Acute molecular response of mouse hindlimb muscles to chronic stimulation

Article (PDF Available)inAJP Cell Physiology 297(3):C556-70 · August 2009with35 Reads
DOI: 10.1152/ajpcell.00046.2009 · Source: PubMed
Stimulation of the mouse hindlimb via the sciatic nerve was performed for a 4-h period to investigate acute muscle gene activation in a model of muscle phenotype conversion. Initial force production (1.6 +/- 0.1 g/g body wt) declined 45% within 10 min and was maintained for the remainder of the experiment. Force returned to initial levels upon study completion. An immediate-early growth response was present in the extensor digitorum longus (EDL) muscle (FOS, JUN, activating transcription factor 3, and musculoaponeurotic fibrosarcoma oncogene) with a similar but attenuated pattern in the soleus muscle. Transcript profiles showed decreased fast fiber-specific mRNA (myosin heavy chains 2A and 2B, fast troponins T(3) and I, alpha-tropomyosin, muscle creatine kinase, and parvalbumin) and increased slow transcripts (myosin heavy chain-1beta/slow, troponin C slow, and tropomyosin 3y) in the EDL versus soleus muscles. Histological analysis of the EDL revealed glycogen depletion without inflammatory cell infiltration in stimulated versus control muscles, whereas ultrastructural analysis showed no evidence of myofiber damage after stimulation. Multiple fiber type-specific transcription factors (tea domain family member 1, nuclear factor of activated T cells 1, peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -beta, circadian locomotor output cycles kaput, and hypoxia-inducible factor-1alpha) increased in the EDL along with transcription factors characteristic of embryogenesis (Kruppel-like factor 4; SRY box containing 17; transcription factor 15; PBX/knotted 1 homeobox 1; and embryonic lethal, abnormal vision). No established in vivo satellite cell markers or genes activated in our parallel experiments of satellite cell proliferation in vitro (cyclins A(2), B(2), C, and E(1) and MyoD) were differentially increased in the stimulated muscles. These results indicated that the molecular onset of fast to slow phenotype conversion occurred in the EDL within 4 h of stimulation without injury or satellite cell recruitment. This conversion was associated with the expression of phenotype-specific transcription factors from resident fiber myonuclei, including the activation of nascent developmental transcriptional programs.


Acute molecular response of mouse hindlimb muscles to chronic stimulation
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
Department of Pathology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine,
Shadyside Hospital, Pittsburgh; and
Department of Pediatrics, Drexel University School of Medicine, Allegheny General
Hospital, Pittsburgh, Pennsylvania;
School of Health Sciences, Exercise Science Division, Central Michigan University,
Mount Pleasant; and Biomedical Imaging Research Center, Departments of
Physiology and
Radiology, Michigan State
University, East Lansing, Michigan
Submitted 23 January 2009; accepted in final form 15 July 2009
LaFramboise WA, Jayaraman RC, Bombach KL, Ankrapp
DP, Krill-Burger JM, Sciulli CM, Petrosko P, Wiseman RW.
Acute molecular response of mouse hindlimb muscles to chronic
stimulation. Am J Physiol Cell Physiol 297: C556 –C570, 2009. First
published July 22, 2009; doi:10.1152/ajpcell.00046.2009.—Stimula-
tion of the mouse hindlimb via the sciatic nerve was performed for a 4-h
period to investigate acute muscle gene activation in a model of muscle
phenotype conversion. Initial force production (1.6 0.1 g/g body wt)
declined 45% within 10 min and was maintained for the remainder of the
experiment. Force returned to initial levels upon study completion. An
immediate-early growth response was present in the extensor digitorum
longus (EDL) muscle (FOS, JUN, activating transcription factor 3, and
musculoaponeurotic fibrosarcoma oncogene) with a similar but attenuated
pattern in the soleus muscle. Transcript profiles showed decreased fast
fiber-specific mRNA (myosin heavy chains 2A and 2B, fast troponins T
I, -tropomyosin, muscle creatine kinase, and parvalbumin) and increased
slow transcripts (myosin heavy chain-1/slow, troponin C slow, and tropo-
myosin 3y) in the EDL versus soleus muscles. Histological analysis of the
EDL revealed glycogen depletion without inflammatory cell infiltration in
stimulated versus control muscles, whereas ultrastructural analysis showed
no evidence of myofiber damage after stimulation. Multiple fiber type-
specific transcription factors (tea domain family member 1, nuclear factor of
activated T cells 1, peroxisome proliferator-activated receptor-coactiva-
tor-1and -, circadian locomotor output cycles kaput, and hypoxia-induc-
ible factor-1) increased in the EDL along with transcription factors charac-
teristic of embryogenesis (Kruppel-like factor 4; SRY box containing 17;
transcription factor 15; PBX/knotted 1 homeobox 1; and embryonic lethal,
abnormal vision). No established in vivo satellite cell markers or genes
activated in our parallel experiments of satellite cell proliferation in vitro
(cyclins A
and MyoD) were differentially increased in the
stimulated muscles. These results indicated that the molecular onset of fast to
slow phenotype conversion occurred in the EDL within4hofstimulation
without injury or satellite cell recruitment. This conversion was associated
with the expression of phenotype-specific transcription factors from resident
fiber myonuclei, including the activation of nascent developmental transcrip-
tional programs.
myofiber; myocyte; fiber type; satellite cell; expression profiling; tran-
scription factor; plasticity; myogenesis; contraction; force; chronic stim-
ulation; histology
ADULT SKELETAL MUSCLES subjected to variations in activity or
loading exhibit striking phenotype plasticity in their ability to
alter contractile and metabolic protein expression over a broad
range of transitional and mature myofiber types (26 –28, 37,
62– 63, 71–73). This phenotype plasticity was originally de-
scribed in cross-innervation (9), chronic nerve stimulation (71),
and denervation studies (41) conducted over 4 decades ago and
led to studies of phenotype conversion during overload via
synergist ablation (32), exercise (28), chronic stretch (17), and
unweighting by hindlimb or whole body suspension (56, 60).
Delineation of fiber type transitions required the detection of
histochemical changes in myofibrillar ATPase activity under
varying pH conditions (41), enzymatic assays of metabolic
enzymes (79), or physiological motor unit analysis (24). How-
ever, identification of the specific myosin heavy chains (MyHCs)
underlying the physiological properties of muscle fiber pheno-
types provided definitive proteins and transcripts as precise
indicators of the conversion process (3, 6, 68). Comprehensive
expression profiling of muscle fiber transformation has now
been performed in rabbit and rodent muscles associated with
aging (61), hindlimb suspension (87), microgravity (2), injury
(80), regeneration (25), and exercise (13, 53). Many of these
transcriptional changes have been individually validated at the
protein level, but the application of high-throughput mass
spectroscopy assays to chronically stimulated fast muscle (23)
has recently added a comprehensive proteomics analysis of the
myofiber transition process to this extensive database of mus-
cle fiber type plasticity. While these studies have necessarily
focused on the outcomes of myofiber phenotype changes in the
days to months after the inductive stimulus, little information is
available regarding the onset of this process. Specifically, the
early molecular events associated with the initiation of the fast
to slow transition induced by a defined physiological stimulus
regimen have yet to be extensively examined at the molecular
Conversion of a fast muscle to a slow phenotype by chronic
low-frequency electrical nerve stimulation (CLFS) has been
well established as a model to study muscle phenotype switch-
ing in vivo (42, 63) and was the focus of the present study.
Unilateral hindlimb stimulation offers several advantages.
First, the contralateral muscle from the same animal provides
an unstimulated control that is genetically identical and sub-
jected to the same circulating plasma hormones and growth
factors. Second, the use of neuronal stimulation rather than
intramuscular electrodes markedly reduces the risk of muscle
damage since the stimulation is indirectly applied and excita-
tion levels can be adjusted to prevent injury. Third, the appli-
cation of supramaximal neural stimuli provides uniform acti-
vation of all motor units and eliminates the complexities
associated with fiber recruitment patterns. Fourth, experiments
can be designed to measure physiological performance (force
per time unit or duty cycle) so that it can be precisely quantified
Address for reprint requests and other correspondence: W. A. LaFramboise, Dept.
of Pathology, Shadyside Hospital West Wing, WG02.11, 5230 Center Ave., Pitts-
burgh, PA 15232 (e-mail: and R. W. Wiseman, Dept. of
Physiology, Michigan State University, 2201 Biomedical and Physical Sciences
Building, East Lansing, MI 48824 (e-mail:
Am J Physiol Cell Physiol 297: C556–C570, 2009.
First published July 22, 2009; doi:10.1152/ajpcell.00046.2009.
0363-6143/09 $8.00 Copyright ©2009 the American Physiological Society http://www.ajpcell.orgC556
and continuously monitored to control the magnitude of the
stimulus and minimize the risk of injury. Finally, the pheno-
typic characteristics of the mouse extensor digitorum longus
(EDL) and soleus muscles have been characterized based on
histochemical, immunohistochemical, and physiological as-
says of the different MyHCs and myosin light chains (MyLCs)
that distinguish individual fiber types (8, 45, 47). Because
of these advantages, this chronic stimulation model was used in
the present study to interrogate the underlying acute transcrip-
tional changes associated with the phenotypic remodeling that
occurs with increased contractile activity.
