The role of myostatin and the calcineurin-signalling pathway in regulating muscle mass in response to exercise training in the rainbow trout Oncorhynchus mykiss Walbaum
Gatty Marine Laboratory, School of Biology, University of St Andrews, St Andrews, Scotland, KY16 8LB, UK
* Author for correspondence (e-mail: iaj{at}st-andrews.ac.uk)
Accepted 15 March 2005
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Summary |
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Key words: rainbow trout, Oncorhynchus mykiss, exercise, muscle, hypertrophy, calcineurin, NFAT2, myostatin
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Introduction |
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Myostatin or growth/differentiation factor 8 (GDF8), a member of the
transforming growth factor-ß (TGF-ß) superfamily, is a negative
regulator of skeletal muscle mass in higher vertebrates
(McPherron et al., 1997). In
myostatin null mice and three breeds of cattle lacking a functional myostatin
protein, a hypermuscular phenotype was observed, the increase in muscle mass
occurring through increased fibre number and muscle fibre hypertrophy
(Kambadur et al., 1997
;
McPherron and Lee, 1997
;
Bass et al., 1999
;
Thomas et al., 2000
; reviewed
by Kocamis and Killefer,
2002
). In studies on humans, myostatin mRNA expression was
downregulated by 37% (Roth et al.,
2003
) and protein concentration was reduced by 20%
(Walker et al., 2004
), in
response to periods of resistance training.
Forced exercise is a powerful stimulus for skeletal muscle hypertrophy in
teleosts (Walker and Pull,
1973; Johnston and Moon,
1980a
,b
;
Totland et al., 1987
).
Comparative studies of the structure of myostatin in teleosts and mammals
indicate a high degree of sequence identity
(McPherron and Lee, 1997
;
Roberts and Goetz, 2003
).
Inhibition of myostatin in transgenic zebrafish Danio rerio Hamilton
resulted in a 20% increase in fast muscle fibre number
(Xu et al., 2003
). Zebrafish
D. rerio embryos treated with a myostatin specific morpholino
exhibited a marked upregulation of myogenic regulatory transcription factors
MyoD and myogenin (Amali et al.,
2004
). These findings imply a pivotal role for myostatin during
embryonic myogenesis in teleosts. In addition, myostatin mRNA expression is
affected by fasting in larval and adult stages of tilapia Oreochromis
mossambicus Peters (Rodgers et al.,
2003
). However, the role of myostatin in regulating muscle mass
during exercise in adult fish has not been investigated.
The main aim of the present study was to use exercise training to investigate the molecular signalling pathways regulating muscle fibre hypertrophy. Specifically, we wanted to test the hypothesis that myostatin and proteins associated with the calcineurin-signalling pathway show altered expression in response to exercise training, consistent with their having a major role in regulating muscle fibre hypertrophy, as is the case in mammals.
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Materials and methods |
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Experimental design
On the basis of fork length (FL), fish were divided into three
groups of 10: TR (tank rested), E15 (slow endurance exercise group
at 15 cm s-1 or 0.8 BL s-1) and E30
(moderate endurance exercise group at 30 cms-1 or 1.6 BL
s-1). The mean FL of the groups was 18.4±0.4 cm
(TR), 18.0±0.3 cm (E15) and 18.2±0.5 cm
(E30) [N=10, mean ± standard error of the mean
(S.E.M.)]. Initial body mass
(Mb) was not measured to minimise the stress experienced
by experimental animals. The exercise groups were placed into the two separate
swimming channels of a purpose-built flume
(Martin, 2003). The TR group
was maintained in a separate 1 m diameter holding tank. Fish were acclimated
to flume conditions at water flow velocities below 5 cm s-1. After
several days the water flow was increased gradually to 15 and 30 cm
s-1 in either swimming channel. Fish were trained continuously for
18 h per day. Experimental animals were fed to satiation once daily during the
6 h rest period. Experimental groups had access to food for the same length of
time each day. Fish were exercised for a period of 30 days. During the
experiment, four fish in the E30 group and one fish from each of
the TR and E15 groups were killed, because of their poor condition,
by a sharp blow to the head. On day 30, all fish from the exercise experiment
were killed, using an overdose of anaesthetic (MS222, tricane
methanesulphonate). The groups were sampled immediately after the last period
of exercise training, the fish were placed on ice after death and all tissue
samples were collected within 5-30 min of death. FL and
Mb were measured and used to calculate condition factor
(CF): CF=100(Mb/FL3).
