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Department of Physiological Science, University of California, Los Angeles, Los Angeles, California 90095-1527, USA
1Correspondence: Department of Physiological Science, 621 Young Dr. S., University of California, Los Angeles, CA 90095-1527, USA. E-mail: jtidball{at}physci.ucla.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: atrophy • myotendinous junction • plantaris muscle • rat • unloading
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
who observed a two- to threefold increase in the muscle mass of
myostatin knockout mice when compared to controls. Myostatin is
transcribed as a 2.9 kb mRNA species and translated as disulfide-linked
dimer that is subsequently secreted, processed, and thought to function
in an autocrine and/or paracrine manner similar to that of other
TGF-ß superfamily proteins (1)
.
The importance of myostatin as a regulator of skeletal muscle mass has
been well established, first in myostatin null mutant mice and
subsequently in Belgian Blue and Peidmontese cattle. The significantly
increased muscle mass observed in Belgian Blue and Peidmontese cattle,
known as the ‘double-muscled’ phenotype, seems to be due to a lack of
functional myostatin protein. The myostatin coding sequence of Belgian
Blue cattle has an 11-nucleotide deletion, which ultimately results in
expression of a truncated protein product; Peidmontese cattle also
express a nonfunctional myostatin protein due to a missense mutation in
the gene sequence (2
3
4)
. Although the mechanism by which
a lack of myostatin results in increased muscle mass is not known,
myostatin null mutants exhibit both muscle hypertrophy and hyperplasia
(1)
.
A functional role for myostatin in fully differentiated muscle has not
yet been established and current data present an unclear picture of
what role the protein may play. The finding that HIV-positive men who
experience severe muscle atrophy express higher levels of a serum and
muscle protein with similar structure to myostatin
(myostatin-immunoreactive protein) supports the possibility that
myostatin may act as a negative regulator of muscle mass during
pathological wasting of muscle (5)
. However, the
relationship between myostatin and myostatin-immunoreactive protein has
not yet been established. Mouse muscles subjected to 1 day of hind limb
muscle unloading showed a significant increase in myostatin mRNA
concentration but no significant decrease in muscle mass, whereas
muscle mass was significantly reduced after 3 and 7 days of unloading,
although myostatin mRNA levels were not significantly elevated at those
times (6)
. Finally, suppression of muscle growth by 3 days
of food deprivation did not affect the expression of myostatin message
in piglets (7)
. Thus, there is no simple relationship
between changes in myostatin mRNA concentration and changes in muscle
mass, and no information concerning changes in myostatin protein
expression and modified muscle use.
In the present investigation, we test the hypothesis that modifications
in muscle loading influence the expression and distribution of
myostatin, and that changes in the expression of myostatin mRNA and
protein relate inversely to changes in muscle mass. Three experimental
perturbations are used to test the hypotheses. First, rat hind limb
muscle unloading for a period of 10 days was used since previous
investigations have shown that this treatment leads to rapid loss of
mass of soleus and plantaris muscles (8)
. Second, 10 days
of hind limb unloading, followed by 4 days of reloading by return to
normal weightbearing, was used to produce a period of rapid increase in
muscle mass. Finally, 10 days of unloading during which rats are
subjected to daily periods of 30 min of muscle loading was used. The
latter treatment was chosen because it subjects hind limb muscles to
dramatically different patterns of loading, with little or no change in
muscle mass (9
10
11)
. This will enable us to determine
whether changes in myostatin expression relate to modifications in
muscle loading or modifications in muscle mass.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Muscle unloading and loading procedures
Rats were subjected to hind limb unloading for 10 days by a
modification of the protocol described by Morey-Holton and Wronski
(8)
according to procedures approved by the University of
California, Los Angeles, Animal Research Committee. Four treatment
groups were analyzed. The first group (n=15) consisted of
ambulatory controls. Animals in the second group (n=15) were
subjected to 10 days of unloading of the hind limb muscles, after which
they were killed without experiencing muscle reloading. In the third
group (n=9), rats were similarly subjected to hind limb
muscle unloading for 10 days, but during this period they were also
subjected to 30 min each day of loading the hind limb muscles by
exercise on a treadmill at 5 m/min on a 20% grade. Rats in the last
group (n=6) were removed from the hind limb unloading
apparatus after 10 days of unloading and allowed to recover for 4 days
with normal cage activity, when hind limb muscles experienced
reloading.