In addition to the remarkable plasticity that mature skeletal
muscles display to altered functional demands, remodeling
often occurs when muscle tissue undergoes activation of an
otherwise quiescent pool of resident progenitor muscle cells
called satellite cells. After injury, adult skeletal muscles un-
dergo repair and regeneration by the activation of these myo-
genic satellite cells, which form new myofibers or fuse with
existing fibers (12, 31, 57, 78). The degree to which satellite
cell activation contributes to muscle remodeling in the absence
of injury remains a source of considerable controversy com-
plicated by the fact that experimental models of myofiber
adaptation, e.g., nerve or muscle stimulation, endurance or
ballistic exercise, and hindlimb loading or unweighting via
synergist ablation, may cause varying degrees of trauma, in-
flammation, and myogenic cell activation as part of the regen-
eration response (1, 34, 49, 91).
The central hypothesis of this study was that muscle fiber
type transformation can occur in the absence of injury and
without the activation of satellite cell nuclei. To test this
hypothesis, the chronic stimulation model was used, and high-
resolution transcriptional profiling was performed to investi-
gate acute changes (4 h) in the mouse EDL muscle, comprised
of fast fiber types (MyHC-2B and 2X), and the soleus muscle,
containing predominantly slow and 2A fiber types (MyHC-1/
slow and 2A) (45, 47, 92). Gene expression of the stimulated
muscles was directly compared with the unstimulated, con-
tralateral muscles for each animal. Physiological responses
were continuously monitored to assess the contractile perfor-
mance of the hindlimb including both the muscle and nerve.
Subsequent histological and ultrastructural analyses were used
to evaluate the morphological integrity of the target muscles.
To delineate transcripts associated with satellite cell activation,
separate mouse EDL and soleus muscles were harvested, and
primary satellite cell cultures were purified and expanded in
culture. Molecular profiles were generated for these cells via
microarray analysis both during proliferation as myoblasts and
after differentiation into myocytes and myotubes in vitro for
comparison with the transcriptional activity of control and
stimulated muscles in vivo.
Exercise protocol and muscle physiology. All mouse care and
experimental protocols were approved by the All University Commit-
tee on Animal Use and Care at Michigan State University. Adult male
Swiss Webster mice (30 –35 g, Harlan, Indianapolis, IN) were main-
tained in a controlled environment with a 12:12-h light-dark cycle and
food and water administered ad libitum. Experiments were performed
on mice anesthetized to a deep plane of surgical anesthesia with an
intraperitoneal injection of pentobarbital sodium (50 mg/kg). Addi-
tional doses were administered as necessary by monitoring respiration
and heart rate in each animal using a pressure transducer placed on the
chest cavity. Core body temperature was maintained at 37°C using a
temperature-controlled environmental chamber and monitored using a
thermocouple placed in the rectum of the animal. Surgical prepara-
tions comprised a small incision (0.5 cm) proximal to the iliac crest to
expose the sciatic nerve as previously described (69). In brief, a pair
of bipolar hook electrodes was implanted adjacent to the nerve, and
the wound was sealed with cyanoacrylate glue. The hindlimb was
subsequently immobilized by positioning the animal prone on a
Plexiglas platform fitted with a patellar brace to fix the knee. The
animal was positioned with the chest wall over an aperture designed
to fit a pressure sensor built into the platform to monitor respiration
and heart rate. A length of 2.0 silk suture was attached to the Achilles
tendon proximal to the calcaneous process, and this ligature was then
connected to a force transducer (Grass FT10, Astromed, West War-
wick, RI) to record the performance of the gastrocnemius-plantarus-
soleus muscle group throughout the stimulation time course. Before
each experiment, the voltage-force relation was determined to estab-
lish supramaximal stimulation conditions, and the length-tension
relation was determined to set the resting length for maximum twitch
tension development. Contractions were induced by sciatic nerve
stimulation using a Grass S48 stimulator (0.5-ms duration, 2–5 V).
Muscles were allowed to rest for 15 min after these preliminary tests
to allow for full metabolic recovery at physiological temperatures
(86). Supramaximal stimulation was applied at a rate of 10 Hz for 4 h
(42, 63, 73). Force was digitally captured using an AT MIO 16E
analog-to-digital board and software (National Instruments, Austin,
TX) and analyzed using custom MatLab software (MathWorks,
Natick, MA) (39). After cessation of the stimulation period, test
twitches were elicited via the sciatic nerve to assess nerve viability
and to confirm recover of muscle force in each animal before tissue
harvest. Four hours of stimulation were selected as a time frame in
which the animals exhibited full recovery after completion of the
stimulation protocol. At the end of each experiment, the EDL and
soleus muscles were carefully dissected and either flash frozen in
liquid nitrogen and stored at 80°C for later analysis of mRNA
expression or fixed at resting length in 10% neutral buffered formalin
for histological processing.
Histological analysis. Whole muscle specimens from matching
stimulated and contralateral resting muscles (n5) were removed
and fixed at resting length in 10% neutral buffered formalin, embed-
ded in paraffin, and cut in serial sections to a thickness of 5– 8 m.
Alternate sections were stained via a standard hematoxylin and eosin
protocol versus periodic acid Shiff (PAS) to detect differences in
intracellular glycogen levels as an index of muscle activity (24). PAS
staining of both experimental and control muscles was performed
concurrently in the same coplin jars, and rinsing times were held
constant to control for methodological artifacts. The PAS protocol
was performed according to the directions specified by the manufac-
turer (no. 24200, Polysciences, Warrington, PA). In brief, slides
containing the muscle sections were deparaffinized by serial washing
with xylene and descending concentrations of alcohol to distilled
water. Slides were placed in 0.5% periodic acid for 5 min followed by
a distilled water rinse (3 times) and then placed in Schiff’s reagent for
15 min. Slides were then washed in 0.55% potassium metabisulfite for
1 min (2 times) and rinsed under gently running tap water for 10 min
to allow the color to develop. Acidified Harris hematoxylin was
applied to each slide for 30 s as a counterstain, after which the
specimens were dehydrated (95% alcohol, 100% alcohol, and xylene)
and a coverslip was applied to each slide.
Electron microscopy analysis. Whole muscles from matched stim-
ulated and control animals (n3 pairs) were rapidly dissected,
mounted on toothpicks at resting length, and placed in 2.5% glutar-
aldehyde in PBS for overnight fixation at 4°C. Postfixation was
performed in aqueous 1% osmium tetroxide for 60 min. Specimens
were dehydrated with ethanol (50% ethanol for 15 min, 70% ethanol
for 15 min, 95% ethanol for 15 min, and 100% ethanol twice for 15
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
min) and propylene oxide (2 15 min), slowly infiltrated with epoxy
resins (Embed 812/Araldite, Electron Microscopy Microscopy Sci-
ences, Hatfield, PA), embedded, and then heated overnight at 60°C.
Thick sections (5–10 m) were obtained from longitudinal cuts along
the length of the muscle and stained with hematoxylin and eosin or
toluidine blue O. Thin sections (90 nm) were cut, stained with
uranium and lead salts, and examined using a transmission electron
microscope (FEI Philips CM12). For scanning electron microsopy, the
specimens were critical point dried, mounted on aluminum stubs,
sputtered with gold coat, and examined under a JEOL 6335F field
emission gun scanning electron microscope.
Satellite cell culture model. The methods for purification and
culture of primary muscle cells have been previously described (45).
Briefly, muscles of the anterior and superficial posterior compartments
were separately dissected, removed aseptically, pooled, and finely
minced with iris scissors to form a suspension that was seeded on
multiple 100-mm tissue culture dishes and left undisturbed overnight
in an incubator at 37°C. Each plate contained 10 ml of growth media
composed of Ham’s F10C (calcium: 1.2 mM, Sigma, St. Louis, MO),
bovine basic fibroblast growth factor [bFGF (6.6 ng/ml), Sigma], 200
l chick embryo extract (CEE; Invitrogen, Carlsbad, CA), and 10 l
Fungizone (GIBCO-BRL, Gaithersburg, MD). The muscle suspension
was transferred to a 15-ml conical tube on ice, triturated, and vortexed
vigorously for 30 s. Tubes were then centrifuged at low speed (Jouan
BH-12, 1,000 rpm for 5 min at 4°C), the supernatant was removed,
and the pellet was resuspended in growth media for transfer to a
100-mm dish. Plates were incubated overnight at 37°C and received
supplemental feeding after 12 h (66 ng bFGF and 200 l CEE). The
entire procedure, beginning with trituration, was repeated each day for
5 days including preplating on plastic plates to remove fibroblasts.
