| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Animal Sciences and
Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA
1Correspondence: 915 W. State St., Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA. E-mail: dgerrard{at}purdue.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: ERK1/2 • MKP-1 • plasticity • signal transduction
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2
3)
. The relative frequency of each muscle fiber type in a muscle defines its overall functional capabilities. In response to a myriad of environmental cues such as aging, atrophy, exercise, and diabetes, however, muscle modifies its functional characteristics by changing its fiber type (4
, 28)
. With minor exceptions to pathological conditions, muscle fibers switch to their "nearest neighbor type" following an obligatory pathway: I 
I/IIa IIa IIa/IIx IIx IIx/IIb IIb (4)
. Understanding the mechanisms responsible for changing muscle fiber type may lead to clinical interventions and treatment for some of the aforementioned maladies.
Nerve-mediated changes in cytosolic calcium alter downstream effector signaling molecules. Molecules such as calcineurin and calcium/calmodulin-dependent protein have been shown to control the slow-fiber phenotype (3
, 5
, 6)
. Peroxisome proliferator-activated receptor-
coactivator-1 
(PGC-1), a potent stimulator of mitochondrial biogenesis, and peroxisome proliferator-activated receptor 
have also been suggested to direct the fast to slow fiber conversion (5
, 7)
. Likewise, several reports have begun to unravel the signaling molecules that regulate the fast fiber phenotype. The mammalian homologues of Drosophila Six and Eya proteins physically interact to form a transcription complex, and overexpression of these proteins in mouse soleus appears to induce de novo synthesis of MyHC IIb, an isoform rarely detected in slow-contracting soleus muscle (8)
. Sox 6, a member of Sox family of transcription factor, is also implied in the regulation of fast-fiber phenotype in the late stage of embryogenesis, Sox 6-null mice lack fast fibers in late fetal skeletal muscles (9
, 10)
. A recent study suggests the transcriptional coactivator PGC-1β drives formation of fast IIx fibers in transgenic mice (11)
. Alternatively, in birds, muscarinic acetylcholine receptor (12)
, ryanodine receptor 1 (13)
, inositol triphosphate receptor 1 (14)
, and PKC (15)
are thought to repress the slow-twitch fiber phenotype in cultured chicken fast pectoralis major myocytes.
The mitogen-activated protein kinase (MAPK) family consists of at least four family members: extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun NH2-terminal kinase (JNK), and ERK5 (16)
. MAPKs are activated via phosphorylation of the threonine and tyrosine residues by MAP kinase kinases, and are inactivated via dephosphorylation by specific phosphatases (17)
. In particular, the MAPK phosphatase 1 (MKP1) is a nuclear phosphatase that selectively dephosphorylates and inactivates JNK, ERK1/2, and p38 MAPKs. Knockout of this gene results in a 5.4-, 3.1-, and 1.8-fold increase in activation of JNK, ERK2, and p38 in skeletal muscle, respectively (18)
. The roles of ERK1/2 (19)
, p38 (20
, 21)
, JNK (22)
, and ERK5 (23)
in muscle growth have been well documented. Moreover, activation of ERK1/2, p38, and JNK signaling cascades has been widely studied in skeletal muscle contraction during exercise and may be involved in the regulation of mechanically induced gene expression (24
, 25)
. However, the role of MAPK signaling cascades in modulating muscle fiber type remains unclear. p38 MAPK has been reported to control myosin heavy chain IIx promoter activity in myotubes (26)
. Moreover, Ras/MEK/ERK has been suggested to play a pivotal role in re-establishing the slow muscle programming in regenerating rat soleus muscle (27)
. Higginson et al. (28)
