Download Immunolocalisation of proto-oncogene expression in mechanically

Biochemical Society Transactions (1995) 23 3293
lmmunolocalisation of proto-oncogene expression in
mechanically stimulated skeletal muscle.
DAVID M. LEE, NICOLA J. D A W S , VALERIE M. COX,
JOHN E. HESKETH' and DAVID F GOLDSPINK.
Muscle Research Unit, Dept. of Clinical Medicine, University of
Leeds, Leeds. LS2 9JT, U.K.
*The Rowett Research Institute, Greenburn Rd, Bucksbum,
Aberdeen, AB2 9SB, U.K.
The products of the proto-oncogenes c7fo.s, c-jiitr and c-myc
are likely to be involved as third messengers in one or more of the
adaptive responses induced by mechanical stimulation of skeletal
muscle. The basal expression of these immediate early genes is low
in post-mitotic striated muscle, but can be rapidly induced within
hours by mechanical stimuli, e.g. pressure overloading the heart
[I]. tenotomy of a hnctional synergistic muscle [2] or increased
neural activity and contractile work in skeletal muscle [3]. Fos and
Jun products form the heterodimeric mammalian transcription
factor AP-I [4]. The c-Myc protein is a nuclear phosphoprotein
which heterodimerises with Max [5]; this dimer has specific
properties including DNA binding and transcriptional activation,
suggesting a role in the regulation of gene expression.
In this study, the expression of these proto-oncogenes was
analysed in skeletal muscle subjected to conditions known to
induce hypertrophy or changes in gene expression. The rabbit
latissimus dorsi (LD), a predominantly fast-twitch muscle [6], was
subjected to either IOHz electrical stimulation or passive stretch.
The mRNA levels of c-mvc were measured using a Reverse
Transcription-Polymerase Chain Reaction (RT-PCR) technique.
Immunolocalisationof c-Fos and cJun proteins was carried out on
transverse sections of experimental muscles, using commercially
available antibodies in conjunction with a peroxidase-labelled
biotidavidin system.
IOHZ electrical stimulation or passive stretch of the LD was
carried out as previously described [ 3 ] . These stimuli were
maintained for varying time periods up to 24 hours. Contralateral
muscles served as internal controls, whilst external controls were
provided from non-operated animals. The animals were killed and
the LD samples immediately dissected out and frozen in liquid
nitrogen. Samples for immunocytochemistry were mounted
between blocks of liver and sectioned to 7pm prior to analysis.
Immuno-sections were pre-incubated in methanoI/H,O, to remove
endogenous peroxidase activity and non-specifically blocked in
10% horse serum. The sections were then incubated overnight at
1 O C with polyclonal antibodies against either c-Fos or c-Jun.
Total RNA was extracted from the muscle according to the
method of Chomczynski and Sacchi [7]. RT-PCR was carried out
as previously described [3] using c - m y primers and 29 cycles of
PCR amplification.
Electrical stimulation of the LD induced a broad expression
of c-nqr mRNG peaking after 4% hours, with an increase of
23*0 3 fold (n-3) over external controls. Following passive stretch
to the LD. c-rnjr mRNA levels were again seen to peak after
hours. *ith an increase of 28*0.6 fold (n=3)
approximately 1%
oker controls. A similar temporal profile of expression was seen
Hith c:fo..c and c-jritr mRNA following lOHz electrical stimulation
to the LD, although stretch to the LD induced an earlier and more
transient peak (after 1 hour) in c:fo.s and c-jrrtr message [3].
C-Fos and c-Jun immunoreactivity was barely detectable in
\ections of external control muscles (Fig. IA). Sections from
muscles which had undergone 1OHz electrical stimulation showed
c-Fos and c-Jun immunoreactivity predominately in non-myofibre
nuclei (Fig. IB). In contrast. sections from muscles which had been
passi\ely stretched showed strong c-Fos and c-Jun
immunoreactivity in nuclei inside the myofibres, as well as
interstitial cells (Fig. 1C). These staining patterns were abolished
when the primary antibodies were either replaced with nonimmune serum or pre-absorbed with their respective peptide
antigens (not shown).
These immunolocalisation studies suggest that the rapid
induction of c-Fos and cJun is an early event in response to
mechanical stretch and might trigger events leading to muscle fibre
hypertrophy. However, the involvement of AP-I in inducing the
phenotypic changes in muscle fibres as a result of electrical
stimulation is less clear.
Fig. 1 , lmmunolocalisation of c-Fos in mechanically stimulated LD.
Sections shown are of control (A), electrically stimulated (B)and
stretched (C) muscles. The section shown in (B) is from a muscle
which was stimulated for 6 hours (4%h & 8h showed the same
pattern). The section shown in (C) is from a muscle which was
stretched for 4% hours (3h and 6h showed the same pattern). Solid
arrows in (C) indicate nuclei within myofibres; the open arrow
shows an interstitial nucleus. Results for c-Jun showed the same
distribution pattern as those for c-Fos (not shown). Bar = 60pm.
This work was supported by the BBSRC and the BHF
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U.S.A 85,339-343.
2. Whitelaw, P. & Hesketh, J. (1992) Biochem. J. 281, 143-147.
3. Osbaldeston, N.J., Lee, D.M., Cox, V.M., Eaves, L., hlomson,
J.F.J. & Goldspink, D.F. ( 1993) J. Physiol. 473, I33P.
4. Curran, T. & Franza, B.R. (1988) Cell 55,395-397.
5 . Kato, G.J. & Dang, C.V. (1992) FASEB. J. 6,3065-3073.
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Goldspink, D.F. (1994) J.Anat. 185, 173-179.
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