Western analysis
Whole muscle extracts of tissues were prepared by homogenizing
samples in sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) reducing buffer (80 mM Tris-HCl pH 6.8, 0.1 M
dithiothreitol, 70 mM SDS, 1.0 mM glycerol). Samples were boiled for 1
min, then centrifuged at 12,000 x g for 1 min. The
supernatant fraction of each sample was removed and used to determine
protein concentration by measuring absorbance at 280 nm. Homogenates
containing 50 µg of total protein were separated on 12% SDS-PAGE
gels according to Laemmli (12)
. Proteins were
electrophoretically transferred onto nitrocellulose membranes while
immersed in transfer buffer (39 mM glycine, 48 mM Tris)
(13)
.
After transfer, membranes were blocked in buffer containing 0.5% Tween-20, 0.2% gelatin, and 3.0% dry milk (blocking buffer) for at least 1 h at room temperature. Membranes were probed with polyclonal anti-myostatin for 2 h at room temperature. Subsequently, membranes were overlaid with alkaline phosphatase-conjugated anti-rabbit IgG (Sigma, St. Louis, Mo.) for 1 h at room temperature. After each incubation, membranes were washed six times for 10 min in wash buffer (0.5% Tween-20, 0.2% gelatin, and 0.3% dry milk). Blots were developed using nitroblue tetrazolium and bromo-chloro-indolyl phosphate.
Relative concentration of myostatin protein in each sample was determined by scanning densitometry (Alpha Innotec, San Leandro, Calif.). Uniformity of protein loading and efficiency of transfer were assessed by staining membranes with Ponceau S (Sigma) after electrophoretic transfer.
Polyclonal anti-myostatin production
A sequence of 20 amino acids (aa #100–119) from the portion of
the myostatin protein sequence encoding the latent peptide, which is
unique to myostatin, was chosen for antibody production based on its
lack of homology to other known proteins. This 20 mer was synthesized
by Research Genetics (Huntsville, Ala.) and subsequently conjugated to
the carrier molecule, hemocyanin keyhole limpet (KLH) (Calbiochem, San
Diego, Calif.). The peptide-KLH conjugate was emulsified in Freund’s
complete adjuvant and injected subcutaneously into a New Zealand White
rabbit after collection of preimmune serum. The rabbit received a
booster injection of the peptide-KLH conjugate emulsified in Freund’s
incomplete adjuvant each 4 wk and serum was collected from an ear vein
10–14 days after each injection.
Northern analysis
RNA was isolated according to Chomczynski and Sacchi
(14)
. The final RNA pellets were resuspended in 10–25
µl of 10 mM Tris, pH 8.0 with 1 mM ethylene diamine tetraacetic acid
(EDTA). The concentration of RNA in each sample was determined by
measuring absorbance at 260 nm. Samples containing 5 µg of total RNA
were loaded onto 1.2% formaldehyde-agarose gels and electrophoresed
overnight at 25 V. The RNA samples were electrophoretically transferred
to uncharged nylon membranes, ultraviolet cross-linked with 150 mJ, and
subsequently stained with methylene blue to verify uniformity of
loading and efficiency of transfer.