showed that ablation of the ERK1/2 pathway increases MyHC IIx and IIb transcripts in cultured rat fetal myocytes, whereas it decreases MyHC I expression. However, in our recent studies, we found that ERK1/2 activity was more than 2-fold higher in fast-twitch muscles than in slow-twitch muscle (29)
, suggesting that ERK1/2 pathway may play an important role in the maintenance of fast fiber phenotype. These findings highlighted the necessity of further investigating the role of MAPK signaling in terminally differentiated muscle fibers. In our studies, we hypothesized that MAPK signaling, especially the ERK1/2 pathway, may be necessary for maintaining the fast-twitch muscle fiber phenotype. To that end, we report herein that inhibition of MAPK signaling, especially the ERK1/2 pathway, decreases fast fiber-specific gene and protein expressions and induces the slow muscle fiber phenotype program in vitro and in vivo.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Fast fiber-specific luciferase reporter constructs harboring the MyHC IIb (–2560 to +13 bp) or sarco(endo)plasmic reticulum calcium ATPase 1 (SERCA1; –1373 to +172 bp) promoters were acquired from Dr. Steven Swoap (Williams College, Williamstown, MA, USA) (31
, 32)
. Constitutively active ERK2 (ERK2-MEK1-LA) was provided as a gift from Dr. Melanie Cobb (University of Texas Southwestern Medical Center, Dallas, TX, USA) (33)
. The enhanced green fluorescent protein (EGFP) -only plasmid pWay21 was obtained from Dr. Thomas Hughes (Montana State University, Bozeman, MT, USA). The pWayMKP1 expression plasmid containing the full length of MKP1 linked to the carboxyl terminus of EGFP in pWay21 was supplied by Dr. Anton Bennett (Yale University, New Haven, CT, USA) (34)
. The pCMV-LacZ, pGL3 containing the minimum TATA promoter, and pRL-SV40 plasmid expressing Renilla luciferase were from Promega (Madison, WI, USA).
Antibodies and reagents
Primary antibodies used for immunoblotting were anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-p38 (Thr180/Tyr182), anti-phospho-JNK (Thr183/Tyr185), anti-ERK1/2, anti-p38, and anti-JNK (Cell Signaling Technology, Beverly, MA, USA); anti-MyHC I (A4.840) (35)
; anti-myogenin, anti-tubulin, anti-fast MyHC antibody (MY-32, immunoreactive with MyHC IIA, IIx, and IIb), anti-SERCA1 (Sigma Chemical, St. Louis, MO, USA), and anti-myoglobin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Primary antibodies used for immunohistochemistry analyses were anti-GFP (Invitrogen, Carlsbad, CA, USA), anti-dystrophin (Millipore, Temecula, CA, USA), anti-MyHC I (A4.840) (35)
, and anti-MyHC IIa (6B8) (36)
. PD98059 was purchased from Cell Signaling Technology. SB203580 and SP600125 were purchased from EMD Biosciences (La Jolla, CA, USA).
Cell culture, transfection, and pharmacological treatment
Mouse C2C12 myoblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. For reporter gene experiments, reporter gene constructs were transfected into myoblasts using FuGene 6 (Roche Applied Science, Indianapolis, IN, USA) for 8 h according to the manufacturer’s recommendations. The pRL-SV40 plasmid expressing Renilla luciferase was cotransfected at a ratio of 1:50 to normalize transfection efficiency. After transfection, medium was shifted to DMEM containing 2% horse serum and 5 µg/ml insulin to induce differentiation. Thirty-six hours after transfection, cells were lysed in passive lysis buffer (Promega), and luciferase was analyzed using the Dual Luciferase Assay Kit (Promega). For fiber-type analysis, when cells reach 90% confluence, the medium was shifted to DMEM containing 2% horse serum to induce differentiation. Five-day differentiated myotubes were used for various treatment combinations. Myotubes were then washed with ice-cold PBS and lysed in modified RIPA lysis buffer (1% Nonidet P-40 in 50 mM NaCl, 20 mM Tris-HCl, pH 7.6) in the presence of 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 50 mM NaF, and 1 mM Na3VO4.
