Animal Genomics, AgResearch Ltd., Ruakura Agricultural Centre, Hamilton 2001, New Zealand
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ABSTRACT |
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Myostatin inhibits skeletal muscle development. Therefore, we sought to determine whether larger body and muscle mass in male mice was associated with lower mRNA and protein expression of myostatin compared with females. Ten male and ten female mice of the C57 strain were killed at 16-18 wk of age, and their biceps femoris, gastrocnemius, and quadriceps femoris muscles were collected. Body and muscle masses were 40% heavier (P < 0.001) in males than in females. Northern analysis showed no difference in mRNA between males and females. In contrast, Western analysis showed that processed myostatin (26 kDa) was 40-60% lower (P < 0.001) in males compared with females. These data show first that decreased processed myostatin is a posttranscriptional and posttranslational event and, second, that decreased abundance of processed myostatin is associated with increased body mass and skeletal muscle mass in male compared with female mice.
sex; body mass
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INTRODUCTION |
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MYOSTATIN IS A
MUSCLE-SPECIFIC growth factor that inhibits muscle development
(15). Remarkable increases (2- to 3-fold) in skeletal
muscle occur in myostatin-null (mstn/
) mice
and in cattle with natural mutations in the myostatin gene that render
the protein inactive (11, 15, 16). Therefore, myostatin
may have a critical role in regulating the amount of muscle mass in individuals.
Myostatin is a 376-amino acid peptide in mice (52 kDa) belonging to the
transforming growth factor- (TGF-
) family and, as such, is
suggested to be secreted as a latent complex consisting of a 40-kDa
latency-associated peptide (LAP) that, after proteolytic cleavage at a
specific RSRR sequence (amino acids 263-266), is noncovalently
attached to the processed COOH-terminal portion, the size of which
varies in reports from 12.5 to 30 kDa, depending on species and
posttranslational modifications (6, 13, 21, 22). Based on
TGF-
, homodimers of LAP and processed myostatin protein are
noncovalently linked to form a latent complex (7, 17,
18). Activation enables processed myostatin to separate from LAP to stimulate activin type II receptors (13).
Male mammals are generally larger than females, and this sexual dimorphism (5) is attributed to the presence of androgens (1), insulin-like growth factor I (14) and growth hormone (23). However, given that myostatin inhibits muscle development, it is possible that myostatin also contributes to sexually dimorphic growth. If true, this would suggest that expression of myostatin is lower in skeletal muscles of males compared with those of females.
The purpose of the present study was to test the hypothesis that larger body and muscle mass in adult male mice is related to decreased expression of myostatin in skeletal muscles of male compared with female mice.
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METHODS |
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Animals.
Ten male and ten female C57 mice were maintained under a photoperiod of
14:10-h light-dark cycle and fed mouse chow (Diet 86, Sharps Grain and
Seed, Carterton, NZ). Mice were killed between 16 and 18 wk by
CO2 asphyxiation followed by cervical dislocation. At
death, mice were weighed, a sample of blood was collected via cardiac
puncture, and the following three hindlimb muscles were dissected free,
weighed, and then frozen in liquid nitrogen: biceps femoris,
gastrocnemius, and quadriceps femoris. Plasma was harvested and stored
at 20°C before analysis by Western blot. This study was approved by
the Ruakura Animal Ethics Committee.
Protein extraction and Western blot analysis.
One milliliter of lysis buffer (PBS, pH 7.2) with 0.5% IGEPAL
detergent (Sigma Chemical, St. Louis, MO) and an enzyme inhibitor (Complete, Roche Diagnostics NZ, Auckland, NZ) was added to 100 mg of
muscle from each animal. Samples were homogenized on ice and then
centrifuged at 11,000 g for 10 min. Supernatant was
recovered, mixed with Laemmli loading buffer (12), boiled
for 5 min, and then stored at 20°C until analysis. The protein
concentration of the supernatant was determined using the bicinchoninic
acid assay (Sigma Chemical).
RNA extraction and Northern blot analyses.