Protein extraction and SDS-PAGE
Fast muscle tissue samples (500 mg) were dissected from a region above the
lateral line at the dorsal fin. Rodents were killed by a sharp blow to the
head and spinal cord section, and the limb extensor muscles sectioned. Salmon
S. salar were killed by a sharp blow to the head and fast muscle
tissue samples (500 mg) were taken as in the trout O. mykiss. Tissue
samples were placed in individual cryovials (Bibby Sterilin, Tilling Drive
Stone, Staffs, UK), snap-frozen in liquid nitrogen and stored at -80°C.
Total cellular protein extracts (Maniatis
et al., 1989) and nuclear protein extracts
(Blough et al., 1999
) were
prepared from individual samples. The Lowry assay determined the protein
concentration in each extract (Lowry et
al., 1951
). For SDS-PAGE, 20 µg of total cellular protein
extracts and 5 µg of nuclear protein extracts were loaded per lane. Each
sample was loaded in triplicate and averaged to give a mean optical density
for the concentration of a particular protein in an individual fish.
The discontinuous Laemmli system
(Laemmli, 1970) was used for
the electrophoresis of proteins. 6%, 12% and 15% resolving gel concentrations
were used. 1 mm thick gels were cast using Mini-Protean III apparatus (Bio-Rad
Laboratories Ltd., Hemel Hempstead, Herts, UK). The molecular masses of
calcineurin catalytic subunit A (CnA), calcineurin regulatory subunit B (CnB)
and myostatin were determined using Cruz Marker standards (Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA), the molecular mass of NFAT2 using
high-range biotinylated standards (Bio-Rad Laboratories Ltd). Standard
electrophoresis conditions were used: 100 V (constant), 30-45 mA (variable).
Resolving gels were stained with Coomassie Blue
(Fazekas de St Groth et al.,
1963
) to compare protein loading and photographed using the
Versadoc 3000 imaging system (Bio-Rad Laboratories Ltd).
Western blotting
Proteins were transferred to PVDF membranes (Hybond-P, Amersham Pharmacia
Biotech., Little Chalfont, Bucks, UK) using a standard technique
(Towbin et al., 1979) and Mini
Trans-Blot Cell (Bio-Rad Laboratories Ltd). Membranes were incubated in
blocking solution (5% milk powder in PBT: phosphate-buffered saline + 0.1%
Tween 20) for 1 h at room temperature (RT) to block non-specific sites. Rabbit
polyclonal antibodies were screened against nuclear protein extracts from
mammalian positive controls and rainbow trout (O. mykiss), and total
protein extracts from Atlantic salmon (S. salar). All antibodies were
diluted in blocking solution. The CnA (sc-9070 H-209, Santa Cruz Biotechnology
Inc.), CnB (PC-359, Calbiochem Inc., Merck Biosciences Ltd, Nottingham, Notts,
UK) and NFAT2 (sc-1149-R K18-R, Santa Cruz Biotechnology Inc.) antibodies each
positively detected a single protein of the expected molecular mass (55 kDa,
19 kDa and 103 kDa, respectively) (Fig.
1A-C). Øivind Andersen (Akvaforsk, PO Box 5010, Aas,
Norway) donated an antibody specific for a 16 amino acid region (347-362) of
the Atlantic salmon (S. salar) myostatin mature peptide. The
myostatin antibody positively detected three proteins: the myostatin precursor
(PC, 53 kDa), latency associated peptide (LAP, 40 kDa) and myostatin mature
peptide (M, 17 kDa monomer) (Fig.