In vivo DNA injection and electroporation
Male imprinting control region (ICR) mice (35 g) or Sprague-Dawley rats (150 g) were used for gene delivery studies. Plasmid DNA was purified using EndoFree plasmid mega kits (Qiagen, Valencia, CA, USA). Plasmid DNA was diluted in sterile 0.9% saline to a final concentration of 0.5 µg/µl. For in vivo reporter assay, reporter constructs were injected into gastrocnemius muscles with either the pWay21 (control) or pWayMKP1 plasmids. Where indicated, minor surgery was performed to expose the soleus muscle and to inject plasmids. pRL-SV40 plasmid DNA was coinjected at a 1:100 ratio to normalize for transfection efficiency. Following injection, electroporation was performed (200 V/cm, 8 pulses, 1 Hz, 20-ms interval) using a BTX ECM 830 electroporator (Genetronics, San Diego, CA, USA). After a specified period of time, electroporated muscles were dissected and homogenized in passive lysis buffer (Promega) and analyzed using a Dual Luciferase Assay Kit (Promega). In overexpression experiments, the pWay21 and pWayMKP1 plasmids were each injected and electroporated into both the superficial medial or lateral gastrocnemius muscles. At indicated times, muscles were dissected and frozen in isopentene cooled in liquid nitrogen. For immunohistochemical analysis, serial transverse muscle sections were cut (10 µm) and stored at –80°C. All procedures were conducted in accordance with guidelines set by the Purdue Animal Care and Use Committee.
Immunohistochemical analysis of muscle fiber types
Frozen serial muscle sections from gastrocnemius muscles were air-dried at room temperature for 30 min and fixed in ice-cold acetone for 6 min. Sections were briefly washed in PBS and blocked in 5% goat serum for 30 min, followed by incubation with primary antibodies in 5% goat serum at room temperature for 2 h. After three 5-min washes, Cy3-conjugated (for GFP) or biotinylated (for dystrophin and MyHC IIa or I) secondary antibodies in PBS were applied on the sections for another 1 h at room temperature. Biotin was detected by using a Cy2-conjugated streptavidin that was incubated on sections for 30 min. Muscle fiber type was assigned for each muscle fiber in a given field based on predominant MyHC isoform. Representative micrographs were taken using Leica DMI6000 B microscope (Leica Microsystems, Bannockburn, IL, USA) with a Photometrics CoolSNAP camera (Roper Scientific, Tucson, AZ, USA). Image color was reassigned using Adobe Photoshop CS2 (ver. 9; Adobe Systems, San Jose, CA, USA) as follows: GFP, green; dystrophin and MyHC IIa and I, red.
Immunoblotting
Protein concentration of cell lysates was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA), and an equal amount of protein was separated on 10% SDS-PAGE gels. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane and blocked with 5% nonfat milk in TBS at 4°C overnight. After two 5-min washes in TBS containing 0.1% Tween-20, membranes were incubated with primary antibodies diluted in 5% nonfat milk and TBS for 1 h at room temperature. After three 5-min washes, blots were incubated with a peroxidase-conjugated secondary antibody for 1 h at room temperature. Bands were visualized using ECL (Amersham, Piscataway, NJ, USA). Where indicated, blots were stripped and reprobed with different antibodies.
Data analysis
Values were expressed as means ± SE (represented as error bars). Statistical analysis was performed using Student’s t test, and the significance level was set as P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. To further investigate the activation of ERK1/2 in slow- and fast-twitch muscles, we chose mouse and rat soleus and extensor digitorum longus (EDL) as representative slow- and fast-twitch muscles, respectively. We observed that phosphorylated ERK1 was similar in mouse and rat soleus and EDL muscles (Fig. 1
A). In contrast, the content of phosphorylated ERK2 was more than 2-fold greater in the EDL than in soleus muscle (Fig. 1A, B
). Analysis of nonphosphorylated ERK1 and ERK2 isoforms revealed that ERK2 was the predominant isoform in the EDL, whereas in the soleus, the abundance of these two isoforms was similar (Fig. 1A, C
). To test whether differences in the activation of p38 and JNK pathways was muscle type dependent, we isolated mouse soleus, gastrocnemius, and TA muscles and subjected muscle homogenates to immunoblotting. No differences in either phosphorylated p38 or phosphorylated JNK content were observed (Fig. 1D-F
). Together, these data suggest that ERK2, rather than p38 and JNK, is differentially activated in slow- and fast-twitch muscles.