Membranes were prehybridized in Denhardt’s solution, 4x SSC (0.03 M
citric acid trisodium-0.3 sodium chloride), 1% SDS, and 100 µg/ml
herring sperm DNA for at least 1 h at 65°C. Membranes were
hybridized in Denhardt’s solution, 4x SSC, 1% SDS, 100 µg/ml
herring sperm DNA, and 10% sodium dextran sulfate with 2 x
106 cpm/ml of
-32P-labeled myostatin cDNA probe (specific
activity > 1x108 cpm/µl) overnight at
65°C. Membranes were washed six times for 20 min with 0.05 M sodium
phosphate, 0.75 M sodium chloride, 5 mM EDTA, and 0.1% SDS and exposed
to autoradiographic film for 1–5 days at -80°C.
Relative concentration of myostatin RNA in each sample was determined by scanning densitometry (Alpha Innotec). Uniformity of loading was verified by stripping blots and reprobing with pTRI RNA 18s control probe (Ambion Inc., Austin, Tex.).
Production of myostatin cDNA probe
After reverse transcriptase-polymerase chain reaction (RT-PCR)
of total RNA isolated from C57 plantaris, the resulting cDNA was
subjected to PCR with the upper primer
5'-GAGGGATGACAGCAGTGATGGCTCTTTGG-3' and the lower primer
5'-CGGTCTACTACCATGGCTGGAATTTTCCC-3', yielding a product of the expected
size of 822 bp. PCR using these primers was carried out at 94°C for
45 s, 56°C for 1 min, and 72°C for 3 min for 35 cycles. The
822 bp product was cloned into the pCR 2.1 vector using the Original TA
Cloning Kit (Invitrogen, San Diego, Calif.). After digestion with
EcoRI, the cloned PCR product was sequenced (UCLA DNA
Sequencing Facility, Los Angeles, Calif.) and confirmed to correspond
to the cDNA sequence for mouse myostatin.
Immunohistochemical analysis
Ten micrometer-thick longitudinal sections were cut from rat
muscles samples, transferred to microscope slides coated with 0.4%
gelatin and 0.04% chromium potassium sulfate, and stored at -20°C.
Prior to staining, slides were air dried for 30 min at room
temperature. Sections were then fixed in ice-cold acetone for 10 min,
air dried, and quenched in 0.3% hydrogen peroxide for 5 min. After
fixation, sections were blocked in a buffer containing 0.05% Tween-20,
0.02% gelatin, and 3% bovine serum albumin. Sections were incubated
either with or without polyclonal anti-myostatin overnight at 4°C.
Sections were then incubated with biotinylated anti-rabbit IgG (Vector
Laboratories, Burlingame, Calif.) and subsequently with horseradish
peroxidase avidin-D (Vector Laboratories) for 30 min each. Slides were
washed three times 5 min in phosphate-buffered saline after each
incubation. Slides were developed using 3-amino-9-ethyl carbazole (AEC,
red) as substrate.
A cross-sectional area of plantaris muscles was measured by counting
the number of intercepts of vertical and horizontal lines in a square
grid of an eyepiece micrometer that overlays muscle tissue when viewed
with a 10x objective. Each intercept of the grid corresponded to 0.33
µM (2)
when viewed at this magnification. All sections
used for analysis were taken from the thickest portion of the belly of
each plantaris muscle.
Statistical analysis
The significance of differences between experimental and control
samples was determined using one-way analysis of variance, with the
confidence limit set at P<0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The concentration of myostatin mRNA was 17-fold greater in plantaris
than in soleus (Fig. 2
). No myostatin mRNA was detected in nonmuscle tissues that were assayed
(Fig. 1)
.
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Protein samples obtained from the same tissues analyzed in Northern
blots were assayed in Western blots using polyclonal anti-myostatin
(Fig. 3
). Anti-myostatin recognized a single polypeptide in plantaris muscle at
~35 kDa and a single polypeptide in soleus muscle at ~37 kDa. As
expected, these masses correspond to the size of the latent myostatin
monomer in reduced form. Similar to the distribution pattern of
myostatin mRNA, the concentration of myostatin protein was 2.5-fold
greater in plantaris as compared to soleus. Because higher levels of
myostatin mRNA and protein expression were observed in plantaris
muscle, it was chosen for all subsequent analyses. No myostatin protein
was detected in nonmuscle tissues that were assayed (Fig. 3)
.