Eventually, a mix of purified myoblast clones remained, which were
seeded on denatured collagen (0.07% gelatin, Difco, Kansas City,
MO)-coated plates. All subsequent media exchanges (every 12 h)
involved complete growth media replacement, but without CEE, and
these plates were expanded to a density of 0.8 10
Myoblasts were either harvested for analysis as proliferating satellite
cells or rinsed twice in PBS and “switched” to a differentiation media
of F10C containing 1 mM insulin and a horse serum concentration of
10% (Hyclone lot no. AFG5429, Logan, UT) without bFGF supple-
mentation. These cultures were left undisturbed for 72 h to undergo
terminal differentiation, at which time they were harvested for RNA
isolation and compared with actively dividing satellite cells.
RNA purification and processing. Frozen muscle specimens were
homogenized and subjected to total RNA purification using the
Qiagen RNeasy Mini Kit and protocol (Purification of Total RNA
from Animal Tissues, Qiagen, Valencia, CA) and suspended in
nuclease-free water. Cultured cells were scraped in TRIzol (5 ml/
100-mm dish, Life Technologies/GIBCO, Gaithersburg, MD), and
RNA was precipitated, washed, and resuspended in nuclease-free
water (44). Criteria for the inclusion in subsequent in vitro transcrip-
tion (IVT) assays was a spectrophotometric absorption ratio of 260/
280 1.8 (NanoDrop, Wilmington, DE) and a RIN value of 8.0 via
electrophoretic analysis (Agilent Bioanalyzer 2100, Agilent Technol-
ogies, Santa Clara, CA). In vitro transcription of total RNA used the
Ambion MessageAmp Premier kit and protocol (Applied Biosystems/
Ambion, Austin, TX) starting with 100 ng of purified RNA. Six
bacterial control mRNAs (Applied Microarrays, Tempe, AZ) were
amplified in parallel to serve as fiducial markers and for intensity
normalization across samples after microarray scanning. Confirmation
of cRNA diversity was obtained using the Bioanalyzer 2100 to
generate an electrophoretogram for each IVT reaction regarding
sample yield, integrity, and size diversity against a laboratory mouse
RNA standard and a Universal Human Reference RNA (Stratagene,
La Jolla, CA). Evaluation of bacterial sequences indicated that the
IVT assay provided 100- to 200-fold amplification, thereby reducing
the variability and potential for errors associated with PCR exponen-
tial amplification (11).
Hybridization procedures. CodeLink fragmentation buffer was
added to the cRNA (5 l buffer/10 g cRNA) and incubated for 20
min at 94°C. Fragmented cRNA was suspended in hybridization
solution such that 250 l loading volume/array contained 10 g
cRNA. The solution was vortexed, heated (5 min at 90°C), and
transferred to ice before being loaded. Each slide contained a remov-
able plastic hybridization chamber with an infusion port that was
sealed immediately after loading. Microarrays were placed in a
mixing incubator (Innova 4080 Shaking incubator, New Brunswick
Scientific, Edison, NJ) for 18 h and 300 rpm at 37°C. CodeLink arrays
(Uniset Mouse 1 Bioarrays, Mouse Whole Genome Bioarrays) were
selected because of their high sensitivity and low error rate compared
with other platforms, especially regarding transcripts expressed at low
copy numbers (11, 76). After hybridization, the array chambers were
removed, and the slides were placed in a reservoir containing 0.75
TNT [0.1 M TrisHCl (pH 7.6), 0.15 M NaCl, and 0.05% Tween 20]
for a 1-h high-stringency wash at 46°C. Arrays were incubated in
Streptavidin Alexa fluor 647 (Molecular Probes) for 30 min (0.2%
Alexa fluor 647 in 0.1 M TrisHCl, 0.15 M NaCl, and 0.5% NEN
blocking reagent; pH 7.6; Perkin-Elmer, Boston, MA). Slides were
subsequently washed (1TNT for 5 min each of 4 washes at room
temperature and then 0.1% SSC and 0.05% Tween 20 for 30 s at room
temperature) and dried by low-speed centrifugation.
Scanning and data processing. Slides were scanned using a 4000B
GenePix scanner (Axon Instruments, Foster City, CA) calibrated
before use. Laser scanning parameters were set at 635 nm, the
photomultiplier tube voltage was 600, and analysis of the array image
was configured using the CodeLink Expression Analysis software
(version 5.0). Data for each spot were determined as intensity per
pixel within the probe zone. Data for each array were then generated
as both raw intensity values and normalized for the large dynamic
range by dividing each spot by the overall median intensity value for
that array. Values derived from questionable spot profiles and all
manufacturing errors designated by the CodeLink manufacturing spot
report were removed from further analysis. The threshold for each
array was subtracted as background from the normalized values for
that array, and these values were subjected to statistical analysis.
Because of the small size of these muscles, four of seven matched
pairs of individual EDL muscles and five of seven matched pairs of
soleus muscles passed all of the rigorous requirements (RNA yield
and quality, IVT yield and profile, and hybridization and scanning
criteria) to undergo statistical analysis. Satellite cells from three mice
were separately expanded under proliferation and differentiation con-
ditions and passed all requirements for statistical evaluation. To
eliminate batch processing effects across the large number of arrays,
each set of EDL, soleus, or satellite cell samples was run from IVT
through hybridization and scanning in a separate batch. Thus, a batch
contained control and stimulated muscles or proliferating and differ-
entiated satellite cells.
Statistical analysis. Data processing followed recommendations for
microarrays using paired sample comparisons. The bacterial standards
provided internal controls spanning the dynamic arrange of the array
as a basis for comparison of all processing steps from the IVT reaction
through the acquisition of final data. Data were subjected to ANOVA
testing to determine whether the stimulation protocol had a significant
effect on gene expression within each muscle group and whether
satellite cell transcripts differed between proliferation and differenti-
ation conditions. Subsequent post hoc analysis was performed on
individual transcripts after removal of 1) transcripts that remained
“off,” i.e., at threshold levels, in both control and stimulated muscles
or in both proliferation and differentiation states for satellite cells and
2) cDNA clones and transcripts lacking a functional gene classifica-
tion. Correction for multiple testing to minimize the false discovery
rate used methods specifically adapted for microarray analysis (66).
Differentially expressed genes were classified into functional groups
based on the National Institute of Allergy and Infectious Diseases
database for annotation, visualization, and integrated discovery
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
(DAVID), and the results were crossed with the GeneCards and
GENATLAS databases between July 1, 2006 and December 1, 2008
(21, 67) and by manual curation of the primary literature. Ingenuity
Pathways Analysis (IPA software version 7.0, Ingenuity, Redwood
City, CA) was used to compare the gene expression results against the
IPA library of canonical functional biological molecules and path-
ways. Transcripts were expressed as fold changes to simplify presen-
tation, but statistical testing was performed on the median normalized
array intensity values after correction for the individual array back-
ground values. The entire compendium of raw intensity and median
normalized data for all probes is provided in the Gene Expression
Omnibus ( under the se-
ries record GSE14421.
Muscle physiology. The force recorded from the Achilles
tendon for a single twitch (0.5-ms stimulation) was 1.4 0.1 g
force/g body wt. This represented the net contractile activity of
anterior and posterior muscle groups contracting in opposition
across the ankle joint. The larger cross-sectional area of the
posterior compartment resulted in a net positive force vector
causing isometric shortening of the posterior compartment of
the hindlimb. The force transients are shown in Fig. 1. The
exercise protocol used stimulation at 10 Hz, consistent with
previous studies using this technique. Under these conditions,
the stimulation protocol produced initial twitch peak values of
1.6 0.1 g force/g body wt but decreased by 45% within 10
min and was maintained at this level for the rest of the
stimulation time course (Fig. 1A). Concomitant with this early
decrease in force output was an increase in half-relaxation
time, but this value recovered progressively toward its initial
value by the end of the exercise period (Fig. 1A). There was no
mechanical activity detected in the contralateral unstimulated
limb as determined by both manual palpation and tension
recordings in control experiments. Single twitch profiles ac-
quired at the onset and end of stimulation are shown for a
representative subject in Fig. 1, Band C.
Histology. Hematoxylin and eosin staining revealed no sig-
nificant differences in the general morphology or cellular
integrity of each pair of matched muscles whether subjected to
stimulation or serving as the contralateral control (Fig. 2, Aand
B). Specifically, there was no visible evidence of myofiber
damage, including disruption of sarcomere organization or
infiltrating inflammatory cells, in cross sections obtained from
either control or stimulated muscles. Differences in PAS stain-
ing were evident among the stimulated EDL muscles, which
demonstrated a marked decrease in overall staining intensity
(Fig. 2D) compared with the contralateral unstimulated muscle
from the same animal (Fig. 2C), consistent with decreased
glycogen content. In both control and stimulated muscles, there
was a mosaic staining pattern that became more pronounced in
the stimulated muscles, suggesting that both initial glycogen
levels and glycogen depletion rates were not uniform among all
Fig. 1. Contractile characteristics for the stimulation
time course. A: temporal changes in twitch force nor-
malized to total body weight (in g/g body wt) and the
associated changes in half-relaxation time (in ms)
throughout a representative experiment reported in 10-
min intervals. There was an initial decline in peak
twitch force output and a concomitant prolongation of
half-relaxation time after the onset of stimulation. Data
are presented as means SE and were averaged for
each 10-s period every 10 min over the time course. B
and C: twitch at the start of the experiment (second
twitch; B) and the next to last twitch at the end of the
experiment (C) for the comparison of changes in the
twitch profile throughout the duration of the experiment.