|
MAPK signaling differentially regulates fiber type-specific reporter gene activities in vitro
To investigate the role of MAPK signaling in the regulation of skeletal muscle fiber-type specification, we transfected slow and fast fiber type-specific reporter gene constructs into C2C12 myoblasts, induced differentiation, and applied specific inhibitors to myotubes to block MAPK signaling. Blocking ERK1/2 signaling with MEK inhibitor PD98059 induced a 3.2-fold increase in MyHC I-Luc activity, whereas the activities of either of the fast fiber type-specific reporters (SERCA1, SERCA1-Luc, and MyHC IIb-Luc) were inhibited (Fig. 2
A). Application of p38 and JNK inhibitors did not elicit detectable changes in reporter gene activities (Fig. 2B
), suggesting that these reporters were not sensitive to blockade of p38 and JNK signaling. Because ERK2 is the predominant isoform and preferentially activated in fast-twitch muscles (Fig. 1A-C
), we postulated that overexpression of a constitutively active ERK2 may affect the activities of the aforementioned reporter constructs. Consistent with the effect of the pharmacological inhibitor PD98059, overexpression of a constitutively active ERK2 down-regulated the MyHC I reporter construct by 83% but up-regulated both fast reporters SERCA1 and MyHC IIb by 2.6- and 2.7-fold, respectively (Fig. 2C
). Collectively, inhibition of ERK1/2 signaling activated slow reporter genes, yet repressed the activities of reporters containing fast promoter elements.
|
Inhibition of ERK1/2 signaling induces the slow fiber phenotype in vitro
To confirm the aforementioned reporter findings, we again utilized the C2C12 cell culture system and applied pharmacological inhibitors to differentiated myotubes. We observed that MyHC I expression in myotubes reaches a plateau at day 5 postdifferentiation (Fig. 3
A) and chose this day for the initial treatment with various MAPK inhibitors. We observed that half dosage of these inhibitors for 3 days had no detectable effect on myotube morphology, whereas full dosage caused myotubes to detach from culture dishes. Thus, we treated cultures with 5 µM of SP600125 or SB203580 on differentiated myotubes (5 day) for 3 days and quantified various proteins to reflect fiber phenotype. We failed to observe any muscle fiber type-specific changes with either inhibitor (Fig. 3B
). In contrast, inhibition of ERK signaling with 25 µM PD98059 for 3 days caused a dramatic decrease in fast fiber type-specific MyHCs and SERCA1 abundance and a significant increase in slow fiber type-specific MyHC I and myoglobin protein production (Fig. 3C
). Taken together, these data show that inhibition of ERK1/2 signaling results in the up-regulation of proteins characteristic of slow fibers and down-regulates those normally associated with fast-contracting fibers, consistent with earlier results.
|
Overexpression of MKP1 induces de novo synthesis of MyHC I or IIa in fast-twitch IIb and IIx fibers in mice
To test the functional role of MAPKs in the regulation of fiber phenotype, we electroporated EGFP-tagged MKP1 plasmids (pWayMKP1) into mouse superficial gastrocnemius muscles. The EGFP-only plasmid (pWay21) was electroporated into the contralateral muscle as a control. The gastrocnemius muscle is ideal for this type of study because the distribution of muscle fiber types in this muscle is unique. Type I and IIa fibers are entirely located in the deep regions of gastrocnemius muscle close to the soleus, whereas the superficial aspects of both medial and lateral gastrocnemius muscles are exclusively composed of type IIb fibers with a scattering of a few type IIx fibers (37)
. As a result, we used the superficial gastrocnemius muscle as the site for pWayMKP1 injection and electroporation, and postulated that fibers expressing plasmid DNA would begin to express proteins characteristic of slow contracting muscle fibers. We observed that most GFP-positive fibers did not undergo degeneration and regeneration, as evidenced by lack of centralized nuclei 7 and 13 days postelectroporation. These fibers also showed no difference in diameter from the surrounding fibers. After 7 days, the electroporated gastrocnemius muscle was dissected and cut into serial sections and stained with GFP, MyHC I/dystrophin, and MyHC IIa/dystrophin antibody combinations. We observed no evidence of fiber type switching with overexpression of the control EGFP plasmid (pWay21) (Fig. 4
A). However, forced expression of EGFP-tagged MKP1 for 1 wk induced the de novo synthesis of type IIa and/or I MyHCs in IIb/IIx fibers (Fig. 4B
). All of the MyHC I- or IIa-positive fibers were GFP positive, but not all of the GFP-positive fibers contained MyHC I or IIa MyHC, suggesting MKP1-overexpression-induced de novo synthesis of slower MyHCs may require at least 1 wk. Together, these findings show that forced expression of MKP1 induces the expression of MyHCs that are characteristic of slow-twitch fibers.