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Myostatin expression is modulated during modified muscle use
The decrease in plantaris muscle wet mass that occurred during
hind limb unloading (-16%) (Table 1
) was accompanied by changes in the levels of myostatin mRNA and
myostatin protein concentrations (Figs. 4
,5
,6
,7
). The reduction of muscle cross-sectional area during unloading (-28%)
was more than sufficient to account for muscle mass loss during the
unloading period and indicates that muscle shortening did not
contribute substantially to the mass losses observed. The 10 day period
of hind limb unloading resulted in a significant increase (+110%) in
myostatin mRNA concentration with a smaller, significant increase in
myostatin protein levels (+37%). Four days of reloading were
sufficient to return plantaris mass (Table 1)
and myostatin mRNA and
protein concentrations (Figs. 4
,5
,6
,7)
to values that were not
statistically different from ambulatory controls.
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Periodic loading during the period of hind limb suspension diminished
or eliminated the changes in plantaris muscle mass and myostatin mRNA
concentration that occurred during hind limb unloading. Those animals
experiencing periodic loading showed no significant difference in
plantaris muscle mass compared to ambulatory controls (Fig. 8
). The concentration of myostatin mRNA in the plantaris muscles of
animals experiencing periodic loading was 55% greater than ambulatory
controls and significantly less than the 110% increase that occurred
in animals that experienced unloading for 10 days without periodic
loading (Fig. 5
and Fig. 9
).
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Muscle unloading increases myostatin concentration at myotendinous
junctions
Myostatin protein was found to be concentrated at the myotendinous
junction (MTJ) in ambulatory control animals (Fig. 10A, B
). A similar distribution of myostatin protein was observed in tissue
samples from animals subjected to hind limb unloading and/or reloading
(Fig. 10D, E
). However, myostatin protein concentration at
the MTJs of unloaded muscles was detectably greater than in ambulatory
controls or animals experiencing reloading (Fig. 10G, H
).
Myostatin was not observed in periodic bands at the lateral surface of
the fibers, called costameres, where proteins enriched at MTJs are
frequently observed (15)
.
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DISCUSSION |
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. Note
that developmental events including satellite cell activation,
formation of new myotubes, and renewed expression of genes that are
normally expressed during development also occur with reloading
(16)
, when myostatin expression decreases. This suggests
that similar mechanisms may underlie myostatin’s influence on muscle
mass in developing and fully differentiated tissue. Although the results presented here show that changes in muscle mass are accompanied by reciprocating changes in myostatin expression, the occurrence of significant elevations of myostatin expression during modified muscle loading is not sufficient to cause significant loss of muscle mass. This conclusion is supported by the finding that animals subjected to prolonged periods of muscle unloading with intermittent muscle loading experienced no significant loss of muscle mass, but showed significant increases in myostatin concentration. Thus, either the increase in myostatin expression that occurs in unloaded muscle subjected to intermittent loading is not sufficient to produce detectable mass loss or other variables override the potential regulatory influence of myostatin on muscle mass during modified muscle use.
Recent findings (5)
showing that myostatin-immunoreactive
protein is present at higher concentrations in the serum and muscle of
HIV-infected men has suggested that myostatin may also play a role in
the pathological wasting of muscle. However, the relationship between
myostatin-immunoreactive protein and myostatin has not yet been
established. The antibody recognizing myostatin-immunoreactive protein
was generated against a peptide present in the mature or active domain
of myostatin, and was shown to bind to 26 kDa polypeptide in
immunoblots. However, SDS-PAGE under reducing conditions of murine
myostatin expressed in CHO cells shows the active myostatin peptide
migrates at a mass of ~15 kDa (1)
. It has been
speculated that the mass discrepancy between myostatin and
myostatin-immunoreactive protein may be due to differential processing
and glycosylation of the human form of the protein (5)
.