Normalized force is presented on the ordinate, and time
(in ms) is presented on the abscissa.
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
fast myofiber types, as has been previously demonstrated in
other studies (24, 40) in both cats and rats.
Ultrastructural analysis. Longitudinal thick sections of
stimulated versus unstimulated muscle specimens revealed
morphological similarity between the paired muscles of each
animal with no obvious signs of inflammation, damage, or
necrosis. Electron microscopic analysis of thin sections dem-
onstrated normal membrane and myofibrillar architecture, in-
cluding intact z-bands from both unstimulated and stimulated
EDL muscles (Fig. 3, Aand E). There were unusual accumu-
lations of mitochondria near the end of EDL muscle fibers in
some but not all animals, but this finding was consistent in both
control and stimulated muscles (Fig. 3, Band F). Figure 3C
shows a satellite cell from an unstimulated muscle identifiable
by the heterochromatic nucleus and absence of myofilaments in
the extensive membrane-bound cytoplasm, whereas the satel-
lite cell nucleus from the stimulated muscle shown in Fig. 3G
is similarly heterochromatic with minimal cytoplasm contained
within its own cell membrane. The resident myofiber nuclei
populating these muscle fibers were similar in appearance and
typically located in juxtaposition to the sarcolemma, consistent
with the morphology of normal skeletal muscle fibers (Fig. 3,
Dand H).
Fiber type-specific transcripts. Four hours of muscle stim-
ulation caused alterations in EDL gene expression in a direc-
tion indicative of long-term molecular and protein transforma-
tion from a fast to slow phenotype (20, 23, 63). The pattern
included decreased MyHC-2B and -2A, fast-phosphorylatable
MyLC, fast troponins T
and I, and muscle creatine kinase in
stimulated versus control muscles (Fig. 4). Parvalbumin was
also significantly decreased after EDL stimulation [control:
1,161 285 vs. stimulated: 824 1.65 intensity units (IU),
means SD, P0.04], consistent with its role as a calcium-
binding protein expressed at highest levels in fast muscles (63).
Each of the animals demonstrated a substantial increase in
MyHC-1/slow expression in stimulated EDL compared with
control muscles. At the same time, transcripts for troponin
C-slow and T
-slow and tropomyosin 3were increased,
consistent with the exercising muscle undergoing conversion
toward a slow phenotype within the time frame of the stimu-
lation protocol. These shifts in phenotype-specific transcripts
were not significant in the soleus muscle, although the de-
creases in fast fiber-specific transcripts followed a similar
pattern in that muscle. The data indicated that the 4-h stimu-
lation protocol induced phenotype transformation in the muscle
fibers of the EDL muscle at the molecular level for transcripts
encoding myofibrillar proteins characteristic of slow myofi-
bers, including MyHC and troponin species not typically ex-
pressed in EDL fibers.
Unstimulated EDL muscles expressed abundant levels of
transcripts for glycolytic enzymes as a signature of fast muscle
fibers, as reflected in phosphofructokinase transcript levels that
were four times higher than isocitrate dehydrogenase, whereas
soleus transcript levels for these two enzymes were nearly
identical. Specifically in the EDL, muscle forms of five critical
glycolytic enzymes [enolase 3 (ENO3; 966 194 IU), phos-
phoglycerate mutase 2 (PGAM2; 816 204 IU), phosphofructo-
kinase (PFKM; 603 54 IU), glucose phosphate isomerase 1
(GPI1; 250 38 IU), and glycogen phosphorylase (PYGM;
155 39 IU)] were expressed at levels approaching that of
myofibrillar proteins. Stimulation caused a decline in the ex-
pression of transcripts for each of these enzymes, but only the
changes in PFKM were statistically significant (P0.02)
despite this common trend. It was interesting to note that in
contrast to all other glycolytic enzymes, hexokinase 2 tran-
scripts increased (control: 150 35 vs. stimulated: 306 67
IU, P0.006), as has previously been reported to occur
transiently using CLFS in fast muscles (85). These data indi-
cated that chronic stimulation of the EDL muscle resulted in a
trend toward a reduction in transcripts encoding glycolytic
enzymes consistent with a shift from a fast to slow phenotype.
Transcripts for these glycolytic enzymes were present in the
control soleus muscles at levels approximately half of those
measured in the EDL muscles (ENO3: 460 78 IU, PGAM2:
293 32 IU, PFKM: 349 53 IU, GPI1: 172 8, and
PYGM: 80 20 IU), as established for slow muscles domi-
nated by oxidative enzymes as a source of metabolic energy
production. No significant changes occurred among these en-
zymes after stimulation.
Fig. 2. Histologic analysis of stimulated and
resting extensor digitorum longus (EDL) mus-
cles. Examples of serial cross sections from both
stimulated and matching unstimulated contralat-
eral EDL muscles are provided, showing the
effects of the experimental protocol on glycogen
content. These muscles exhibited a profound fast
fiber type-specific response of glycogen deple-
tion associated with chronic stimulation. Aand
C: representative sections stained with hematox-
ylin and eosin (H&E; A) and periodic acid Schiff
(PAS; C) from a control EDL, respectively. B
and D: results obtained from the matched con-
tralateral EDL from the same animal subjected
to stimulation (with H&E stain in Band PAS
stain in D). The PAS (Cand D) stain is specific
to the presence of glycogen, which was dimin-
ished in Dfrom the stimulated EDL muscle.
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
Early response genes. There was an “immediate-early
growth response” or “stress-adaptive response” pattern indic-
ative of a primary reaction to extracellular stimulation partic-
ularly within the EDL muscle, where 11 transcripts reached
levels 1.5- to 10-fold of normal values (Fig. 5). The response
within the soleus muscle was comparatively smaller, with five
transcripts elevated above 1.5-fold. FOS and JUN-B have been
defined as canonical genes of the early response pathway and
were similarly elevated in both EDL and soleus muscles,
whereas Dmel/homer1 (HOMER) and early growth response 2
(EGR2) were significantly increased in the EDL muscle (Fig.
5) (16, 55). In addition, activating transcription factor 3
(ATF3), B cell translocation gene 2 (BTG2), musculoaponeu-
rotic fibrosarcoma oncogene (MAFK), immediate-early re-
sponse 5 (IER5), ephrin A
(EFNA1), JUN protooncogene
related D
(JUND), and heterogenous ribonucleoprotein D
(HNRPD) have been associated with both an immediate-early
gene response as well as a stress response, and these transcripts
were all increased in the EDL muscle (Fig. 5).
Transcription factors. Gene ontology (GO) and pathway
analysis of differentially expressed genes revealed that “tran-
scriptional regulators” was the highest ranked category under
the GO term “molecular function.” The most significant “mo-
lecular and cellular functions” among these transcription fac-
tors derived from the GO category of “cellular development”
(28 genes) with several genes overlapping the classification of
“cell growth and proliferation” comprising 39 genes signifi-
cantly overrepresented among all transcription factors (IPA
version 7.0: P5.97 10
to 2.58 10
). Genes within
these categories were further classified by manual curation of
the primary literature.
Among the altered transcription factors and cofactors were
several transcripts that have been previously implicated di-
rectly or indirectly in fiber type transformation. Intensity val-
ues for these 11 genes were significantly elevated in the EDL
muscle, whereas 2 genes were increased in the soleus muscle
(Fig. 6). Peroxisome proliferator-activated receptor (PPAR)-
and its coactivators, PPAR-coactivator (PPARGC)-1and
Fig. 3. Ultrastructural analysis of stimulated and con-
trol EDL muscles. A–D: scanning electron micrographs
obtained from thin sections of unstimulated EDL mus-
cles; E–H: comparable regions from contralateral stim-
ulated EDL muscles. All images were obtained at the
same original direct magnification (2,650). Scale
bars 2m, which applies to each image. Aand
E: membrane and myofibrillar architecture from both
EDL muscles. There were unusual accumulations of
mitochondria (Mito) near the end of EDL muscle fibers
in some but not all animals, as demonstrated in Band F.
Cand G: satellite cells commonly identified in both
muscles [satellite cell nucleus (SatN) and satellite cell
membrane (SCM)]. The resident myofiber nuclei
(MyoN) populating these muscle fibers were similar in
appearance and located in juxtaposition to the sarco-
lemma, consistent with the morphology of normal skel-
etal muscle fibers (Dand H).