|
Overexpression of MKP1 induces slow fiber-specific reporter gene activity, whereas fast fiber-specific reporters are inhibited in vivo
To investigate whether the de novo synthesis of MyHC I/IIa in IIb/x fibers induced by overexpression of MKP1 is mirrored by changes in the transcription of fiber type-specific genes, we electroporated slow and fast fiber-specific reporters with either pWay21 (control) or pWayMKP1 (treatment) plasmids into the superficial aspects of mouse gastrocnemius muscles. Forced expression of MKP1 induced a 4.7- and 8.2-fold increase in the slow reporter MyHC I-Luc by 1 and 2 wk postelectroporation, respectively (Fig. 5
A). Overexpression of MKP1 markedly inhibited both SERCA1-Luc and MyHC IIb-Luc reporters by 75 and 73%, respectively (Fig. 5B, C
). These findings corroborate our immunohistochemical staining data, suggesting that fiber type phenotype is regulated at both the protein and gene expression levels.
|
Forced expression of MKP1 induced similar phenotype in rat muscle in a time-dependent manner
We determined whether the altered fiber type phenotype observed in mice could be replicated in rats. We electroporated the pWayMKP1 construct into the superficial rat gastrocnemius muscle. As in mice, rat superficial gastrocnemius muscles are composed exclusively of IIb and IIx fibers. Forced expression of MKP1 in the rat superficial gastrocnemius muscle induced a similar fiber phenotype as that observed in mouse muscle (Fig. 6
A, B). To investigate whether the de novo synthesis of MyHC I/IIa in IIb/x fibers was time dependent, we obtained muscles from 7 and 13 days postelectroporation. After 7 days, however, a portion of fast fibers contained immunoreactive type I or IIa MyHC (Fig. 6A
). By 2 wk, a greater number of IIb or IIx fibers contained detectable levels of type I and IIa MyHCs (Fig. 6B
). To capture this event quantitatively, we enumerated all GFP-(MKP1), MyHC IIa-, and MyHC I-positive fibers and expressed these data on a percentage basis (Table 1
). At day 7 postelectroporation, 43 and 20% of GFP-positive fibers were IIa and I positive, respectively, whereas at day 13 postelectroporation, these figures increased to 74 and 58%, respectively. Granted, there was some overlap in the number of fibers expressing both MyHC IIa and I. Another caveat to this methodology resides in the fact that some IIa and I staining accounts for only a very small portion of the whole cross-sectional area of some GFP-positive fibers, although we observed an increase in the percentage of fibers that expressed MyHC I/IIa between the two time points. These data proved that the similar effect of forced expression of MKP1 on fiber type phenotype occurred in rats as observed in mice and suggested that time-dependent changes may reflect the necessity of continued cell-signaling events to alter muscle fiber phenotype.
|
|
Overexpression of MKP1 differentially regulates slow and fast fiber-specific reporter activities in rat muscles
As in mice, we also posed the question whether we could detect a change in fiber type-specific reporter activity by forced expression of MKP1 in rat muscles. After minor surgery to expose the soleus, we injected slow MyHC I reporter gene constructs with either the pWay21 (control) or pWayMKP1 (treatment) plasmids. MKP1 overexpression induced nearly a 12-fold increase in MyHC I reporter activity in soleus (Fig. 7
A), much greater than the induction of the same reporter in rat gastrocnemius muscle (5.8-fold increase) (Fig. 7B
). Consistent with that shown in the mouse, overexpression of MKP1 in the rat gastrocnemius dramatically decreased fast SERCA1-Luc reporter activity by 84% (Fig. 7C
). These findings in rats are consistent with those of mice, confirming a role of MAPK signaling in the maintenance of fast fiber phenotype.
