Cultured slow vs. fast skeletal muscle cells differ in physiology and
responsiveness to stimulation
Yen-Chih Huang1, Robert G. Dennis4,2,3, and Keith Baar5,‡.
1Department of Biomedical Engineering, and 2Department of Mechanical Engineering,
3Harvard-MIT HST, Cambridge, MA 02139, 4Department of Biomedical Engineering,
University of North Carolina at Chapel Hill. and 5Division of Molecular Physiology,
University of Dundee.
Running Title: Engineering skeletal muscle
Correspondence should be addressed to:
Keith Baar, Ph.D.
Division of Molecular Physiology
University of Dundee
MSI/WTB Dow Street
DUNDEE DD1 5EH
Articles in PresS. Am J Physiol Cell Physiol (January 25, 2006). doi:10.1152/ajpcell.00366.2005
Copyright © 2006 by the American Physiological Society.
In vitro studies have used protein markers to distinguish between myogenic cells
isolated from fast and slow skeletal muscles. The protein markers provide some
support for the hypothesis that satellite cells from fast and slow muscles are different,
but the data are equivocal. To test this hypothesis directly, 3-dimensional skeletal
muscle constructs were engineered from myogenic cells isolated from fast tibialis
anterior (TA) and slow soleus (SOL) muscles of rats and functionality was tested. Time
to peak twitch tension (TPT) and 1/2 relaxation times (1/2RT) were ~ 30% slower in
constructs from the SOL. The slower contraction and relaxation times for the SOL
constructs resulted in left shift of the force-frequency curve compared to those from the
TA. Western blot analysis showed a 60% greater quantity of fast myosin heavy chain in
the TA constructs. 14 days of chronic low frequency electrical stimulation (CLFS)
resulted in a 15% slower TPT and a 14% slower 1/2RT, but no change in absolute force
production in the TA constructs. In SOL constructs, slow electrical stimulation resulted
in an 80% increase in absolute force production with no change in TPT or 1/2RT. The
addition of Cyclosporine A did not prevent the increase in force in SOL constructs
following CLFS suggesting that calcineurin is not responsible for the increase in force.
We conclude that myogenic cells associated with a slow muscle are imprinted to
produce muscle that contracts and relaxes slowly and that calcineurin activity cannot
explain the response to a slow pattern of electrical stimulation.
Keywords: Tissue engineering, calcineurin, electrical stimulation, engineered muscle,
Skeletal muscle is a highly plastic tissue that changes its phenotype in response to
changes in loading and recruitment (18, 19). One of the models that have been used to
experimentally alter muscle phenotype is chronic low frequency electrical stimulation
(CLFS). A tonic stimulus that mimics the impulse pattern of a slow motor neuron slows
the speed of both contraction and relaxation in fast-twitch muscles (36). The importance
of electrical activity on the phenotype of skeletal muscle was reinforced when Salmons
and Sréter showed that the slow-to-fast muscle transition following cross reinnervation
of the soleus with the peroneal nerve could be reversed by CLFS (35). This experiment
showed conclusively that the role of the nerve in determining muscle phenotype was
largely dependent on activation patterns, that could be emulated by electrical
stimulation, and not chemical signals derived from the nerve. Since that time, the CLFS-
induced transition has been shown to result in coordinated changes in the isoforms of
the contractile (5), regulatory (22, 34, 37)), and calcium sequestering proteins (20, 32)
as well as inducing a concomitant mitochondrial biogenesis (38).
Even though muscle is highly plastic, it is generally accepted that the myoblasts within a
muscle are patterned to reflect the phenotype of that muscle (33). This assumption has
been made on the basis of the expression of protein markers that are thought to
represent the phenotype of muscle such as myosin heavy chain (MHC), myosin light
chain (MLC), and the myogenic regulatory factors (MRF, see table 1). However,
depending on the protein analyzed, the culture conditions, and the organism studied this
difference has not always been observed (3, 4, 12-14, 30).
Regardless of whether there are differences in the expression of a single protein, the
rate of contraction and relaxation of muscle is the result of numerous protein systems -
calcium release, regulatory proteins (MLC, troponins, and tropomyosin), myosin heavy
chain, and calcium sequestering - all working together to produce a faster muscle.
Therefore, analyzing a single protein or even representative proteins could produce
erroneous conclusions. We have recently reported a model system that permits the
functional analysis of skeletal muscle engineered from isolated cells (24). Using this
model, we tested the hypothesis that 3D engineered muscles generated from myogenic
cells isolated from a slow muscle (soleus) would contract and relax more slowly than
those generated from a fast muscle (tibialis anterior) and that electrical stimulation
would modulate the contractility of both engineered constructs similarly.