Alternatively, anti-myostatin immunoreactive protein may recognize a
nonmyostatin protein. For example, the peptide used to generate
anti-myostatin-immunoreactive protein is highly homologous to a
sequence in mature GDF-11, another TGF-ß superfamily protein
(17)
. Although the presence of GDF-11 in skeletal muscle
has not yet been confirmed, it is expressed in a number of other
tissues that may potentially secrete the protein into serum, thereby
creating the possibility of cross-reactivity with
anti-myostatin-immunoreactive protein.
The antibody used in the current study was generated against a unique
peptide present in the precursor, or latent domain, of myostatin.
Although it was anticipated that the antibody used in the present
investigation would recognize both the latent domain of cleaved
myostatin at 37 kDa and the intact precursor at 52 kDa, only a 37 kDa
polypeptide was identified. This may indicate that the intact precursor
is not present in the tissue or, more likely, that sample processing
for Western analysis dissociated the intact complex. Dissociation of
active and latent peptides of other members of the TGF-ß superfamily
by chaotrophic agents such as SDS has been demonstrated previously
(18
, 19)
. We have been unable to confirm this speculation
experimentally because the anti-myostatin used here appears to
recognize the antigen only under denaturing and reducing conditions; it
has not been used successfully for immunoprecipitation or affinity
chromatographic isolation of either the latent or intact myostatin.
Presumably, the binding of the antibody to myostatin in tissue sections
occurs because acetone fixation and peroxide treatment denature the
protein and thereby expose the epitope to which the antibody binds.
The higher concentration of myostatin mRNA (ref 6
; present
investigation) and myostatin protein (present investigation) in
fast-twitch muscle than in slow twitch muscle indicates that
myostatin’s function is likely to be more prominent in fast muscle. In
addition, the present finding that the molecular mass of myostatin
differs in plantaris and soleus muscles, suggests that there may be
fiber type-specific, posttranslational modifications in myostatin that
relate to fiber type-specific functions. Modifications in myostatin
glycosylation may underlie the observed mass differences because
glycosylation is a common posttranslational modification in TGF-ß
superfamily members (20
, 21)
. An intriguing hypothesis put
forward by Carlson et al. (6)
that may relate to the
elevated expression in fast-twitch muscles is that myostatin may
function as an inhibitor of satellite cell proliferation. This
speculation is supported by several previous observations that include
1) myostatin null mutants display muscle hypertrophy and
hyperplasia (1)
, both of which may relate to elevated
satellite cell activity, 2) hind limb unloading causes
suppression of satellite cell proliferation (22)
, which
would coincide with increased myostatin expression, and 3)
fast-twitch muscles have lower concentrations of satellite cells
(23)
, which would coincide with the higher level of
expression of myostatin in fast muscle. If myostatin is found to
function in regulating satellite cell proliferation and activity, the
protein’s elevated concentration at MTJs, where satellite cells are
also present in high numbers (24)
, may reflect that
function.
An additional possibility that may underlie the elevated
concentration of myostatin at MTJs is that this may reflect the
elevated concentration of myostatin ligands at these sites. Proteins in
the TGF-ß superfamily are typically stored in an inactive form in the
extracellular matrix (ECM) after their processing and secretion
(25)
. Release of these proteins from the ECM is thought to
lead to their activation. For example, TGF-ß1 is bound in an inactive
form to collagen IV and fibronectin and is activated upon release
(19
, 26
, 27)
. The interaction between TGF-ß1 and the ECM
is mediated by LTBP (latent TGF-ß1 binding protein), which associates
with the latent domain of TGF-ß1 via disulfide bonds and the ECM via
other covalent interactions (19
, 27)
. The structural
similarities between other TGF-ß superfamily proteins and myostatin
suggests that a similar regulatory mechanism may exist for myostatin.