|
Activation of ERK2 signaling induced differential response of slow and fast reporters in slow and fast muscle
To address the question whether the MKP1-overexpression-induced fiber type change is via inhibition of ERK2 signaling, we electroporated constitutively active ERK2 into the soleus muscle together with slow and fast reporters. Activation of ERK2 induced a 4.8-fold increase in fast MyHC IIb reporter activity, whereas it had no effect on slow MyHC I reporter activity (Fig. 8
A). In the fast-contracting gastrocnemius muscle, activation of ERK2 signaling had no effect on either slow or fast reporters (Fig. 8B
). Together, the results indicate that overexpressing ERK2 induced the fast fiber type program in the slow-contracting soleus muscle.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Furthermore, constitutively active MAP kinase kinase 6, an upstream kinase of p38, is reported to promote fast MyHC expression in the presence of forced MyoD expression (38)
. Meissner (26)
also reported that p38 MAPK can regulate MyHC IIx promoter activity yet has little effect on fast MyHC IIa promoter activity (39)
. In addition, muscle contains all three p38 isoforms (, β, and ) and we inhibited only the /β isoforms with SB203580. Thus, it is possible that the isoform may play a significant role in controlling muscle fiber type.
Although MKP-1 dephosphorylates and thus inactivates ERK1/2, p38, and JNK, we postulated that overexpression of MKP1-induced alteration in fiber type phenotype is mainly through ERK1/ERK2 MAPK signaling pathways because no detectable effects of p38- and JNK-specific inhibitors were observed on reporter activities and proteins characteristic of slow and fast fibers. Granted, it is possible that the observed MKP-1 effect is mediated through the interaction of ERK1/2, p38, or JNK MAPKs. For example, though each of these three MAPKs has distinct downstream effector molecules, they share common substrates such as Elk-l (40)
, which provides a platform for an integration of these signaling pathways. Nonetheless, how these signaling pathways interact to fine-tune fiber type-specific programs remains to be investigated.
Our findings support the hypothesis that MAPK signaling, especially the ERK1/2 pathway, is necessary for the maintenance of fast fiber phenotype, a process that may involve the repression the slow fiber phenotype. There are several lines of evidence to support this hypothesis. First, fast muscles, composed mainly of type II fibers, have a higher level of ERK2 activation than those muscles predominantly composed of type I fibers. Second, overexpression of a constitutively active ERK2 enhanced fast fiber type-specific reporter activities, whereas it inhibited slow reporter activity. Likewise, inhibition of ERK signaling repressed the fast fiber phenotype, whereas it up-regulated the slow phenotype. Third, forced expression of MKP1 in vivo induced de novo synthesis of MyHC I/IIa in fast IIb/x fibers in both mouse and rat fast muscles. Finally, overexpression of a constitutively active ERK2 induced the fast fiber type-associated MyHC IIb reporter activity by 4.8-fold in soleus muscle. Lack of an effect of ERK2 on the MyHC IIb reporter construct in the fast-contracting gastrocnemius muscle may be partially due to the fact that MyHC IIb is already highly expressed in this muscle. Surprisingly, constitutively active ERK2 had no effect on slow reporter in vivo compared to its inhibition of slow reporter activity in cell culture experiments. This discrepancy suggests that other factors, such as neuronal activity, may also play a pivotal role in controlling the slow fiber type program in muscles such as the soleus. Nonetheless, when ERK2 signaling is activated, the fast fiber type program is up-regulated in slow muscle.