The data presented here show that functional muscles engineered from myogenic cells
of the slow soleus muscle contract and relax 30% slower than similar tissues
engineered from the fast TA muscle. Two weeks of chronic low frequency electrical
stimulation resulted in a slowing of time to peak tension and half relaxation without
altering force production in TA constructs while in the SOL constructs there was no
change in the rate of contraction and relaxation but force increased 2-fold. Fast-
patterned electrical activity had no effect on any of the constructs. To evaluate the
potential role of calcineurin in the increase in SOL construct force production, constructs
were stimulated in the presence or absence of cyclosporine A. Cyclosporine A
treatment resulted in a 28% faster TPT without altering the 1/2RT of the SOL
constructs. Treatment with cyclosporine A alone increased force production and when
combined with CLFS augmented the increase in force production induced by
stimulation. These data suggest that, in rats, myogenic cells from a slow muscle are
imprinted not only to produce muscle that contracts and relaxes more slowly but also
responds to chronic low frequency electrical stimulation differently than myogenic cells
from fast muscle.
Material and Methods
Materials. The SDS-PAGE gels were from Cambrex Bioscience (Rockland, ME). The
horseradish peroxidase-conjugated secondary antibodies and WestDura
chemiluminescent reagents were purchased from Pierce (Rockford, IL). SYLGARD
(polydimethylsiloxane – PDMS, type 184 silicone elastomer) is from Dow Chemical
Company (Midland, MI). Antibodies raised against eukaryotic initiation factor 2 were
from cell signaling (Danvers, MA). The total (MF 20) and the type II (F59) myosin heavy
chain antibodies were developed by Dr. D.A. Fischman and F.E. Stockdale,
respectively, and obtained from the Developmental Studies Hybridoma Bank developed
under the auspices of the NICHD and maintained by the Department of Biological
Sciences, at the University of Iowa. All other reagents were from Sigma (St. Louis,
Isolation of myogenic cells from muscle. Myoblasts were isolated as described
previously (24). Briefly, Hind limb muscles (Soleus and TA) from Sprague-Dawley rats
were dissected and washed with PBS 3~4 times to remove hair and other debris. The
muscles were cut into small pieces and cleaned of excess connective tissues and
tendons. Dissociation of muscles was carried out in 10ml of digestion buffer (F12
medium containing 0.1% collagenase and 0.05% dispase) per 100mg tissue. The
digested tissues were filtered through a 100µm cell strainer (Becton Dickinson, Franklin
Lakes, NJ) and centrifuged at 1500 x g for 6 min. The supernatant was discarded, and
the cell pellet was resuspended in growth medium (10% heat-inactivated FBS and
5ng/ml FGF-2 in F12 or F12K medium with penicillin/streptomycin 100U/100 mg/ml and
fungizone 2.5 µg/ml). After preplating overnight, the myoblast enriched supernatant was
transferred to a new plate and expanded for 5-6 days.
Formation of 3D engineered skeletal muscle. 3D muscles were engineered as
described previously (24). Briefly, two 1.0 gauge sutures were secured to a SYLGARD
(polydimethylsiloxane - PDMS) coated 35mm plate. To form the fibrin gel the plates
were coated with 700µl of growth media containing 5 units of thrombin and 4mg of
fibrinogen. One hundred thousand cells were added to the top of the gel and the plates
were placed in a standard incubator (37°C and 5% CO2). Following 14 days in culture,
the 3D muscle constructs had completely formed and were ready for stimulation.
Determination of engineered muscle contractility. All contractile properties were
measured 28 days after the cells were plated. The temperature of the engineered
muscles was maintained at 37±1°C during the measurement of contractile properties
using a heated aluminum platform. For measurements of contractility, one of the
sutures was freed from the PDMS substrate and a force transducer was attached to its
minutien pin using canning wax. The custom-built force transducer has a resolution
range of 1µN to 2000µN and a sampling rate of 1kS/sec (10), allowing the determination
of time-to-peak tension (TPT) and half-relaxation time (1/2RT). Constructs were
stimulated three times with either a single twitch pulse of 4ms at 15 volts or a one
second tetanus at 5, 10, 20, 40, 60, 80, 100, and 150 Hertz 1.2ms pulses at 15 volts
and the average force recorded over the three trials was determined. Baseline force
was measured as the average baseline passive force preceding the onset of
Determination of myosin heavy chain protein levels. 4 weeks after plating, SOL and TA
constructs were frozen, sonicated in 100µl of 1X Laemmli sample buffer, and boiled for
10 minutes to denature and free intracellular proteins. Protein concentration was
determined using the non-interfering protein assay (Upstate, Waltham, MA) and equal
aliquots of protein were separated by SDS-PAGE, transferred to nitrocellulose, and
blocked in 5% milk. Blots were exposed to primary antibodies (eEF2 at 1:1000; MF20
and F59 at 1:10 of unpurified hybridoma supernatant) overnight at 4°C and the bound
antibody was detected chemiluminescently using WestDura chemiluminescent reagent
and a Syngene Chemigenius 2 gel documentation system.