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
PPARGC-1, have been found to play a central role in mito-
chondrial biogenesis, inflammation, and metabolism (65, 88),
and a recent study (33) in knockout mice has established
PPARGC-1as a critical factor in the formation and transfor-
mation of the myofiber phenotype. All three genes were sig-
nificantly increased in the EDL muscle by the stimulation
paradigm (Fig. 6). Similarly, eyes absent 1 homolog (EYA1)
and tea domain family member 1 (TEAD1) have been associ-
ated with muscle fiber type development and transformation
and were substantially increased in the stimulated EDL muscle
(Fig. 6) (29, 30, 83). The calcium/calcineurin nuclear factor of
activated T cells (NFATc) transcription complex has been
reported to modulate phenotype specification during both mus-
cle development and fiber transformation, and both NFATc1
and NFATc4 were significantly increased in the EDL muscle
(43, 52, 54). Hypoxia-inducible transcription factors have been
implicated in fiber type transformation through an impact on
metabolic genes (74, 81) or through an interaction with circa-
dian clock genes (14, 51). Transcripts for hypoxia-inducible
factor (HIF)-1, a master transcriptional regulator of the hy-
poxic response, and HIF-3were significantly increased in the
EDL muscle (Fig. 6), whereas circadian locomotor output
cycles kaput (CLOCK) transcript levels increased along with
its modulator, the basic helix-loop-helix domain containing B2
(BHLHB2) gene (Fig. 6).
A surprising number of the transcription factors significantly
elevated in this study have been characteristically associated
with functional roles during embryogenesis, mesodermal pat-
tern formation, and somitogenesis under the classification of
“growth and differentiation” based on IPA pathway analysis
(Fig. 7). For example, Kruppel-like transcription factors (KLF4
and KLF5) (18), SRY box-containing genes (SOX 17) (64),
transcription factor 15 (TCF15) (35), embryonic lethal, abnor-
mal vision (ELAV) (15), and tuberous sclerosis factor 1
(TSC1) (59) were significantly increased in the stimulated but
not resting EDL (Fig. 7; not all shown). In addition, PBX/
Fig. 5. Immediate-early gene/stress response transcripts.
Each of the bars indicates an increase of 1.5-fold change in
the EDL muscle for the mean value SE of transcripts
associated with an immediate-early gene response, including
FOS, JUN-B, and early growth response 4 (EGR4). B cell
translocation gene 2 (BTG2), musculoaponeurotic fibrosar-
coma oncogene (MAFK), Dme1/homer1 (HOMER1), im-
mediate-early response 5 (IER5), ephrin A
(JUND), and heterogenous ribonucleoprotein D
(HNRPD) were transcripts associated with a stress-activated
response to extracellular stimulation. *Statistically significant
change in either the EDL or soleus muscles.
Fig. 4. Myofiber phenotype-specific transcripts altered
by stimulation. Fast and slow muscle contractile gene
transcripts obtained after4hofstimulation are shown
and were associated with the process of myofiber phe-
notype transformation. Data are presented as fold
changes (means SE) relative to the contralateral
muscle (resting control) for each animal in paired com-
parison. Decreased values are depicted as 1/%change
to compare fold changes in equivalent scalar magnitude
regardless of directional change. *Statistically signifi-
cant fold changes as detected by ANOVA followed by
a post hoc paired t-test except for myosin heavy chain
(MyHC)-2B, where all subjects demonstrated a de-
crease (P0.057), and MyHC-1/slow, where all
subjects demonstrated increased transcript levels (P
0.103). MyHC-1/slow was detected at background
levels in the control muscle, and the stimulated values
reflected “turn on” of this transcript. TROPN, troponin;
TROPOMY 3G, tropomyosin 3; MYLC, myosin light
chain; mCK, muscle creatine kinase; SOL, soleus mus-
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
knotted 1 homeobox (PKNOX1) binds the E2A/myogenic
factor complex in competition with inhibitor of DNA binding
(ID) during myogenesis (22), and both were elevated within
the stimulated EDL muscle (Fig. 7). Figure 7 also shows
significant changes in CRIPTO and TWISTED as mouse ho-
mologs for Drosophila genes critical to dorsal-ventral meso-
dermal pattern formation during gastrulation, although these
gene products are not transcription factors (48, 89). The stim-
ulated soleus muscle also exhibited the activation of a small
number of developmental transcripts but within a subset of
factors that demonstrated little overlap to the EDL muscle.
Satellite cell gene expression in vitro and in vivo. A previous
study (45) has established that primary mouse hindlimb muscle
satellite cells differentiated into myocytes and myofibers that
coexpressed MyHC-1/slow and -2A in culture by PCR and
immunocytochemistry. In the present study, both MyHC-1/
slow and -2A transcripts were significantly increased in differ-
entiated myocyte cultures along with muscle creatine kinase
based on microarray analysis, thereby corroborating previous
PCR findings (Table 1). The microarray methodology provided
the capability to identify a large number of additional pheno-
type specific transcripts that were found to be significantly
increased in our differentiated myocyte cultures, including fast
phosphorylatable MyLC, troponins C, T
and T
, skeletal
muscle actin, and dystrophin (Table 1). Myogenin was also
significantly higher after myocyte maturation and myotube
fusion compared with proliferating satellite cells in the present
study, as has been reported previously (12, 91). Overall, 412
different transcripts were detected at significantly different
levels between the proliferating satellite cells and differentiated
muscle cell cultures.
Transcripts significantly elevated in the proliferating satellite
cell-derived cultures compared with the differentiated myo-
cytes comprised a large number of genes associated with cell
cycling, including multiple DNA polymerase-,-, and -ε, cyclin A
, C, and E
, cyclin-dependent kinases 1 and 4, and cell
Fig. 6. Transcription factors associated with myofiber trans-
formation. All displayed values (means SE) were statisti-
cally significant (P0.05) in the EDL muscle, including a
cutoff of 1.5-fold change. The value for eyes absent 1
homolog (EYA1) represents a graphical maximum where
fold change values were 20-fold based on expression levels
in the unstimulated EDL muscle at or below threshold levels.
PPARG, peroxisome proliferative activated receptor-;
PPARGC1a, PPAR-coactivator 1; PPARGC1b, PPAR-
coactivator 1; CLOCK, circadian locomotor output cycles
kaput; BHLHB2, basic helix-loop-helix domain containing
B2; HIF1a and HIF3a, hypoxia-inducible factor 1and 3;
NFATc1 and NFATc4, nuclear factor of activated T cells 1
and 4; TEAD1, tea domain family member 1. *Statistically
significant changes for transcripts in the soleus muscle.
Fig. 7. Transcription factors associated with organo-
genesis/histogenesis induced by sciatic nerve stimula-
tion. The classification of cellular development was
significantly overrepresented among all transcription
factors. These genes are characteristically involved in
functions associated with tissue and organismal devel-
opment, cell proliferation, and embryo formation. The
transcripts presented were significantly elevated in the
stimulated EDL muscle by 1.5-fold change but not in
the soleus muslce. Kruppel-like factor 4 (KLF4), SRY
box-containing gene 17 (SOX 17), inhibitor of DNA
binding (ID), transcription factor 15 (TCF15), embry-
onic lethal, abnormal vision (ELAV), PBX/knotted 1
homeobox (PKNOX1), and tuberous sclerosis factor 1
(TSC1) were significantly elevated in stimulated EDL
muscles. CRIPTO and TWISTED are murine homologs
of Drosophila genes critically associated with meso-
derm pattern formation, and transcripts for these mole-
cules were significantly increased by stimulation.
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
division cycle transcripts 6 and 20 (Table 1). In addition,
multiple developmental transcription factors and coregulators
were significantly elevated in these cells, including transcrip-
tion factors involved in morphogenesis, e.g., Hedgehog-
interacting protein, Distal-less, SLUG, Cornichon-like, MyoD,
Frizzled 3, Paired-box transcription factor 4 (PAX4), Odd-
skipped related 2 (OSR2), and Paired-like homeodomain tran-
scription factor 3 (PITX3). None of these cyclins and cyclin-
dependent kinases elevated in the proliferating satellite cell
cultures were increased in either stimulated EDL or soleus
muscles. Furthermore, none of the developmental transcription
factors expressed in either the stimulated muscles or prolifer-
ating satellite cells overlapped in their expression pattern. Only
HIF-1, F box 10, and IDB3 transcription factors were com-
monly increased in both stimulated EDL and proliferating
satellite cells, suggesting that the contribution of satellite cell
transcripts as defined by the in vitro experiments for the in vivo
stimulated muscles was minimal, if present at all. Other studies
have identified satellite cell markers including several mem-
bers of the PAX gene family (PAX3 and PAX7) and myogenic
determination factors (MEF2A, MEF2D, Myf6, and myoge-
nin) (12, 91). These factors were detected in all control and
stimulated muscles, but none of these satellite cell markers
were significantly altered by the chronic stimulation protocol.