1D). Primary antibodies were used at 1:500 (NFAT2), 1:1000 (CnA),
1:4000 (CnB), and 1:20,000 (myostatin). Following primary antibody incubation
(4 h at RT), membranes were washed in PBT for 30 min. A horseradish peroxidase
(HRP)-conjugated goat anti-rabbit IgG (sc-2030, Santa Cruz Biotechnology Inc.)
and ExtrAvidin HRP-conjugated secondary antibody (E2886, Sigma-Aldrich,
Dorset, UK) were used at 1:1000 dilutions to detect conjugated primary
antibodies and markers. Following secondary antibody incubation (1 h at RT),
membranes were washed in PBT for 1 h. Proteins were visualised using the
Immun-Star HRP Chemiluminescence kit (Bio-Rad Laboratories Ltd) and
photographs taken using the Versadoc 3000 Imaging System (Bio-Rad Laboratories
Ltd). The optical density and molecular mass of proteins were calculated using
Quantity One (v. 4.4.1) image analysis software (Bio-Rad Laboratories
Ltd).
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Muscle fibre cellularity
A 5 mm thick steak was dissected from each fish immediately anterior to the
anal fin. The steak outline was traced onto an acetate sheet for measurement
of the total muscle cross-sectional area (TCSA). The right
hand side of the myotome was divided into four blocks of tissue. Each tissue
block was placed on a cork tile, covered in cryomatrix, snap frozen in liquid
nitrogen cooled isopentane (-159°C) and stored at -80°C. Transverse
serial cryostat sections (7 µm) were cut at right angles to the long axis
of the fish (Cryocut 1800, Reichert-Jung, Leica, Deerfield, IL, USA). Tissue
sections were stained with Mayer's Haematoxylin and photographed using a frame
capture camera (TK-F7300U, JVC Ltd, London, UK) connected to a light
microscope with a x10 objective (Laborlux S, Leitz, Rockleigh, NJ, USA).
1200 fast muscle cross-sectional areas (FCSA) were
measured per fish, from images of random fields of view, using Sigma Scan Pro
image analysis software (v. 5.0.0, SPSS Inc., USA). Muscle fibre diameter
(D) was expressed as the diameter of the equivalent circle from the
FCSA measurement:
D=2(FCSA/)0.5.
TCSA was measured using Sigma Scan Pro.
Statistical analysis
The distribution of the data was assessed using the Anderson-Darling test
of normality. Mb, TCSA and
FCSA were compared by analysis of covariance (ANCOVA) and
Tukey's tests (Zar, 1996),
using FL as the covariate. CF was analysed using a one-way analysis
of variance (ANOVA) and Tukey's tests. Non-parametric smoothing and
bootstrapping techniques were employed to compare distributions of muscle
fibre diameters between experimental groups, using an in-house statistical
program written in the open source software R (v. 1.4.1, R Foundation for
Statistical Computing, Vienna, Austria). The statistical methods used are
described in Johnston et al.
(1999
). Muscle fibre diameter
distributions were compared using a non-parametric Kolmogorov-Smirnov test. If
the distributions were significantly different, the 5th,
10th, 50th, 95th and 99th
percentiles of fibre diameter were calculated from the fitted curves and
compared using a Mann-Whitney Rank Sum test
(Zar, 1996
).
The group mean protein concentration (± S.E.M.) was reported as the percentage of the TR group. Mean optical densities were compared using one-way ANOVA and Tukey's tests (or the non-parametric equivalents). The correlation between mean FCSA and the concentration of a particular protein was assessed using a Spearman Rank Correlation. All statistical tests were performed using Minitab software (v 13.2, Minitab Inc., IA, USA) unless otherwise stated.
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Results |
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Fast muscle fibre growth
Compared with the TR group, mean FCSA relative to
FL was 24% and 30% higher in the E15 and E30
groups, respectively (Table 1; ANCOVA, N=24, P<0.001). The average probability density
of muscle fibre diameter of the TR and exercised groups fell outside the 100
bootstrap estimates of the exercised and TR groups combined
(Fig. 2A,B). The distributions
in exercised groups were shifted to the right relative to the TR group,
indicating a significant increase in fast fibre diameter in response to
exercise training (Kolmogorov-Smirnov, N=24, P<0.05).