Electrical stimulation of engineered muscles. SOL and TA constructs were electrically
stimulated for 14 days starting 2 weeks after seeding using a protocol that mimicked
either fast or slow motor neuron activity. The slow protocol delivered 5 pulses at 20Hz
every 4 seconds, while the fast protocol delivered 5 pulses at 100Hz every 100
seconds. Both protocols used the same voltage (5V) and pulse width (1.5ms).
Cyclosporine A treatment. Control and chronically stimulated SOL constructs were
treated with 500ng/ml CsA for 2 weeks. At the end of the 2-week incubation the
constructs were stimulated as described above.
Statistics. Data is presented as mean ± S.E.M for 4 to 6 engineered muscle constructs
per group. Differences in mean values were compared within groups and significant
differences were determined by ANOVA with posthoc Tukey-Kramer HSD. The level of
significance was set at p<0.05.
Isolation of myoblasts from fast and slow muscle. Myoblasts isolated from both the TA
and SOL muscles grew well in culture. Digestion of the soleus muscle tended to
provide more myoblasts per milligram of muscle than the TA muscle as has been
reported previously (16). After expansion, equal numbers of myoblasts isolated from
the SOL or TA were seeded on the fibrin gels.
Contractility of the engineered muscles. To determine whether the cells isolated from
the soleus muscle were imprinted to be slower than those of the TA, the rate of
contraction and relaxation of the engineered tissues was determined. The TPT and
1/2RT of the SOL constructs were 30% and 31% slower than the TA constructs,
respectively (SOL construct TPT=52±0.3 1/2RT=45.3±2.3; TA construct
TPT=39.8±0.5ms 1/2RT=35.3±1.7, Figure 1A). As a result of the slower twitch kinetics,
the force frequency curve of the SOL constructs was shifted to the left compared to the
TA constructs (Figure 1B). The SOL constructs had a significantly higher percentage
of maximal force production from 20 Hertz to 60 Hertz.
Myosin heavy chain isoform expression. To assess whether the rate of contraction
reflected differences in the contractile proteins expressed by the constructs, the level of
fast myosin heavy chain was determined by western blotting (Figure 2). Using an
antibody to detect all forms of the fast myosin heavy chain, the TA constructs had 57%
more fast MHC than the SOL constructs (TA=580±13.0, SOL=370±35.9).
Stimulation alters the function of engineered muscle. Both SOL and TA constructs were
stimulated with either a slow or a fast paradigm of electrical activity (see methods) to
determine the effect on contractile function. Two weeks of slow electrical activity
resulted in a 14.5% longer TPT and a 13.5% longer 1/2RT in the TA constructs (Figure
3B). This increase in TPT and 1/2RT was not accompanied by a change in the amount
of force produced by the constructs (Figure 4B). In contrast, the TPT and 1/2RT of the
SOL constructs was unaffected by CLFS (Figure 3A) but this paradigm of stimulation
increased the force produced by the SOL constructs by 80.4% (Figure 4A). The
increase in force production following CLFS was not the result of an increase in myosin
heavy chain in the stimulated SOL constructs. Total MHC as determined by western
blotting showed a trend towards decreasing being 49.2 and 53.6% of control in two
separate experiments. The change in MHC did not reflect a change in the number of
cells in the constructs as the amount of elongation factor 2 was unchanged following
stimulation. The fast paradigm of electrical activity had no effect on the force production
or the contractile dynamics of either the SOL or TA constructs (Figure 3 and 4).