Even though our results suggest that MAPK signaling is required for the maintenance of fast fiber phenotype, some observations differ from those reported by Murgia et al. (27)
and Higginson et al. (28)
. Using regenerating soleus, Murgia et al. suggested that Ras/MEK/ERK is required for the re-establishment of slow fiber program. The requirement of ERK signaling in the regenerating soleus may be due to its role in promoting muscle precursor cell proliferation (41
42
43)
. Likewise, ERK may also be utilized to reconstruct fast muscles during regeneration. Our results suggest a complementary role of ERK1/2 in fiber type specialization in fast muscles. Therefore, it is reasonable that select pathways may be tailored to suit the various roles that different muscles (slow vs. fast) may need depending on their physiological status (for example, regeneration vs. maintenance). In another study, Higginson et al. (28)
investigated the effect of MEK inhibitor U0126 on MyHC expression in cultured rat fetal myotubes and showed that blocking MEK/ERK pathway increased MyHC IIx and IIb transcripts, yet decreased MyHC I mRNA. Curiously, no change in MyHC IIa mRNA was observed in this study. However, the mitochondrial enzymes malate dehydrogenase (MDH) and β-hydroxy-acyl-CoA dehydrogenase (HAD) were up-regulated. In addition, these researchers did not monitor fiber type-specific contractile and regulatory protein abundance to verify that changes in gene expression paralleled changes in protein content. Regardless of these differences, our data consistently point to the role of MAPK signaling in the regulation of muscle fiber type and provide a molecular mechanism responsible for driving fast fiber-specific gene expression.
Although our findings reveal a novel role of MAPKs, especially ERK signaling, in the maintenance of fast fiber phenotype, the upstream signals and the downstream effectors have yet to be identified. In view of distinct neuronal firing patterns in slow and fast fibers (44
, 45)
, it is intriguing to investigate whether the pattern of calcium oscillation in fast fibers favors the activation of ERK1/2. The activated ERK signaling regulates fast fiber-specific gene expression, likely through 1) phosphorylation and activation of transcription factors (16
, 46
, 47)
; 2) enhanced translation initiation (48)
; and 3) (satellite) cell cycle promotion (49
50
51
52)
. Furthermore, our data do not rule out the roles of other pathways or mechanisms in the regulation of fast fiber phenotype. For example, it would be interesting to study whether there is any interaction between ERK1/2 and such transcription factors as Six1/Eya1 (8)
and Sox6 (9
, 10)
in adult and fetal skeletal muscles. On the basis of the reported mechanisms and pathways that are involved in the control of muscle fiber type, it is reasonable to speculate that fiber phenotype is regulated and/or maintained by the interaction of a myriad of these intracellular events, among which MAPK signaling plays an integral role in defining muscle fiber identity.
Received for publication September 13, 2007. Accepted for publication March 27, 2008.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B signaling in skeletal muscle. J. Appl. Physiol. 103,388-395, 2007This article has been cited by other articles:
|
|
 |
|
  T. Yamaguchi, T. Suzuki, H. Arai, S. Tanabe, and Y. Atomi Continuous mild heat stress induces differentiation of mammalian myoblasts, shifting fiber type from fast to slow Am J Physiol Cell Physiol, January 1, 2010; 298(1): C140 - C148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.-U. Bae, Y.-J. Yang, G. Jiang, M. Hong, H.-J. Lee, M. Tessier-Lavigne, J.-S. Kang, and R. S. Krauss Neogenin Regulates Skeletal Myofiber Size and Focal Adhesion Kinase and Extracellular Signal-regulated Kinase Activities In Vivo and In Vitro Mol. Biol. Cell, December 1, 2009; 20(23): 4920 - 4931. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-A. Caron, R. Debigare, P. N. R. Dekhuijzen, and F. Maltais Comparative assessment of the quadriceps and the diaphragm in patients with COPD J Appl Physiol, September 1, 2009; 107(3): 952 - 961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Shi, J. M. Scheffler, C. Zeng, J. M. Pleitner, K. M. Hannon, A. L. Grant, and D. E. Gerrard Mitogen-activated protein kinase signaling is necessary for the maintenance of skeletal muscle mass Am J Physiol Cell Physiol, May 1, 2009; 296(5): C1040 - C1048. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. A. LaFramboise, R. C. Jayaraman, K. L. Bombach, D. P. Ankrapp, J. M. Krill-Burger, C. M. Sciulli, P. Petrosko, and R. W. Wiseman Acute molecular response of mouse hindlimb muscles to chronic stimulation Am J Physiol Cell Physiol, January 1, 2009; 297(3): C556 - C570. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