If myostatin binds the basement membrane in a manner similar to
TGF-ß1, one would expect to see concentration of the protein at the
MTJ due to folding of the basement membrane at this site. Additional
studies in this area will contribute to understanding the regulation of
myostatin activity.
The results of the current study support the hypothesis that myostatin
plays a role in the loss of skeletal muscle mass due to unloading.
However, previous investigations have provided data to show that other
processes may also function in an important way in the loss of muscle
mass. For example, Ca2+-dependent proteases,
lysosomal proteases, and the ATP-ubiquitin-proteasome pathway can also
contribute to proteolysis induced by muscle unloading. A period of 2–3
days of hind limb unloading is sufficient to induce an 8.5% loss in
total protein concentration of soleus muscle that may be attributable
to Ca2+-dependent proteases (28)
. In
addition, the concentration of lysosomal proteases increases during
muscle unloading (29)
, although it is reported that
calpain concentrations may increase (29)
or not change
significantly during 10 days of unloading (30)
. When
soleus muscle is excised from a rat after 9 days of suspension and
subsequently incubated with or without inhibitors of lysosomal and
Ca2+-dependent proteases for 2 h, it appears
that only 18% of the overall proteolysis observed can be attributed to
lysosomal and Ca2+-dependent mechanisms
(29)
. This finding indicates that other mechanisms must be
contributing to the majority of proteolysis and atrophy observed during
muscle unloading. Although the significant increase in transcription of
the proteasome subunits involved in the ATP-ubiquitin-proteasome
pathway that occurs within 9 days of unloading suggests that the
proteasome pathway may contribute to muscle mass loss, it has not been
demonstrated that this increase in mRNA results in increased
proteolysis in this model (29)
.
The possibility that myostatin may modulate the expression or activity
of proteases has not been examined, although this would provide a
straightforward explanation for its proposed role in regulating muscle
mass. This potential role in regulating the activity or expression of
proteases would be consistent with known functions of TGF-ß, which
can modulate the expression of proteases and their inhibitors
(31
, 32)
. Alternatively, myostatin may play a broader role
in regulating protein balance in muscle by exerting more general
controls on translation or transcription, either directly or
indirectly, through modulating the expression of growth factors. This
latter possibility is also consistent with current knowledge of TGF-ß
functions (33)
. Continuing studies are directed at
identifying through which of these mechanisms myostatin may function in
regulating muscle mass during unloading.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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S. Reisz-Porszasz, S. Bhasin, J. N. Artaza, R. Shen, I. Sinha-Hikim, A. Hogue, T. J. Fielder, and N. F. Gonzalez-Cadavid Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin Am J Physiol Endocrinol Metab, October 1, 2003; 285(4): E876 - E888. [Abstract] [Full Text] [PDF]
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 S. McCroskery, M. Thomas, L. Maxwell, M. Sharma, and R. Kambadur Myostatin negatively regulates satellite cell activation and self-renewal J. Cell Biol., September 15, 2003; 162(6): 1135 - 1147. [Abstract] [Full Text] [PDF]
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 E. J Stevenson, P. G Giresi, A. Koncarevic, and S. C Kandarian Global analysis of gene expression patterns during disuse atrophy in rat skeletal muscle J. Physiol., August 15, 2003; 551(1): 33 - 48. [Abstract] [Full Text] [PDF]
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K. Ma, C. Mallidis, S. Bhasin, V. Mahabadi, J. Artaza, N. Gonzalez-Cadavid, J. Arias, and B. Salehian Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression Am J Physiol Endocrinol Metab, August 1, 2003; 285(2): E363 - E371. [Abstract] [Full Text] [PDF]