Role of calcineurin in the increase in SOL construct force following CLFS. Since
calcineurin may play a role both in the control of muscle fiber type and muscle
hypertrophy, we sought to determine whether the increase in force production in the
SOL constructs following CLFS was the result of the activation of calcineurin in the SOL
constructs. Control and chronically stimulated SOL constructs were treated with
500ng/ml CsA for 2 weeks, in an effort to prevent calcineurin activation, and the effect
on contractile dynamics and force production were determined. Two weeks of CsA
treatment alone increased force production 230% (CTL=203±31.4µN;
CsA=471±71.0µN; Figure 5). As before, CLFS increased force production
(326±201.0µN). The combination of CsA and CLFS resulted in an additive effect on
force production to a level 339% of control (690±85.1µN). Unlike electrical stimulation,
CsA treatment increased total MHC within the SOL constructs (Figure 6). This increase
in MHC reflects not only an increase in the total amount of MHC but also a 3.4-fold
increase in the percentage of the fast isoform. Concurrent treatment with CsA and
CLFS had the greatest increase in total MHC (9.5-fold). However, the increase in the
proportion of fast MHC was prevented by CLFS.
Role of calcineurin in altering TPT and 1/2RT. Determination of TPT and 1/2RT showed
that both the CsA alone and the CsA with CLFS groups showed a 25% decrease in TPT
(CTL=52.7±1.8ms; CsA=39.6±1.2ms; CsA+CLFS=39.3±0.6ms; Figure 7), while there
was no change in TPT with CLFS alone (53.6±2.7ms). In contrast to TPT, 1/2RT was
unchanged for any of the groups studied (CTL=45.9±1.8ms; CsA=41.8±3.6ms;
This is the first demonstration that myogenic cells isolated from a slow muscle are
functionally distinct from those isolated from a fast muscle, contracting and relaxing
30% slower. The differences between myogenic cells are much greater than simply
differential expression of myosin heavy chain, myosin light chain, or myogenic
regulatory factors as previously described in the rodent (6, 7, 12, 26, 33, 39). Not only
are the rate of contraction and relaxation different, but the engineered muscles also
show a differential response to electrical stimulation. This indicates that the plasticity of
rat muscle is dependent on a long-term reprogramming of the composite cells. The first
essential steps that are required for this reprogramming remain to be identified.
Before discussing the significance of the findings we must first discuss one issue with
the techniques. The variability in the basal measurements from experiment to
experiment reflect the fact that each experiment is generated from a separate isolated
muscle cell population. As a result, there may be differences in the total number of cells
in the constructs and in the ratio of myoblasts to fibroblasts from experiment to
experiment. For this reason, all of the experiments have been repeated at least 3 times
and the percent change between the trials are equivalent (compare figure 4A slow
stimulation to figure 5 slow stimulation, each shows an 87% increase in force).
As was expected from the seminal work by Salmons (36) and Pette (39), electrical
stimulation altered the contractility of the muscle constructs. Unexpectedly, the
changes in contractility depended on the muscle used to generate the constructs.
CLFS slowed the rate of both contraction and relaxation in the TA constructs without
affecting force production. In contrast, neither the TPT nor the 1/2RT were effected in
the SOL constructs. Instead, CLFS of the SOL constructs resulted in a 61-80%
increase in force production without an increase in total myosin heavy chain. The shift
in dynamics in the TA constructs is likely due to the shift in myosin heavy chain and
SERCA to a slower isoform as has been reported both in vivo (21) and in vitro (39).
The increase in force production in the SOL constructs is more difficult to explain and
could not have been observed in the 2-dimensional culture systems that have
previously been used. The increase in force in the stimulated SOL constructs may
reflect the fact that, to slow myogenic cells, chronic low-frequency stimulation promotes
the reorganization of sarcomeres within the SOL constructs resulting in greater force
production with the same or fewer myosin molecules or that CLFS results in an
alteration in the matrix produced by the SOL muscle cells allowing for better
transmission of the force to the anchors. We initially thought that hypertrophy was
occurring within the SOL constructs and that this was due to calcineurin, which has
been hypothesized to be involved in both the development of slow fiber phenotype and
muscle growth (9, 31). However, treatment with cyclosporine A, which should inhibit
calcineurin, did not prevent the increase in force production in the stimulated SOL
In contrast to the effects of CLFS, stimulating the constructs with a paradigm modeled
after a fast nerve did not change contractility. This likely reflects the fact that the fast
paradigm of stimulation was not appropriate for ex vivo stimulation. Previous work from
our laboratory has shown that the maintenance of the mass and force production of a
denervated fast muscle, the extensor digitorum longus, in vivo requires between 200
and 800 contractions a day (11). Greater than 800 contractions a day resulted in a loss
of muscle mass and force production. In this study the fast protocol delivered one
contraction every 100 seconds. This amounts to over 850 contractions per day for an
isolated engineered muscle construct. Though this pattern may be within the normal
range for fast twitch muscle in vivo, it is likely that in culture this pattern was
inappropriate. The fast protocol of electrical stimulation resulted in the loss of force
within 24 hours (data not shown). This is likely the result of electrically induced tissue
damage. The effect of excessive electrical stimulation was also observed in slow
protocols of electrical stimulation. The CLFS protocol adopted for this study (250ms
train duration every 4 seconds) produced only 6% active time compared with 25~30%
active time in the adult soleus (23). In preliminary experiments, doubling the train
duration to 500ms (bringing the active time to ½ that of the adult soleus) induced a
decrease in force production within a couple of days. This suggests that electrical
stimulation ex vivo does not precisely emulate the stimulation of muscle in vivo.