Similar results were obtained for both exercised groups
(Fig. 2C). The 50th,
95th and 99th percentiles of fast fibre diameter were
significantly greater in the E15 group compared to the TR group
(Mann-Whitney Rank Sum Test, N=24, P<0.01 and
P<0.05, respectively; Table
2). The 5th to 99th percentiles of fast
fibre diameter were significantly greater in the E30 group compared
to the TR group (Mann-Whitney Rank Sum Test, N=24, P<0.01
and P<0.05, respectively; Table
2). These data indicate that the exercise-training regime used was
sufficient to induce a marked hypertrophy of fast muscle fibres.
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Calcineurin signaling
Nuclear localisation of CnA was 5% and 7% higher in the E15 and
E30 groups, respectively, compared to the TR group (Figs
3,
4A; one-way ANOVA,
N=24, P<0.001). CnB nuclear localisation was 8% higher in
both exercised groups relative to the TR group (Figs
3,
4B; Kruskal-Wallis test,
N=24, P<0.01). Increased CnA and CnB nuclear localisation
were significantly correlated with mean FCSA (Spearman
Rank Correlation, N=24, P<0.001). CnA and CnB nuclear
localisation was similar in the two exercised groups. The overall
concentration of both calcineurin subunits in total cellular extracts was
similar between groups (Figs 3,
4A,B).
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|
Nuclear localisation of NFAT2 was 8% and 10% higher in the TR group, relative to the E15 and E30 groups, respectively (Figs 3, 4C; one-way ANOVA, N=24, P<0.001). The nuclear localisation of NFAT2 protein was significantly inversely correlated with mean FCSA (Spearman Rank Correlation, N=24, P<0.05). The overall concentration of NFAT2 in total cellular extracts was 15% and 11% higher in the TR group, relative to the E15 and E30 groups (Figs 3, 4C; one-way ANOVA, N=24, P<0.001). The overall concentration of NFAT2 was significantly negatively correlated with mean fast FCSA (Spearman Rank Correlation, N=24, P<0.01). Nuclear localisation of NFAT2 and overall NFAT2 concentration were similar in the two exercised groups.
Myostatin expression
The overall concentration of the 53 kDa myostatin precursor protein (PC)
was reduced by 5-9% in the E15 and E30 groups, relative
to the TR group; however, the reduction was not significant. The 40 kDa
latency-associated peptide (LAP) concentration was also similar between groups
(Fig. 5A,B). The overall
concentration of the myostatin active peptide was reduced by 6-7% in the
E15 and E30 groups relative to the TR group
(Fig. 5A,B) (one-way ANOVA,
N=24, P<0.05). The overall myostatin active peptide
concentration was inversely correlated with mean FCSA
(Spearman Rank Correlation, N=24, P<0.05). The
concentration of the active peptide was similar in the two exercised
groups.
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Discussion |
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Calcineurin - a regulator of muscle fibre hypertrophy in teleosts?
The calcineurin signaling pathway is thought to be one of several
intracellular pathways in higher vertebrates that regulate hypertrophic growth
of skeletal muscle (Musaro et al.,
2001). In vivo calcineurin inhibition prevented
compensatory hypertrophy of the plantaris muscle in the rat hind limb,
stimulated by functional overload (Dunn et
al., 1999
). The co-localisation of calcineurin and NFAT2 proteins
has been demonstrated in subsets of myonuclei in mammalian myofibres and the
association is thought to synergistically initiate muscle-specific gene
expression and muscle fibre hypertrophy
(Musaro et al., 1999
;
Semsarian et al., 1999
). In
exercised rainbow trout O. mykiss, a slight but significant increase
in calcineurin nuclear localisation was observed relative to tank-rested
controls. Increased calcineurin nuclear localisation was significantly
positively correlated with mean FCSA, which implied that
calcineurin might regulate exercise-induced muscle fibre hypertrophy. However,
in rainbow trout nuclear localisation of NFAT2 was not associated with
exercise-induced muscle growth. Furthermore, overall NFAT2 protein
concentration was markedly reduced in response to exercise, in contrast to the
upregulation of NFAT2 mRNA observed with exercise in humans
(Hitomi et al., 2003
). A
persistent non-catalytic association between calcineurin and NFAT2 is required
to maintain NFAT2 in the myonuclei (Zhu
and McKeon, 1999
). Uncoupling of the calcineurin/NFAT2 complex or
rephosphorylation of NFAT2 by vigorous kinases such as glycogen synthase
kinase-3 (GSK-3) results in nuclear export and cessation of NFAT2 mediated
transcription (Beals et al.,
1997
). These data suggest that after the initial dephosphorylation
and nuclear translocation of the calcineurin/NFAT2 complex, the association
between these proteins was disrupted, leading to rephosphorylation and nuclear
export of NFAT2. Since nuclear localisation of the NFAT2 protein is required
to mediate transcription of muscle-specific genes, these results imply that
muscle fibre hypertrophy in rainbow trout O. mykiss is potentially
calcineurin dependent, but NFAT2 independent. Interestingly, the reduced
concentration of NFAT2 observed in response to exercise implies that this
transcription factor may fulfil an alternative role in teleost muscle.