Signal transduction transcripts. Table 2 shows a list of
transcripts associated with signal transduction pathways that
were significantly altered in the EDL and/or soleus muscles by
the stimulation protocol. Compared with the transcription fac-
tor analysis, this category was notable for small numbers of
significant genes and relatively small fold changes in transcript
levels. The stimulation regimen increased EDL transcript lev-
els of MAPK6 (ERK3), a critical skeletal muscle MAPK,
whereas MAP2K4, MAP3K4, and MAP-binding protein-inter-
acting protein (MAPBPIP) fell significantly in the soleus mus-
cle during stimulation. Transcripts representing four distinct
serine/threonine kinase pathways were affected, with home-
odomain-interacting protein kinase 3 (HIPK3) increasing in the
EDL muscle, whereas Fas-activated serine/threonine kinase
(FASTK), PKC-, and serine/threonine protein kinase 11
(STK11) all significantly decreased in the EDL muscle but
remained unchanged in the soleus muscle. Calcium/calmodu-
lin-dependent serine protein kinase (CASK) was activated in
the stimulated EDL muscle, indicative of a potentially impor-
tant role for calcium and calcineurin in modulating the muscle
remodeling process, as has been previously demonstrated in
animals and muscle fiber cultures (4, 58). Macrophage-stimu-
lating protein receptor (MST1R or RON protein tyrosine ki-
nase) and salt-inducible kinase 1 (SNF1-like kinase) increased
significantly in the stimulated EDL muscle, indicating roles for
these two receptor tyrosine kinase pathways in the response to
chronic stimulation.
The results obtained in this study revealed that 10-Hz stim-
ulation of the murine hindlimb for 4 h produced MyHC
transcripts in the EDL muscle, reflecting the molecular onset of
the conversion from a fast to slow phenotype. To our knowl-
edge, this is the earliest time point in a chronic stimulation
model demonstrating the initiation of these phenotype changes
in contractile protein transcripts. In contrast to the EDL mus-
cle, the stimulated soleus muscle, with an intrinsically slower
myofiber composition, exhibited comparatively few pheno-
type-specific changes after stimulation. The chronic stimula-
Table 1. Primary muscle cell cultures
Proliferating Primary Cells Differentiated Muscle Cells
PValueMean SD Mean SD
Elevated during proliferation
MyoD 144.4 10.3 85.9 2.0 0.013
CDK1 35.1 7.0 2.1 1.3 0.016
CDK4 16.0 4.0 5.1 1.4 0.024
Cyclin A
10.6 1.4 0.3 0.3 0.009
Cyclin B
9.4 1.7 0.4 0.5 0.018
CDC20 8.8 2.6 0.5 0.5 0.041
Cyclin C 6.5 0.4 3.3 0.7 0.007
Cyclin E
4.4 0.7 0.6 0.1 0.013
CDC6 3.2 1.0 0.0 0.0 0.001
Elevated after differentiation
Muscle actin 259.5 237.0 620.5 269.6 0.003
Fast-phosphorylatable myosin light chain 168.9 130.8 562.3 225.9 0.019
Troponin C-fast 93.5 86.4 345.4 98.2 0.014
Troponin T
-fast 71.0 62.2 257.5 64.6 0.046
Myogenin 68.2 65.1 134.9 68.5 0.001
Troponin C-slow 31.4 22.7 121.1 8.4 0.021
Troponin T
-slow 20.9 17.3 73.4 30.5 0.023
Myosin heavy chain-slow 8.2 12.2 41.6 20.1 0.047
Myosin heavy chain-2A 0.1 0.2 28.8 7.5 0.023
Dystrophin 0.9 1.2 7.2 3.5 0.041
Mean values indicate the median normalized expression intensity values SD for 3 independent satellite cell harvests from the anterior and posterior
compartments of the hindlimb. Myoblast cultures were studied under high-serum conditions supporting proliferation and were switched to low-serum conditions
to induce myocyte differentiation and myotube formation. CDK, cyclin-dependent kinase; CDC, cell division cycle. All values were statistically significant at
the delineated Pvalue including a cutoff of 1.5-fold change.
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
tion protocol produced an increase in the EDL muscle of
transcription factors previously implicated in myofiber pheno-
type conversion in other experimental models, including EYA1,
TEAD1, and CLOCK and members of the NFAT, PPAR-,
and HIF-1 gene families. Surprisingly, multiple genes charac-
teristically associated with morphogenesis and mesoderm pat-
tern formation were concurrently activated in the stimulated
EDL muscle. Finally, the transcriptional response of the stim-
ulated muscles bore little similarity to the expression profile of
cyclins and myogenic factors detected in parallel proliferating
satellite cells, nor were there increases in other established in
vivo satellite cell markers (PAX3 and PAX7) (12, 91). The
absence of indicators of satellite cell activation and the lack of
transcripts associated with injury and inflammation of the
stimulated EDL muscles suggested that the molecular response
originated within resident nuclei of the differentiated myofi-
bers. Histological and ultrastructural analyses confirmed that
the stimulated muscles showed no sign of damage, inflamma-
tion, and/or regeneration, adding further support to the hypoth-
esis that mature skeletal muscle nuclei retained the plasticity to
reactivate otherwise quiescent transcription programs typically
associated with development and morphogenesis.
Chronic stimulation model. Chronic electrical stimulation
has been used broadly for the activity-induced conversion of
muscle phenotype, and unilateral sciatic nerve stimulation has
been used in acute and long-term studies of both unanesthe-
tized and sedated animals (63, 73). The absence of quantitative
information regarding force output and mechanical loads in
most of these studies prevented a comparison of the model
across laboratories as well as the evaluation of parameters such
as energetic demands and calcium handling. The present study
continuously monitored mechanical performance from individ-
ual twitches throughout the entire time course with muscles
held at resting length and under loaded conditions, thus opti-
mizing the maximal initial force output and standardizing the
physiological readout for the working muscle preparation
across all of the subjects. Supramaximal stimulation intensities
were applied to ensure that all motor units excited by the sciatic
nerve were activated during the stimulation protocol. The
recovery of initial force after the stimulation protocol indicated
that no profound neuronal or mechanical damage occurred
within the stimulation paradigm, as confirmed by the histolog-
ical and ultrastructural analyses. In addition to mechanical
output, the physiological homeostasis of each animal was
monitored with respect to body temperature, respiration, and
heart rate to maintain consistency both within and among all
Sciatic nerve stimulation at 10 Hz caused glycogen depletion
among EDL myofibers based on the diminution of PAS stain-
ing versus unstimulated muscles. There was no indication of
tissue or motor nerve damage based on poststimulation recov-
ery to initial force values, microscopic evaluation of hematox-
ylin and eosin-stained specimens, and ultrastructural analysis
using scanning electron microscopy. These findings were con-
sistent with the microarray results, which revealed no signifi-
cant expression of markers associated with injury or inflam-
mation in the muscles. An obligatory role for muscle injury and
fiber replacement in the process of fast to slow phenotype
conversion remains uncertain. Fiber degeneration and regener-
ation have been reported in chronically stimulated rabbit fast
muscles (49) but did not occur in rat fast muscles subjected to
the same protocol (20). In the absence of physiological data
from these other studies, we can only speculate as to whether
these results were attributable to species differences or differ-
ential mechanical demands on the muscles in those studies. In
other studies where contractile forces were quite large, signif-
icant damage has been noted. For example, Yamasaki et al.
(90) demonstrated damage to rat masseter muscles stimulated
in the same time frame as the present study. Based on their
physiological measurements, the masseter muscles generated
five times the net force output compared with the hindlimbs in
the present study. Thus, differences in mechanical strain asso-
ciated with the higher magnitude of force generated by the
masseter muscles may account for the injury associated with
chronic stimulation in some cases. Precise delineation of the
minimal contractile activity and activation time required for the
Table 2. Signal transduction pathways
Extensor Digitorum Longus Muscle Soleus Muscle
Mean SD Pvalue, stimulated vs. control Mean SD Pvalue, stimulated vs. control
MAPK6 2.85 1.38 0.028 1.61 1.52 NS
MAP2K4 1.01 0.46 NS 0.75 0.18 0.05
MAP3K4 0.95 0.20 NS 0.84 0.13 0.05
MAPBPIP 0.76 0.27 NS 0.76 0.12 0.02
Serine/threonine kinase related
HIPK3 1.29 0.19 0.007 0.94 0.27 NS
FASTK 0.86 0.10 0.048 0.95 0.27 NS
PKCA 0.80 0.07 0.004 1.07 0.43 NS
STK11 0.74 0.04 0.008 0.84 0.16 NS
Calcium related
CASK 1.93 0.61 0.0015 1.08 0.40 NS
Tyrosine kinase
MST1R 1.29 0.12 0.004 1.36 0.80 NS
SNF1LK 1.81 0.53 0.037 0.96 0.24 NS
Values indicate fold changes for the median normalized expression intensity of stimulated muscles versus unstimulated muscles. A fold change cutoff was not
included because of the small numbers of transcripts detected in this category. Pvalues are reported for each comparison. MAPK, mitogen-activated protein
kinase; MAPBPIP, mitogen-activated protein-binding protein-interacting protein; HIPK3, homeodomain-interacting protein kinase 3; FASTK, Fas-activated
serine/threonine kinase; PKC, PKC-; STK11, serine/threonine protein kinase 11; CASK, calcium/calmodulin-dependent serine protein kinase; MST1R,
macrophage-stimulating protein receptor; SNF1LK, salt-inducible kinase 1.