Electrical stimulation is potentially destructive, both in vivo and in vitro, due to hydrolytic
degradation of the fluids and tissues surrounding the electrodes, electrode breakdown,
and the large field of stimulation compared with the smaller area directly involved in an
intact neuromuscular junction. Overall, the greater energy flux associated with electrical
stimulation means that activity patterns derived from normal nerve-muscle activity may
be more than the tissue can endure via field stimulation, resulting in collateral tissue
damage (11). Therefore, for both protocols used in this study the amount of electrical
stimulation still needs to be optimized further in terms of frequency, pulse width, and
train duration to minimize tissue damage and maximize phenotypic alterations.
The data also suggest that calcineurin has a direct effect on the rate of muscle
contraction but has limited effect on the rate on relaxation. Our data are consistent with
others that have shown that calcineurin directly alters the expression of myosin heavy
chain away from slow type I myosin (9, 29) towards the fast type II isoform (8, 25). In
the data presented here, the shift towards a fast muscle is reflected not only in an
increase in fast MHC levels, but for the first time both a decrease in the TPT and an
increase in the force of contraction were observed in cell culture. Interestingly, there
was no change in the rate of relaxation of the SOL constructs when they were incubated
with CsA in spite of the decrease in the TPT. This suggests that calcineurin has no
direct effect on the expression of the calcium sequestering machinery and that the fast-
to-slow transformation that occurs in vivo (8) requires other factors such as thyroid or
other circulating hormones. A similar effect has been seen in regenerating soleus
muscle where inhibition of calcineurin in isolated fibers prevented the accumulation of
type I MHC but had no effect on the expression of the slow sarco/endoplasmic reticulum
calcium pump SERCA2 (40).
The effects of cyclosporine A on myosin heavy chain expression are more difficult to
explain. The increase in total myosin heavy chain following treatment with CsA
suggests that a target of CsA, possibly calcineurin, inhibits myosin heavy chain
expression. It is noteworthy that the increase in MHC in the CsA treated constructs was
primarily the result of an increase in the fast isoform of the protein. This is consistent
with the in vivo work of Giger et al (17) who showed that nine days of CsA treatment
increased the expression of both the type IIa and IIx isoforms. In contrast, Meissner
and colleagues did not see any change in the mRNA of either type IIa or IIx MHC in
rabbit primary bead cultures treated with CsA for 14 days. The difference between the
studies likely reflects differences between the models. The engineered model
described here is in agreement with the data collected in vivo and suggests that a CsA-
inhibited process blocks normal expression of fast MHC isoforms.
The fact that the ability of CsA to drive the expression of fast MHC was reduced by slow
electrical stimulation in the SOL constructs may indicate that slow electrical activity is
dominant over CsA in determining MHC isoform or that the amount of CsA used was
not sufficient to block calcineurin, or other downstream targets, when CLFS was added.
It is interesting to note that even though the proportion of fast MHC was not increased in
these constructs the TPT was faster. This likely reflects the fact that there is an
increase in the total amount of fast MHC in these constructs. In small muscles like
those engineered here, any increase in the amount of fast myosin may be sufficient to
It is clear from the data presented here that the development of a muscle and the
functional determination of what constitutes a fast muscle cell is more complex than
simply the expression of myosin heavy chain. The identification of why SOL constructs
increase force production while decreasing MHC in response to CLFS and why CsA
treatment and CLFS increase total MHC more than just CsA treatment alone may give
us some insight into how adult muscle fibers develop and determine skeletal muscle
Barjot C, Cotten ML, Goblet C, Whalen RG, and Bacou F. Expression of
myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit
muscle satellite cell cultures. J Muscle Res Cell Motil 16: 619-628, 1995.
Barjot C, Rouanet P, Vigneron P, Janmot C, d'Albis A, and Bacou F.