The present study suggests that calcineurin plays some role in the regulation of muscle fibre hypertrophy. Further work, such as the pharmacological inhibition of calcineurin through administration of cyclosporin A or FK506, or morpholino knockdown experiments targeting a calcineurin substrate such as NFAT2, could provide stronger causal evidence for the involvement of calcineurin. Until that point, however, this experiment provides an interesting insight into a potential regulatory pathway.
Myostatin - a negative regulator of muscle growth in teleosts?
An inverse relationship between muscle mass and the overall concentration
of myostatin active peptide has been demonstrated in several studies with
mammals (Gonzalez-Cadavid et al.,
1998; Schulte and Yarasheski,
2001
; McMahon et al.,
2002
). Moreover, myostatin expression was significantly
upregulated in response to muscle damage and an atrophic stimulus
(Wehling et al., 2000
;
Peters et al., 2003
), but
downregulated in response to a hypertrophic stimulus
(Roth et al., 2003
;
Walker et al., 2004
). A
significant reduction of the myostatin active peptide concentration was
observed in the groups of rainbow trout that displayed marked exercise-induced
muscle fibre hypertrophy and this inverse relationship was significant.
However, it is questionable whether this relatively small reduction, of less
than one third that found in humans
(Walker et al., 2004
), could
on its own account for the degree of exercise-induced hypertrophy observed.
The double-muscled phenotype is only observed in three of the six breeds of
cattle that possess a functional mutation in the myostatin protein, which
suggests that dysfunction of one major gene may not entirely account for the
increase in muscle mass (reviewed by
Kocamis and Killefer, 2002
).
Similarly, inhibition of myostatin in transgenic zebrafish only resulted in a
20% increase in muscle fibre number (Xu et
al., 2003
).
In conclusion, it is likely that myostatin and the calcineurin signaling pathway play relatively minor roles in the intracellular signaling network regulating muscle fibre hypertrophy in this species and further work is required to elucidate the function of myostatin and calcineurin in adult fish.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amali, A. A., Lin, C. J.-F., Chen, Y.-H., Wang, W.-L., Gong, H.-Y., Lee, C.-Y., Ko, Y.-L., Lu, J.-K., Her, G. M., Chen, T. T. and Wu, J.-L. (2004). Upregulation of muscle-specific transcription factors during embryonic somitogenesis of zebrafish (Danio rerio) by knockdown of myostatin-1. Dev. Dyn. 229,847 -856.[CrossRef][Medline]
Bass, J., Oldham, J., Sharma, M. and Kambadur, R. (1999). Growth factors controlling muscle development. Domest. Anim. Endocrinol. 17,191 -197.[CrossRef][Medline]
Beals, C. R., Sheridan, C. M., Turck, C. W., Gardner, P. and
Crabtree, G. R. (1997). Nuclear export of NF-ATc enhanced by
glycogen synthase kinase-3. Science
275,1930
-1933.
Blough, E., Dinnen, B. and Esser, K. (1999). Extraction of nuclear proteins from striated muscle tissue. BioTechniques 26,202 -206.[Medline]
Dolmetsch, R. E., Lewis, R. S., Goodnow, C. G. and Healy, J. I. (1997). Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 386,855 -858.[CrossRef][Medline]
Dunn, S. E., Burns, J. L. and Michel, R. N.
(1999). Calcineurin is required for skeletal muscle hypertrophy.
J. Biol. Chem. 274,21908
-21912.