Frozen muscle samples were homogenized on ice in TRIzol reagent
(GIBCO-BRL, Gaithersburg, MD) for 30 s at 13,500 rpm using an
Ultra Turrax homogenizer. Debris was removed by centrifugation for 10 min at 10,000 g, and total RNA was isolated using the TRIzol protocol (GIBCO-BRL). RNA was resuspended in diethyl
pyrocarbonate-treated water, and the final concentration was determined
by measuring absorbance at 260 nm. Ten micrograms of total RNA were
separated in a 1.2% formaldehyde-agarose gel and then transferred to
an uncharged nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Auckland, NZ) by capillary action. Membranes were cross-linked using
ultraviolet irradiation and stained with methylene blue to verify the
uniformity of loading and transfer. The primers to generate
myostatin-specific DNA probe were 5'-GGTATTTGGCAGAGTATTGAT-3'and 5'-ATCTACTACCATGGCTGGAAT-3'. First-strand cDNA synthesis was performed using a Superscript II Pre-Amplification kit (GIBCO-BRL) and 5 µg of
total RNA from mouse skeletal muscle, according to the manufacturer's protocol. PCR was carried out with 2 µl of the reverse transcriptase reaction (94°C for 30 s, 55°C for 1 min, and 72°C for 1 min)
for 35 cycles and a final extension of 5 min at 72°C. Radiolabeled cDNA probes were prepared using [-32P]dCTP and the
Rediprime II Labeling Kit (Amersham Pharmacia Biotech) according
to the manufacturer's instructions. Membranes were prehybridized in
Church and Gilbert buffer for 2 h (0.5 M
Na2HPO4, pH 7.2, 7% SDS, 1 mM EDTA) at 55°C.
Membranes were then hybridized at 55°C overnight in fresh buffer with
the radiolabeled probe. After hybridization, they were washed at 55°C
for 15 min in each of 2× SSC-0.5% SDS and 1× SSC-0.5% SDS and
exposed against BioMax X-ray film (Eastman Kodak). The intensity of
each band was measured and analyzed as per Western blot analysis.
Statistical analysis. Data were subjected to t-tests and are presented as means ± SE or the pooled SE.
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RESULTS |
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Body and muscle masses were over 40% heavier (P < 0.001) in male than in female mice (Table
1). A representative Northern blot is
shown for biceps femoris, gastrocnemius, and quadriceps femoris muscles
from two male and two female mice (Fig.
1). There was no difference between sexes
in steady-state mRNA expression of myostatin, although there was a
tendency (P = 0.07) for lower expression of myostatin
mRNA in female gastrocnemius (Fig. 2). Three immunoreactive bands were identified for myostatin protein on
Western blots that corresponded to precursor, LAP, and processed myostatin. A representative Western blot is shown for biceps femoris, gastrocnemius, and quadriceps femoris muscles for three male and three
female mice (Fig. 3). Protein expression
of processed myostatin was consistently lower (P < 0.001) by 40-60% in all three muscles collected from males than
from females despite there being no difference in expression of LAP
(Fig. 4). Expression of precursor myostatin was more variable, being higher in male gastrocnemius and
quadriceps femoris muscles, but not different between sexes in biceps
femoris (Fig. 4). To determine whether the reduced abundance of
processed myostatin in muscles of males was associated with increased
secretion into blood, we measured processed myostatin in plasma by use
of Western blot analysis. A representative Western blot is shown for
processed myostatin in plasma (Fig.
5A), and abundance was not
different (P = 0.17) between sexes (Fig.
5B).
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DISCUSSION |
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We demonstrate here that increased body and muscle mass in male compared with female mice is associated with decreased expression of processed myostatin. Interestingly, this decreased expression of processed myostatin occurs after translation and, presumably, after secretion, because there was no difference in steady-state mRNA or in the LAP form of myostatin, the latter of which is cosecreted with the processed COOH-terminal fragment of myostatin in equimolar proportions.