Transformation of slow- or fast-twitch rabbit muscles after cross-reinnervation or low
frequency stimulation does not alter the in vitro properties of their satellite cells. J
Muscle Res Cell Motil 19: 25-32, 1998.
Bonavaud S, Agbulut O, Nizard R, D'Honneur G, Mouly V, and Butler-
Browne G. A discrepancy resolved: human satellite cells are not preprogrammed to fast
and slow lineages. Neuromuscul Disord 11: 747-752, 2001.
Boudreau-Lariviere C, Parry DJ, and Jasmin BJ. Myotubes originating from
single fast and slow satellite cells display similar patterns of AChE expression. Am J
Physiol Regul Integr Comp Physiol 278: R140-148, 2000.
Brown WE, Salmons S, and Whalen RG. The sequential replacement of
myosin subunit isoforms during muscle type transformation induced by long term
electrical stimulation. J Biol Chem 258: 14686-14692, 1983.
Bryla K and Karasinski J. Diversity of myosin heavy chain expression in
satellite cells from mouse soleus and EDL muscles. Folia Histochem Cytobiol 39: 295-
Cantini M, Fiorini E, Catani C, and Carraro U. Differential expression of adult
type MHC in satellite cell cultures from regenerating fast and slow rat muscles. Cell Biol
Int 17: 979-983, 1993.
Chin ER, Olson EN, Richardson JA, Yang Q, Humphries C, Shelton JM, Wu
H, Zhu W, Bassel-Duby R, and Williams RS. A calcineurin-dependent transcriptional
pathway controls skeletal muscle fiber type. Genes Dev 12: 2499-2509, 1998.
Delling U, Tureckova J, Lim HW, De Windt LJ, Rotwein P, and Molkentin JD.
A calcineurin-NFATc3-dependent pathway regulates skeletal muscle differentiation and
slow myosin heavy-chain expression. Mol Cell Biol 20: 6600-6611, 2000.
Dennis RG and Kosnik PE. Excitability and isometric contractile properties of
mammalian skeletal muscle constructs engineered in vitro. In Vitro Cell Dev Biol Anim
36: 327-335, 2000.
Dow DE, Cederna PS, Hassett CA, Kostrominova TY, Faulkner JA, and
Dennis RG. Number of contractions to maintain mass and force of a denervated rat
muscle. Muscle Nerve 30: 77-86, 2004.
Dusterhoft S and Pette D. Satellite cells from slow rat muscle express slow
myosin under appropriate culture conditions. Differentiation 53: 25-33, 1993.
Dusterhoft S, Yablonka-Reuveni Z, and Pette D. Characterization of myosin
isoforms in satellite cell cultures from adult rat diaphragm, soleus and tibialis anterior
muscles. Differentiation 45: 185-191, 1990.
Edom F, Mouly V, Barbet JP, Fiszman MY, and Butler-Browne GS. Clones of
human satellite cells can express in vitro both fast and slow myosin heavy chains. Dev
Biol 164: 219-229, 1994.
Feldman JL and Stockdale FE. Skeletal muscle satellite cell diversity: satellite
cells form fibers of different types in cell culture. Dev Biol 143: 320-334, 1991.
Gibson MC and Schultz E. The distribution of satellite cells and their
relationship to specific fiber types in soleus and extensor digitorum longus muscles.
Anat Rec 202: 329-337, 1982.
Giger JM, Haddad F, Qin AX, and Baldwin KM. Effect of cyclosporin A
treatment on the in vivo regulation of type I MHC gene expression. J Appl Physiol 97:
Gollnick PD, Armstrong RB, Saltin B, Saubert CWt, Sembrowich WL, and
Shepherd RE. Effect of training on enzyme activity and fiber composition of human
skeletal muscle. J Appl Physiol 34: 107-111, 1973.
Gollnick PD, Armstrong RB, Saubert CWt, Piehl K, and Saltin B. Enzyme
activity and fiber composition in skeletal muscle of untrained and trained men. J Appl
Physiol 33: 312-319, 1972.
Green HJ, Klug GA, Reichmann H, Seedorf U, Wiehrer W, and Pette D.
Exercise-induced fibre type transitions with regard to myosin, parvalbumin, and
sarcoplasmic reticulum in muscles of the rat. Pflugers Arch 400: 432-438, 1984.
Hamalainen N and Pette D. Coordinated fast-to-slow transitions of myosin and
SERCA isoforms in chronically stimulated muscles of euthyroid and hyperthyroid
rabbits. J Muscle Res Cell Motil 18: 545-554, 1997.