Fazekas de St Groth, S., Webster, R. G. and Daytner, A. (1963). Two new staining procedures for quantitative estimation of proteins on electrophoretic strips. Biochim. Biophys. Acta 71,377 -391.[CrossRef]
Gonzalez-Cadavid, N. F., Taylor, W. E., Yarasheski, K.,
Sinha-Hikim, I., Ma, K., Ezzat, S., Shen, R., Lalani, R., Asa, S., Mamita, M.,
Nair, G., Arver, S. and Bhasin, S. (1998). Organization of
the human myostatin gene and expression in healthy men and HIV-infected men
with muscle wasting. Proc. Natl. Acad. Sci. USA
95,14938
-14943.
Guerini, D. (1997). Calcineurin: Not just a simple protein phosphatase. Biochem. Biophys. Res. Comm. 235,271 -275.[CrossRef][Medline]
Hitomi, Y., Kizaki, T., Katsumura, T., Mizuno, M., Itoh, C. E., Esaki, K., Fujioka, Y., Takemasa, T., Haga, S. and Ohno, H. (2003). Effect of moderate acute exercise on expression of mRNA involved in the calcineurin signaling pathway in human skeletal muscle. IUBMB Life 55,409 -413.[Medline]
Johnston, I. A. and Moon, T. W. (1980a). Exercise training in skeletal muscle of brook trout (Salvelinus fontinalis). J. Exp. Biol. 87,177 -194.[Abstract]
Johnston, I. A. and Moon, T. W. (1980b). Endurance exercise training in the fast and slow muscles of a teleost fish (Pollachius virens). J. Comp. Physiol. 135A,147 -156.
Johnston, I. A., Strugnell, G., McCracken, M. and Johnstone,
R. (1999). Muscle growth and development in normal-sex-ratio
and all-female diploid and triploid Atlantic salmon. J. Exp.
Biol. 202,1991
-2016.
Kambadur, R., Sharma, M., Smith, T. P. L. and Bass, J. J.
(1997). Mutations in myostatin (GDF8) in double-muscled
Belgian Blue and Piedmontese cattle. Genome Res.
7, 910-915.
Kocamis, H. and Killefer, J. (2002). Myostatin expression and possible functions in animal muscle growth. Domest. Anim. Endocrinol. 23,447 -454.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Lowry, O. H., Rosebrough, N. J., Lewis Farr, A. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 19,265 -275.
Maniatis, T., Sambrook, J. and Fritsch, E. F. (1989). Molecular Cloning: A Laboratory Manual (2nd edn), pp. 18.38-18.39. New York: Cold Spring Harbour Laboratory Press.
Martin, C. I. (2003). The effect of exercise on muscle growth and muscle-specific gene expression in the common carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum). PhD thesis, University of St Andrews, p138.
McMahon, C. D., Popovic, L., Jeanplong, F., Oldham, J. M., Kirk, S. P., Osepchook, C. C., Wong, K. W. Y., Sharma, M., Kambadur, R. and Bass, J. J. (2002). Sexual dimorphism is associated with decreased expression of processed myostatin in males. Amer. J. Physiol. 284,E377 -E381.
McPherron, A. C., Lawler, A. M. and Lee, S.-J. (1997). Regulation of skeletal muscle mass in mice by a new TGF-ß superfamily member. Nature 387, 83-90.[CrossRef][Medline]
McPherron, A. C. and Lee, S.-J. (1997). Double
muscling in cattle due to mutations in the myostatin gene. Proc.
Natl. Acad. Sci. USA 94,12457
-12461.
Musaro, A., McCullagh, K. J. A., Francisco, J. N., Olson, E. N. and Rosenthal, N. (1999). IGF-1 induces skeletal muscle hypertrophy through calcineurin in association with GATA-2 and NF-ATc1. Nature 400,581 -585.[CrossRef][Medline]
Musaro, A., McCullagh, K., Paul, A., Houghton, L., Dobrowolny, G., Molinaro, M., Barton, E. R., Sweeney, H. L. and Rosenthal, N. (2001). Localized IGF-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat. Genet. 27,195 -200.[CrossRef][Medline]
Norrbom, J., Sundberg, C. J., Ameln, H., Kraus, W. E., Jansson,
E. and Gustafsson, T. (2004). PGC-1alpha mRNA expression is
influenced by metabolic perturbation in exercising human skeletal muscle.