This is the first report to our knowledge showing that processed myostatin is reduced in skeletal muscle of adult male compared with female mammals that exhibit sexually dimorphic growth. Others have reported that myostatin mRNA does not differ between sexes in humans (9) or in swine (10). Our data are consistent with these findings, but we also observed a tendency for lower mRNA in female gastrocnemius compared with male. This difference is apparent in the precursor form but does not affect the abundance of the LAP form of myostatin. There is also a higher abundance of the precursor form of myostatin in male quadriceps femoris, whereas there is no difference in the LAP form. These data suggest to us that there are other sex-related differences in transcription and translation of myostatin than processed myostatin, and their function is not immediately apparent.
At present, it is unclear what happens to processed myostatin after it
is secreted. On the basis of TGF- as the best studied member of this
superfamily, the processed moiety is proteolytically cleaved from LAP
in the Golgi apparatus and then recombines in a noncovalent manner with
LAP before secretion (2-4). The binding of the
processed form of myostatin to the LAP form and to large latent
TGF-
-binding proteins is crucial for correct folding and secretion
of TGF-
(7, 18). Therefore, the decrease in abundance of processed myostatin in muscle likely occurs after secretion and
could have resulted from a higher rate of secretion from muscles to
blood of males. One problem with this possibility is that processed myostatin circulates bound to the LAP form (24), and we
observed that the LAP form remained in muscle in similar abundance
between sexes, whereas the processed myostatin was 50% lower in
muscle. Therefore, without increased abundance of processed myostatin in blood, it is more likely that processed myostatin is degraded in
skeletal muscle at a higher rate in males than in females. We cannot,
however, rule out the possibility that processed myostatin is cleared
faster from blood of males than females. Whatever mechanism is
involved, a reduction in the abundance of processed myostatin in the
skeletal muscles of males suggests reduced bioactivity and, thereby,
reduced inhibition on myogenesis. It should be noted that although
myostatin-null mice have two to three times more muscle mass than
wild-type controls, males remain heavier than females
(15). Therefore, a reduction in myostatin does not
regulate sexual dimorphism per se. Rather, it may enable
anabolic factors to enhance growth. In this regard, sexually dimorphic
growth of males also requires insulin-like growth factor I
(14), growth hormone mediated by signal transducer and
activator of transcription 5b (23), and testosterone
(1).
Growth rates are similar for male and female mice up to 3 wk of age, after which time growth rates accelerate in males. Growth rates decrease in both sexes after 60 days and reach a plateau in females after 100 days, but males continue growing slowly from 100 to 200 days (data not recorded after 200 days) (8). Thus, at 16-18 wk (112-126 days), the age at which muscles were collected from mice in the present study, most growth had ceased. Therefore, it is not clear whether reduced processed myostatin is important for maintaining the greater absolute muscle mass in adult males or contributes to accelerated growth in male compared with female adolescent mice. Furthermore, increased muscle mass in males is relative to the increased skeletal frame rather than increased mass on the same-sized skeletal frame and is attributed to the greater duration and rate of growth compared with females before skeletal maturation (8, 20). These data suggest that reduced abundance of processed myostatin in male mice may play a role in sexually dimorphic growth and/or maintaining the greater muscle mass at maturity. We are currently examining the expression of myostatin during various stages of postnatal growth from birth to adulthood and through to old age in male and female mice.
In summary, skeletal muscles of adult male C57 mice have a reduced abundance of processed myostatin compared with females. We suggest that a reduced abundance of processed myostatin reflects reduced inhibition on myogenesis and contributes to the greater muscle mass in males compared with females.
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ACKNOWLEDGEMENTS |
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We thank Rick Broadhurst and the staff of the Small Animal Colony at AgResearch, Ruakura, for care and maintenance of mice during this study. We also thank Dr. Neil Cox for statistical advice.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. D. McMahon, AgResearch Ltd., Ruakura Agricultural Centre, Private Bag 3123, Hamilton, 2001 New Zealand (E-mail: chris.mcmahon{at}agresearch.co.nz).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 15, 2002;10.1152/ajpendo.00282.2002
Received 26 June 2002; accepted in final form 10 October 2002.
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