Hartner KT and Pette D. Fast and slow isoforms of troponin I and troponin C.
Distribution in normal rabbit muscles and effects of chronic stimulation. Eur J Biochem
188: 261-267, 1990.
Hennig R and Lomo T. Firing patterns of motor units in normal rats. Nature 314:
Huang YC, Dennis RG, Larkin L, and Baar K. Rapid formation of functional
muscle in vitro using fibrin gels. J Appl Physiol 98: 706-713, 2005.
Kubis HP, Hanke N, Scheibe RJ, Meissner JD, and Gros G. Ca2+ transients
activate calcineurin/NFATc1 and initiate fast-to-slow transformation in a primary skeletal
muscle culture. Am J Physiol Cell Physiol 285: C56-63, 2003.
Lagord C, Soulet L, Bonavaud S, Bassaglia Y, Rey C, Barlovatz-Meimon G,
Gautron J, and Martelly I. Differential myogenicity of satellite cells isolated from
extensor digitorum longus (EDL) and soleus rat muscles revealed in vitro. Cell Tissue
Res 291: 455-468, 1998.
Martelly I, Soulet L, Bonnavaud S, Cebrian J, Gautron J, and Barritault D.
Differential expression of FGF receptors and of myogenic regulatory factors in primary
cultures of satellite cells originating from fast (EDL) and slow (Soleus) twitch rat
muscles. Cell Mol Biol (Noisy-le-grand) 46: 1239-1248, 2000.
Matsuda R, Spector DH, and Strohman RC. Regenerating adult chicken
skeletal muscle and satellite cell cultures express embryonic patterns of myosin and
tropomyosin isoforms. Dev Biol 100: 478-488, 1983.
Meissner JD, Gros G, Scheibe RJ, Scholz M, and Kubis HP. Calcineurin
regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow
transformation in rabbit skeletal muscle cell culture. J Physiol 533: 215-226, 2001.
Mouly V, Edom F, Barbet JP, and Butler-Browne GS. Plasticity of human
satellite cells. Neuromuscul Disord 3: 371-377, 1993.
Musaro A, McCullagh KJ, Naya FJ, Olson EN, and Rosenthal N. IGF-1
induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2
and NF-ATc1. Nature 400: 581-585, 1999.
Ohlendieck K, Briggs FN, Lee KF, Wechsler AW, and Campbell KP. Analysis
of excitation-contraction-coupling components in chronically stimulated canine skeletal
muscle. Eur J Biochem 202: 739-747, 1991.
Rosenblatt JD, Parry DJ, and Partridge TA. Phenotype of adult mouse muscle
myoblasts reflects their fiber type of origin. Differentiation 60: 39-45, 1996.
Roy RK, Mabuchi K, Sarkar S, Mis C, and Sreter FA. Changes in tropomyosin
subunit pattern in chronic electrically stimulated rabbit fast muscles. Biochem Biophys
Res Commun 89: 181-187, 1979.
Salmons S and Sreter FA. Significance of impulse activity in the transformation
of skeletal muscle type. Nature 263: 30-34, 1976.
Salmons S and Vrbova G. Changes in the speed of mammalian fast muscle
following longterm stimulation. J Physiol 192: 39P-40P, 1967.
Sreter FA, Gergely J, Salmons S, and Romanul F. Synthesis by fast muscle of
myosin light chains characteristic of slow muscle in response to long-term stimulation.
Nat New Biol 241: 17-19, 1973.
Takahashi M and Hood DA. Chronic stimulation-induced changes in
mitochondria and performance in rat skeletal muscle. J Appl Physiol 74: 934-941, 1993.
Wehrle U, Dusterhoft S, and Pette D. Effects of chronic electrical stimulation on
myosin heavy chain expression in satellite cell cultures derived from rat muscles of
different fiber-type composition. Differentiation 58: 37-46, 1994.
Zador E, Fenyvesi R, and Wuytack F. Expression of SERCA2a is not regulated
by calcineurin or upon mechanical unloading in skeletal muscle regeneration. FEBS Lett
579: 749-752, 2005.
This work was supported by a grant from the United States Defense Advanced
Research Projects Agency (DARPA), to R.G. Dennis with subcontract to K. Baar, Navy
Tables and Figures
Table 1. A selection of the previous reports on the similarities and/or differences
between muscle cells isolated from human (3, 14, 30), fowl (15, 28), rabbit (1, 2), rat (4,
7, 12, 13, 26, 27, 39), and mouse (6, 33) fast and slow muscle.