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C. D. McMahon, L. Popovic, J. M. Oldham, F. Jeanplong, H. K. Smith, R. Kambadur, M. Sharma, L. Maxwell, and J. J. Bass Myostatin-deficient mice lose more skeletal muscle mass than wild-type controls during hindlimb suspension Am J Physiol Endocrinol Metab, July 1, 2003; 285(1): E82 - E87. [Abstract] [Full Text] [PDF]
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 S. M. Roth, G. F. Martel, R. E. Ferrell, E. J. Metter, B. F. Hurley, and M. A. Rogers Myostatin Gene Expression Is Reduced in Humans with Heavy-Resistance Strength Training: A Brief Communication Experimental Biology and Medicine, June 1, 2003; 228(6): 706 - 709. [Abstract] [Full Text] [PDF]
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 J. J. Hill, Y. Qiu, R. M. Hewick, and N. M. Wolfman Regulation of Myostatin in Vivo by Growth and Differentiation Factor-Associated Serum Protein-1: A Novel Protein with Protease Inhibitor and Follistatin Domains Mol. Endocrinol., June 1, 2003; 17(6): 1144 - 1154. [Abstract] [Full Text] [PDF]
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 B. D. Rodgers, G. M. Weber, K. M. Kelley, and M. A. Levine Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae; short-term fasting elevates Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2003; 284(5): R1277 - R1286. [Abstract] [Full Text] [PDF]
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 B. W. C. Rosser and E. Bandman Heterogeneity of protein expression within muscle fibers J Anim Sci, February 1, 2003; 81(14_suppl_2): E94 - 101. [Abstract] [Full Text] [PDF]
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 M. P. Spiller, R. Kambadur, F. Jeanplong, M. Thomas, J. K. Martyn, J. J. Bass, and M. Sharma The Myostatin Gene Is a Downstream Target Gene of Basic Helix-Loop-Helix Transcription Factor MyoD Mol. Cell. Biol., October 15, 2002; 22(20): 7066 - 7082. [Abstract] [Full Text] [PDF]
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F. W Booth, M. V Chakravarthy, and E. E Spangenburg Exercise and gene expression: physiological regulation of the human genome through physical activity J. Physiol., September 1, 2002; 543(2): 399 - 411. [Abstract] [Full Text] [PDF]
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P.-Y. Rescan, I. Jutel, and C. Ralliere Two myostatin genes are differentially expressed in myotomal muscles of the trout (Oncorhynchus mykiss) J. Exp. Biol., March 12, 2002; 204(20): 3523 - 3529. [Abstract] [Full Text] [PDF]
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K. Ma, C. Mallidis, J. Artaza, W. Taylor, N. Gonzalez-Cadavid, and S. Bhasin Characterization of 5'-regulatory region of human myostatin gene: regulation by dexamethasone in vitro Am J Physiol Endocrinol Metab, December 1, 2001; 281(6): E1128 - E1136. [Abstract] [Full Text] [PDF]
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T. J. Marcell, S. M. Harman, R. J. Urban, D. D. Metz, B. D. Rodgers, and M. R. Blackman Comparison of GH, IGF-I, and testosterone with mRNA of receptors and myostatin in skeletal muscle in older men Am J Physiol Endocrinol Metab, December 1, 2001; 281(6): E1159 - E1164. [Abstract] [Full Text] [PDF]
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C. Huet, Z.-F. Li, H.-Z. Liu, R. A. Black, M.-F. Galliano, and E. Engvall Skeletal muscle cell hypertrophy induced by inhibitors of metalloproteases; myostatin as a potential mediator Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1624 - C1634. [Abstract] [Full Text] [PDF]
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P. O. Mitchell and G. K. Pavlath A muscle precursor cell-dependent pathway contributes to muscle growth after atrophy Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1706 - C1715. [Abstract] [Full Text] [PDF]
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W. E. Taylor, S. Bhasin, J. Artaza, F. Byhower, M. Azam, D. H. Willard Jr., F. C. Kull Jr., and N. Gonzalez-Cadavid Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells Am J Physiol Endocrinol Metab, February 1, 2001; 280(2): E221 - E228. [Abstract] [Full Text] [PDF]
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