slow vs. fast muscles. We then analyzed ERK activation by immunoblotting for phosphorylated ERK1/2 and ERK activity by immunoprecipitation kinase assay. Clenbuterol induced the phosphorylation of ERK1/2 to a greater extent in fast muscle TA than in slow muscle soleus (Fig. 6A). Quantitative kinase assay indicated that although a 39% increase of ERK activity was observed in soleus in response to clenbuterol, there was a 2.3- and 2.5-fold increase in ERK activity in TA and gastrocnemius muscles compared with the corresponding control (Fig. 6B). These findings indicate that ERK signaling is differentially involved in -adrenergic receptor signaling in slow and fast muscles. Of note is the basal level of phosphorylated ERK and ERK activity in slow and fast muscles. Although no differences in the amount of immunoreactive ERK1/2 were detected between slow and fast muscles, the amount of phospho-ERK was much higher in fast TA muscle than in slow soleus muscle (Fig. 6A). ERK activity was 2.3- and 2-fold higher in TA and gastrocnemius muscles, respectively, compared with the soleus muscle (Fig. 6B). Together, these findings show that the ERK signaling is activated to a higher level in fast muscles than in slow muscles both at the basal level and in response to BA stimulation.

View larger version (31K): [in this window] [in a new window]   Fig. 6. ERK signaling is greater in fast muscles and in response to clenbuterol. Mice were fed with 20 ppm clenbuterol for 2 wk. Soleus, TA, and gastrocnemius (Gas) muscles were dissected and homogenized. A: representative immunoblots show ERK1/2 phosphorylation (pERK1/2). Blots were first probed with antibodies specific for phospho-ERK1/2 and then stripped and reprobed with ERK1/2 and tubulin antibodies. B: quantification of ERK activity by immunoprecipitation kinase assay (see MATERIALS AND METHODS). Open bars, controls; solid bars, clenbuterol. a,b,cP < 0.05, different letters indicate significantly different means.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Using both in vitro and in vivo approaches, we have demonstrated that the slow and fast fiber-specific genes respond differentially to BA receptor signaling. Although blocking PKA abolishes effects of Iso on both slow and fast fiber-specific reporter gene expression, blocking ERK signaling with PD98059 elicits a differential response by these genes in vitro. Specifically, ablation of ERK has little, if any, effect on BA-induced slow fiber-specific gene expression, whereas it inhibits BA-induced fast fiber-specific gene expression. This observation was confirmed using another MEK inhibitor, U0126, a c-Raf inhibitor (unpublished observation), and genetically by using MKP-1 in vitro and in vivo. However, blocking p38 and JNK MAPK signaling with specific inhibitors had no effect on Iso-induced fast fiber-specific gene expression. Furthermore, the hypertrophic effect of BA could be reversed by forced expression of GFP-tagged MKP-1 in vivo. Although MKP-1 may dephosphorylate and thus inactivate p38 and JNK MAPK, we speculate that the observed effect of MKP-1 is mainly through blocking ERK signaling, considering the fact that blocking these latter two pathways had no effect on BA-induced gene expression. Together, these findings suggest that ERK signaling may convey the hypertrophic effect of BA receptor signaling by enhancing fast fiber-specific gene expression and/or the associated changes in protein synthesis.

Investigation of the BA-induced activation of ERK in slow and fast muscles revealed that ERK signaling is differentially activated in these muscles. BA induces greater amounts of activated ERK1/2 in fast than in slow muscles, whereas the basal level of activated ERK1/2 is greater in fast than in slow muscles. The cumulative difference of the basal and induced activation of ERK between slow and fast muscles is about fivefold, an observation that may be sufficient for the differential responses of different muscles to BA stimulation. The level of the BA-induced activation of ERK1/2 in slow muscles, although less than that in fast muscles, may help drive the fiber type transition toward a faster phenotype. Further studies may be required to address whether ERK signaling is sufficient to induce the skeletal muscle hypertrophy or whether this pathway works synergistically with other signaling cascades (19, 45) to induce hypertrophy. However, ERK is necessary in the hypertrophic process, because genetically blocking this pathway by overexpression of MKP-1 antagonizes BA-induced increases in fast muscle-specific gene expression and muscle fiber hypertrophy in fast muscles.

The notion that ERK signaling is involved in BA-induced skeletal muscle hypertrophy is further supported by the fact that overexpression of a constitutively active ERK2 increased fast fiber-specific gene expression, mimicking the effect of BA-stimulation (unpublished observation). Working with rat ventricular cardiomyocytes, Gillespie-Brown et al. (15) showed that constitutively active MEK1 stimulates the expression of genes characteristic of hypertrophy. Although the mechanism of the involvement of ERK signaling in skeletal muscle hypertrophy remains unknown, we speculate that it may work through 1) protein accretion by mediating the phosphorylation of eukaryotic initiation factor eIF4E (43) and 2) the activation and recruitment of satellite cells to participate in the hypertrophic process in skeletal muscles. Satellite cells activation contributes to skeletal muscle hypertrophy (2), whereas ERK signaling is necessary for myoblast proliferation (4, 23).

Although BA receptor signaling and IGF-I signaling are distinct and in this study we failed to observe any significant changes in the activation of the Akt/Gsk3 signaling, a well-known pathway shown to participate in IGF-1-induced hypertrophy and antagonize muscle atrophy (6, 16, 33), blocking ERK signaling seems to result in similar reversion of the hypertrophic effect of both BA and IGF-I. Haddad and Adams (17) reported that blocking ERK signaling in vivo blunted IGF-I-induced skeletal muscle hypertrophy in rats. This further supports the hypothesis that ERK signaling is actively participate in skeletal muscle hypertrophy.

The observations that basal and BA-induced ERK MAPK activation are higher in fast muscles suggest that this signaling pathway may play an important role in fast muscle or muscle fiber type establishment and(or) maintenance. Whether downstream ERK signaling events are involved in regulation of fast fiber-specific gene expression or other components that may be controlling muscle protein turnover is not known but is intriguing.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. E. Gerrard, 915 W. State St., Dept. of Animal Sciences, Purdue Univ., West Lafayette, IN 47907 (e-mail: dgerrard{at}purdue.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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