Figure 1. Contractile dynamics of SOL and TA constructs. (A) Time-to-peak tension
(TPT) and half-relaxation time (1/2RT) were determined in engineered muscles
generated from either soleus or tibialis anterior muscles. Constructs were stimulated
three times with a single twitch pulse of 4ms at 10 volts and the average force recorded
over the three trials was determined. (B) The force-frequency relationship was
determined in SOL and TA constructs using a one second tetanus at 5, 10, 20, 40, 60,
80, 100, and 150 Hertz 1.2ms pulses at 10 volts. * represents a significant difference
between the SOL and TA constructs (p<0.05, n=6).
Figure 2. Expression of Type II myosin heavy chain in SOL and TA constructs.
Western blots using the N3.36 antibody were performed on muscles engineered from
either soleus or tibialis anterior muscles. (A) representative western blot from 4 SOL
and 4 TA constructs. (B) Quantification of the reactive bands demonstrated a 57%
greater level of fast MHC in the TA constructs. * indicates significantly difference
between SOL and TA constructs (p<0.05, n=4).
Figure 3. Effect of electrical stimulation on contractility in (A) SOL and (B) TA
constructs. Time-to-peak tension (TPT) and half-relaxation time (1/2RT) were
determined following 14 days of electrical stimulation using either a slow or a fast
paradigm of electrical stimulation. (A) The effects of stimulation on the SOL constructs.
(B) TA construct TPT and 1/2RT after stimulation. * indicates a significant effect of
electrical stimulation (p<0.05, n=6).
Figure 4. Effect of electrical stimulation on force production in (A) SOL and (B) TA
constructs. Force of contraction was determined following 14 days of electrical
stimulation using either a slow or a fast paradigm of electrical stimulation. The effects of
stimulation on (A) SOL construct and (B) TA construct force were determined. *
indicates a significant effect of electrical stimulation (p<0.05, n=6).
Figure 5. Effect of electrical stimulation and cyclosporine A on force production of SOL
constructs. Force of contraction was determined following 14 days of chronic low-
frequency electrical stimulation, treatment with 500ng/ml CsA, or both interventions. *
indicates a significant difference from control constructs, # indicates significantly
different from CsA alone, and † indicates significantly different than slow stimulation
alone (p<0.05, n=6).
Figure 6. Effect of electrical stimulation and cyclosporine A on the levels of total and
fast myosin heavy chain. The amount of myosin heavy chain within a construct was
determined by western blotting following 14 days of chronic low-frequency electrical
stimulation, treatment with 500ng/ml CsA, or both interventions. (A) Representative
blots for total MHC, fast MHC, and elongation factor 2. (B) Quantification of total MHC
and (C) the percentage of fast MHC relative to total MHC. * indicates a significant
difference from control constructs and # indicates significantly different from CsA alone
Figure 7. Effect of electrical stimulation and cyclosporine A on contractility of SOL
constructs. Time-to-peak tension (TPT) and half-relaxation time (1/2RT) were
determined following 14 days of chronic low-frequency electrical stimulation, treatment
with 500ng/ml CsA, or both interventions. * indicates a significant difference from control
constructs (p<0.05, n=6).
Table 1. Studies looking at the imprinting of skeletal muscle cells to fast or slow lineage
Study Marker Used to Determine Fast or SlowSource of Myoblasts Fast and Slow Different?
Barjot et al. 1995, 1998 MHC RabbitYes
Bonavaud et al. 2001MHCHumanNo
Boudreau-Lariviere et al. 2000Acetylcholinesterase (AChE)Rat and mice No
Bryla and Karasinski 2001 MHCmouseYes
Cantini et al. 1993 MHCratYes
Dusterhoft et al. 1990MHC and MLC ratNo
Dusterhoft and Pette 1993 Slow MHCrat Soleus Yes
Edom et al. 1994MHC and MLC humanNo
Feldman and Stockdale 1991 MHCchicken/quail Yes
Lagord and Soulet 1998MyoD and myogenin rat Yes
Martelly et al. 2000 FGF receptors and MRFsrat Yes
Matsuda et al. 1983NHC,MLC and tropomyosin chickenYes
Mouly et al. 1993 MHC and MLC humanNo
Rosenblatt et al. 1996MHCmouseYes
Wehrle et al. 1994MHCratYes
5hz10hz20hz40hz60hz 80hz100hz 150hz
CTLCsASlow StimulationSlow Stimulation +
CTLStimCsACsA + Stim
CTLStim CsACsA + Stim
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CTLCsASlow Stimulation Slow Stimulation +