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
induction of phenotype remodeling remains to be defined, but
potentially confounding effects of injury, inflammation, myo-
genic cell activation, and other factors may be eliminated by
establishing these parameters.
The electrode was positioned on the sciatic nerve to activate
both the anterior and posterior hindlimb muscle compartments
containing the EDL and soleus muscles, respectively. While
previous studies have demonstrated the efficacy of the stimu-
lation protocol for the transformation of fast muscles, the
phenotype conversion of the soleus muscle typically required
high-frequency tetanic stimulation (100 Hz) plus the removal
of tonic activity by anatomic or functional denervation (27, 63,
73). Thus, the changes observed in the stimulated soleus
muscle may have reflected an adjustment toward a slower
phenotype within the limited fast myofiber complement of
MyHC-2X fibers (45, 47, 92). However, it is also important to
consider that the EDL muscle differs from the soleus muscle in
both lower mitochondrial density and capillary-to-fiber ratios
(19). Therefore, we cannot rule out that the higher metabolic
cost of contraction in the EDL muscle contributed to the
observed differences. It is also possible that the soleus muscle
response reflects its smaller contribution to the synergistic
muscles of the posterior compartment in contrast to the EDL
muscle, which contributes relatively more of the output of the
anterior compartment. Based on the present results, additional
studies will be required to delineate the precise physiological
signaling mechanisms underlying the differential response of
the EDL and soleus muscles.
Fiber type transformation. Prototypical fast to slow pheno-
type conversion of MyHCs in response to chronic stimulation
has been detected in transcripts as early as 1 day after stimu-
lation onset with RNA and protein changes reaching a steady
state around 35 days (38, 82). It has been established that the
chronic molecular adaptations to spaceflight and hindlimb
suspension undergo rapid reversal (3.5– 4 h) when exposed to
reloading, but changes in major myofibrillar proteins were not
detected (2). In this study, decreased transcripts for fast muscle
contractile proteins (MyHC-2B and -2A, fast phosphorylatable
MyLC, and fast troponin isoforms I and T
) were observed
after only4hofstimulation along with increased slow muscle
specific transcripts (MyHC-1/slow and slow troponin C and
). The rapidity of this response was reflected in the fact that
it occurred within the same time frame as the immediate-early
growth response typically observed shortly after a cellular
stress. These data emphasized the remarkable lability of the
transcriptional programs controlling adult myofibrillar proteins
despite their abundant contribution to the overall anatomic
muscle hierarchical structure and relatively long half-life
within myofibers (7–10 days) (5, 38). There was a concomitant
decline in transcripts encoding glycolytic enzymes, consistent
with the shift in myofibrillar proteins. While the pattern of
these changes in metabolic enzymes was consistent and the
magnitude was substantial, the variability was sufficiently
large so that few of these changes achieved statistical signifi-
cance. This may reflect a wider variability in the transcriptional
control or functional reserve of these proteins versus that of the
myofibrillar apparatus but fits the overall pattern observed in
the contractile proteins of a rapid shift in EDL fiber phenotype.
Transcripts associated with phenotype transformation were
detected without satellite cell activation as indicated by 1) the
lack of overlap of stimulated EDL and soleus expression
profiles with transcripts expressed in parallel proliferating
satellite cell cultures and 2) the absence of changes in tran-
scripts indicative of in vivo satellite cell activation, including
myogenic determination factors (MEF2A, MEF2B, MEF2C,
Myf6, and myogenin) and PAX transcription factors (PAX3
and PAX7). These satellite cell markers were present at low
levels in both control and stimulated muscles but were not
altered by stimulation. Controversy exists regarding the role of
satellite cells in phenotype conversion. The application of
gamma-irradiation has been shown to inhibit satellite cell
proliferation and fusion but did not alter the process of fast to
slow phenotype conversion in the EDL muscle of mice sub-
jected to synergist ablation (70), whereas significantly dimin-
ished fiber type transformation occurred in gamma-irradiated
rat tibialis anterior muscles after chronic stimulation (50). The
results of the present study agree with those obtained in the
mouse EDL muscle via synergist ablation, albeit in an earlier
time frame: that satellite cell activation was not a prerequisite
for the induction of transcripts for contractile protein pheno-
type conversion.
An unexpected finding was the detection of several tran-
scription factors and coactivators previously observed among
various cell types associated with morphogenesis, somitogen-
esis, mesodermal patterning, and lineage differentiation. These
transcription factors included multiple members of the Kruppel
family (KLF4, KLF5, and GLI5), SOX 17, PKNOX1, ID,
TCF15, and ELAV (18, 22, 35, 59, 64) and other transcripts
characteristic of tissue formation and organogenesis (CRIPTO,
TWISTED, and ZYXIN) (36, 48, 89). All were present at
significantly increased levels in stimulated EDL muscles. The
presence of these transcripts indicated that unique developmen-
tal pathways were reactivated in the myonuclei of otherwise
differentiated myofibers by the stimulation paradigm. These
results raise other interesting biological questions regarding the
capacity for reprogramming of mature myonuclei within dif-
ferentiated myofibers. For example, is there a unique subset of
nuclei apart from the satellite cells but within the mature
muscle myonuclear population that may undergo reprogram-
ming associated with the expression of slow muscle tran-
scripts? Mechanistic studies will be required to establish both
the role of these transcription factors as well as to locate the
precise nuclear domains where they were present. Neverthe-
less, these data provide evidence of unique plasticity in the
nuclei of mature muscles to recapitulate or reprogram a pattern
of expression of factors typically associated with early embry-
Phenotype conversion transcription factors. TEAD1,
PPARGC1, and NFATc1 have separately been implicated in
direct transcriptional activation of the fast to slow phenotype
switch, and each was elevated by4hofchronic stimulation.
The overexpression of TEAD1 under the control of the muscle
creatine kinase promoter in transgenic mice increased the
population of MyHC-1/slow fibers at the virtual exclusion of
MyHC-2X fibers (83). TEAD1 binds to an A/T-rich element
critical for MyHC-1/slow transgene expression in response to
mechanical overload. PPARGC1binds with MEF2C to in-
duce a transcriptional cascade including the coactivation of
several nuclear factors (nuclear response factors 1 and 2)
leading to the activation of mitochondrial transcription factors,
e.g., TFAM (33). The overexpression of PPARGC1in trans-
genic mice increased mitochondria numbers accompanied by
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
increased slow and type 2A fiber numbers. The transcriptional
basis for an effect of NFATc1 on myofiber transdifferentiation
has been obtained predominantly from transfection experi-
ments in cultured myotubes that undergo a calcium ionophore-
induced fast to slow phenotype shift. NFATc1 translocated to
the nucleus under the influence of calcium transients, where it
bound to a consensus sequence in the MyHC-1/slow pro-
moter in a complex including MEF2D, MYOD, and p300 (54).
The finding that each of these factors increased in the stimu-
lated EDL muscle was consistent with the initiation of pro-
cesses leading to the established end result of chronic stimu-
lation, i.e., wholesale remodeling of the myofiber structure
from mitochondria to sarcomeres. The present study indicated
that these transcriptional programs were acutely activated
within the first4hofstimulation.
Transcription factors with indirect effects on myofiber phe-
notype were significantly increased by chronic stimulation in
the EDL muscle, including CLOCK, PPARGC1, and HIF-1.
The recent discovery of a circadian-regulated muscle-specific
transcriptome associated with mitochondrial number and met-
abolic function (51) suggested that significant changes in the
CLOCK gene associated with chronic stimulation play an
important role in phenotype conversion. PPARGC1was one
of those circadian-regulated genes but also played a role in the
basic energetic balance of muscles expressing MyHC-1/slow,
since PPARGC1knockout mice demonstrated a reduced
mitochondrial complement in both the soleus and heart mus-
cles (46). Thus, increased PPARGC1and PPARGC1indi-
cated the activation of pathways leading to the remodeling of
oxidative enzyme content and the mitochondrial compartment
associated with the availability of ATP. In contrast, members
of the HIF family were first discovered in association with
hypoxia-induced angiogenesis, where HIF-1binds to a hy-
poxic response element within the VEGF promoter (7, 81). We
cannot rule out focal ischemia as the stimulus leading to
increased HIF-1and HIF-3levels during CLFS despite the
presence of an intact circulation. However, it has recently been
shown that HIF-1and VEGF play a direct role in embryonic
development and wound repair under physiological conditions
other than hypoxia (7). HIF-1transcripts have also been
detected bound to glucose transporter 4 E box-binding sites in
isolated, incubated soleus muscles subjected to a brief bout of
high-frequency stimulation (77), indicative of a role in modu-
lating glucose uptake. Consequently, increased HIF-1tran-
script levels may represent a response to changes in energy
The role of increased EYA1 and NFATc4 transcripts in the
present study was unclear. The Six1/EYA1 pathway has been
implicated in the maintenance of the fast fiber phenotype, and
forced expression converts the slow twitch muscle of the soleus
to a fast phenotype (30), opposite of the effect in the present
study. However, EYA1 has been subsequently demonstrated to
be critical for somitogenesis (29), and it may be that this
distinct role was enlisted by chronic stimulation in the present
study. Similarly, the forced expression of NFATc4 in cardio-
myocytes indicated a role in calcineurin-induced hypertrophy
(84), and, by virtue of its sequence similarity to NFATc1, it has
been implicated in muscle fiber-specific expression pathways
through conserved NFAT consensus binding sites. Similarly,
knockout of NFATc3 and NAFATc4 in mice resulted in
embryonic lethality secondary to aberrant mitochondrial bio-
genesis and cardiac morphogenesis (10). Therefore, the role of
increased transcripts for these two genes in the stimulated EDL
muscle remains to be determined regarding their participation
in potentially discreet transcriptional programs associated with
phenotype conversion versus muscle morphogenesis.