J. Appl. Physiol. 96,189
-194.
Peters, D., Barash, I. A., Burdi, M., Yuan, P. S., Mathew, L.,
Friden, J. and Lieber, R. L. (2003). Asynchronous functional,
cellular and transcriptional changes after a bout of eccentric exercise in the
rat. J. Physiol. 553,947
-957.
Roberts, S. B. and Goetz, F. W. (2003). Myostatin protein and RNA transcript levels in adult and developing brook trout. Mol. Cell. Endocrinol. 210, 9-20.[CrossRef][Medline]
Rodgers, B. D., Weber, G. M., Kelley, K. M. and Levine, M. A. (2003). Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae; short-term fasting elevates. Am. J. Physiol. 284,R1277 -R1286.
Roth, S. M., Martel, G. F., Ferrell, R. E., Metter, E. J.,
Hurley, B. F. and Rogers, M. A. (2003). Myostatin gene
expression is reduced in humans with heavy-resistance strength training: a
brief communication. Exp. Biol. Med. (Maywood)
228,706
-709.
Schulte, J. N. and Yarasheski, K. E. (2001). Effects of resistance training on the rate of muscle protein synthesis in frail elderly people. Int. J. Sport Nut. Exerc. Metab. 11 Suppl.,S111 -S118.
Semsarian, C., Wu, M.-J., Ju, Y.-K., Marciniec, T., Yeoh, T., Allen, D. G., Harvey, R. P. and Graham, R. M. (1999). Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signalling pathway. Nature 400,577 -581.
Shaw, K. T.-Y., Ho, A. M., Raghavan, A., Kim, J., Jain, J.,
Park, J., Sharma, S., Rao, A. and Hogan, P. G. (1995).
Immunosuppressive drugs prevent a rapid dephosphorylation of transcription
factor NFAT1 in stimulated immune cells. Proc. Natl. Acad. Sci.
USA 92,11205
-11209.
Sugiura, R., Sio, S. O., Shuntoh, H. and Kuno, T. (2001). Molecular genetic analysis of the calcineurin signalling pathways. Cell. Mol. Life Sci. 58,278 -288.[Medline]
Thomas, M., Langley, B., Berry, C., Sharma, M., Krik, S., Bass,
J. and Kambadur, R. (2000). Myostatin, a negative regulator
of muscle growth, functions by inhibiting myoblast proliferation.
J. Biol. Chem. 275,40235
-40243.
Totland, G. K., Kryvi, H., Jodestol, K. A., Christiansen, E. N., Tangeras, A. and Slinde, E. (1987). Growth and composition of the swimming muscle of adult Atlantic salmon (Salmo salar L.) during long-term sustained swimming. Aquaculture 66,299 -313.[CrossRef]
Towbin, H., Staehelin, T. and Gordon, J.
(1979). Electrophoretic transfer of proteins from polyacrylamide
gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76,4350
-4354.
Walker, K. S., Kambadur, R., Sharma, M. and Smith, H. K. (2004). Resistance training alters plasma myostatin but not IGF-1 in healthy men. Med. Sci. Sports Exerc. 36,787 -793.[Medline]
Walker, M. G. and Pull, G. (1973). Skeletal muscle function and sustained swimming speeds in the coalfish Gadus virens L. Comp. Biochem. Physiol. 44A,495 -501.[CrossRef]
Wehling, M., Cai, B. and Tidball, J. G. (2000).
Modulation of myostatin expression during modified muscle use.
FASEB J. 14,103
-110.
Xu, C., Wu, G., Zohar, Y. and Du, S. J. (2003).
Analysis of myostatin gene structure, expression and function in zebrafish.
J. Exp. Biol. 206,4067
-4079.
Zar, J. H. (1996). Biostatistical Analysis (3rd edn), pp. 147-156, 353-369. London: Prentice-Hall International Inc.
Zhu, J. and McKeon, F. (1999). NF-AT activation requires suppression of Crm1-dependent export by calcineurin. Nature 398,256 -260.[CrossRef][Medline]