Changes in transcripts associated with signal transduction
were surprisingly few, suggesting that signal transduction re-
sponses were mediated at nontranscriptional levels during the
acute phase of stimulation, such as through posttranslational
modifications, e.g., phosphorylation. Elevated MAPK path-
ways have been associated with myofiber transdifferentiation,
as occurred with MAPK6 in this study (75). Also, increased
CASK transcripts in the stimulated muscle supported a role for
calcium as a specific trigger in the early activation of transcrip-
tion factors associated with contractile protein remodeling (4).
However, the transduction pathways whereby electrically in-
duced, neural stimulation was translated into transcriptional
events were not reflected at the RNA level despite the abun-
dance of these transcripts detected by the arrays. Furthermore,
transcripts for critical energy regulators such as AMP kinase,
mammalian target of rapamycin, AKT, and transcripts associ-
ated with mitochondrial biogenesis (TFAM) were not signifi-
cantly altered after4hofstimulation, suggesting that signals
driving the activation of transcription factors may have arisen
from extracellular sources such as cytokines, chemokines,
metabolic substrates, and growth factors or through changes in
the intracellular ionic equilibrium involving calcium, potas-
sium, or other critical electrolytes.
Methodological considerations. The present study used an
established chronic stimulation protocol for the induction of
fiber type transformation in the hindlimb. We delivered supra-
maximal sciatic nerve stimulation and adjusted muscle length
to obtain consistent, comparable force output in each animal.
The contralateral muscles provided identically matched genetic
controls for comparison with the stimulated muscles regarding
histological properties and transcript profiles. The microarray
platform comprised oligonucleotide probes (30-mer) with ap-
proximately equivalent target avidities, thereby scaling tran-
script output levels based on biological abundance. Low array
background levels (average background intensity: 0.29 0.15
IU) resulted in a high signal-to-noise ratio from transcription
factors to myofibrillar proteins (5- to 5,000-fold background,
respectively) (11, 76). Transcript levels for MyHC proteins and
myogenic determination factors from control EDL, soleus, and
satellite cell cultures correlated with results obtained previ-
ously using PCR and immunocytochemical assays (46).
Conclusions. In conclusion, the onset of fast to slow myo-
fiber transformation of critical myofibrillar proteins was de-
tected in the EDL muscle after4hofchronic stimulation in the
absence of ultrastructural, histological, and molecular signs of
muscle damage or satellite cell activation. Multiple fiber type-
specific transcription factors were increased in synchrony with
the process of phenotype conversion along with a surprising
subset of transcription factors characteristic of embryogenesis.
Based on these findings, the mature myonuclei of differentiated
muscle fibers or a subset of these nuclei were the source of
transcriptional plasticity including the recapitulation of devel-
opmental morphogenic programs. The distinct subsets of tran-
scription factors activated by chronic stimulation may provide
insight into the separate pathways and transductive signals
responsible for the delineation of the myofiber phenotype and
AJP-Cell Physiol VOL 297 SEPTEMBER 2009
phenotype conversion as well as potential new therapeutic
targets for muscle repair and regeneration.
The authors express special thanks to Dr. Sheldon Bastacky, Ardith Reis,
and Donna Ziesmer for the excellent work in providing the electron micro-
scopic analysis and Dr. Marcia Ontell for the insightful review of these images.
This work was supported by National Space Biomedical Research Institute
Grant MA 00210, Michigan State University Investigator-Initiated Research
Project Grant 41006, National Cancer Institute Cancer Center Support Grant
P30-CA-47904, funding through the Pasquerilla Foundation, and with support
from Allegheny Heart Institute Grant 49399109.
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AJP-Cell Physiol VOL 297 SEPTEMBER 2009
    • "We have identified the Pbx/ Knotted homeobox binding site in the promoter region of a number of upregulated genes. This agrees with the finding in skeletal muscle, in which chronic hypoxia leads to conversion of fast to slow fibers with increased expression of Pbx/Knotted homeobox [59]. Pbx also has been shown to be involved in cardiac muscle differentiation [60]. "
    [Show abstract] [Hide abstract] ABSTRACT: In humans and other species, long-term hypoxia (LTH) during pregnancy can lead to intrauterine growth restriction with reduced body/brain weight, dysregulation of cerebral blood flow (CBF), and other problems. To identify the signal transduction pathways and critical molecules, which may be involved in acclimatization to high altitude LTH, we conducted microarray with advanced bioinformatic analysis on carotid arteries (CA) from the normoxic near-term ovine fetus at sea-level and those acclimatized to high altitude for 110+ days during gestation. In response to LTH acclimatization, in fetal CA we identified mRNA from 38 genes upregulated >2 fold (P<0.05) and 9 genes downregulated >2-fold (P<0.05). The major genes with upregulated mRNA were SLC1A3, Insulin-like growth factor (IGF) binding protein 3, IGF type 2 receptor, transforming growth factor (TGF) Beta-3, and genes involved in the AKT and BCL2 signal transduction networks. Most genes with upregulated mRNA have a common motif for Pbx/Knotted homeobox in the promoter region, and Sox family binding sites in the 3' un translated region (UTR). Genes with downregulated mRNA included those involved in the P53 pathway and 5-lipoxygenase activating proteins. The promoter region of all genes with downregulated mRNA, had a common 49 bp region with a binding site for DOT6 and TOD6, components of the RPD3 histone deacetylase complex RPD3C(L). We also identified miRNA complementary to a number of the altered genes. Thus, the present study identified molecules in the ovine fetus, which may play a role in the acclimatization response to high-altitude associated LTH.
    Full-text · Article · Dec 2013
    • "In response to various contractile demands such as exercise, skeletal muscle demonstrates remarkable adaptability or plasticity that is largely dictated by changes in motor neuron activity. For example, chronic low-frequency electrical stimulation (CLFS; 10 Hz) of the motor nerve mimics the tonic firing pattern typical of slow motor neurons (Hennig & Lomo, 1985) and induces maximal faster-to-slower fibre type transformations in the absence of skeletal muscle injury in the rat model (Putman et al. 1999Putman et al. , 2000Putman et al. , 2001 Martins et al. 2006; LaFramboise et al. 2009). This fibre type transformation generally follows the 'next nearest-neighbour' rule where fibre types undergo a predictable pattern of transformation in the direction of fast type IIB→IID(X)→IIA→ slow type I (Pette & Vrbová, 1999; Pette & Staron, 2000). "
    Full-text · Article · Jan 2012
    • "However, the role of immediate early genes has not been fully elucidated in skeletal muscle. In particular c-Fos has previously been shown to respond rapidly to various types of stimuli, including hypoxia (Yuan et al. 2004), growth factors (Greenberg and Ziff 1984; Kruijer et al. 1984), and contractile activity (Xia et al. 1997; Neufer et al. 1998; Puntschart et al. 1998; LaFramboise et al. 2009). As a result, c-Fos is a potential transcription factor that may help facilitate mitochondrial biogenesis by binding to the promoter region of nuclear genes that encode mitochondrial proteins. "
    [Show abstract] [Hide abstract] ABSTRACT: Many proteins that function as transcription factors regulate the transcriptional activity of nuclear genes encoding mitochondrial proteins. Several of these are rapidly inducible with contractile activity, followed by a recovery phase. The aim of the present study was to evaluate the expression of a number of rapidly responding gene products to an acute bout of contractile activity followed by a recovery period in both slow- and fast-twitch muscle. Using an in vitro isolated muscle preparation, extensor digitorum longus (EDL) and soleus muscles were stimulated for 15 min, followed by 30 min recovery. Following stimulation, ATP levels were decreased in both the EDL and soleus (25% and 32%, respectively). We found that phosphorylation of p38 MAP kinase was elevated in both muscle types, with a more dramatic 3.5-fold increase observed in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA expression was unchanged as a result of stimulation and recovery, while c-Fos transcript levels were decreased as a result of stimulation, but returned to resting values following recovery. Interestingly, nuclear respiratory factor 1 mRNA levels were unaffected by stimulation, but increased significantly (34%) during the recovery phase. These data suggest that the extent of the induction of transcription factor mRNA to acute exercise, which leads to subsequent muscle adaptations, is transcript specific and dependent on (i) the activation of upstream kinases, (ii) the muscle phenotype, and (iii) the duration of the recovery period.
    Article · Apr 2011
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