The temporal expression of cellular oncogenes in ...

Biochemical Society Transactions ( 1 993) 21

The ternpod expression of eellulrr oncogenes in m e d m i d l y stimulated muscle. NICOLA J. OSBALDESTON, DAVID M. LEE, VALERIE M. COX, LINDA EAVES,JOHN F.J.MORRISON, JOHN HESKETH' & DAVID F. OOLDSPINK. Department of Clinical Medicine, Unimity of Leeds, Leah, LS2 9JT, U.K. *TheRowelt Research Institute, Bucksburn, Aberdeen, AB2 9SB, U.K.

The cellular omoge!nef4,cyos and c-jm, are associated with early signals fm hypertrophic or mitogenic growth (1) and with the biochemical signalhgmedmwma ' whereby hormones or growth factors control gene expression (2). Fos and jun products ,fmmthehetemdkaiC l U l i D 'l h tianscsiption factor AP-1. The expression of these early mponse genes is low in postmitotic striated muscle, but can be induced within a few hours by mechanical stimuli,e.g. pressure overloadmg the heart (3), increasing c~onary perfusion pressure ( 4 , tenotomy of a fu~~tional synergistic muscle or administration of p-agonists (5). In this study skeletal muscle was subjected to varying mechanical stimuli i.e. electrical stimulation, stretch or a combination of the two.The mRNA levels of c-fos and c-jun were measured using a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technique. Stretch was imposed upon the extensor digitorum longus (EDL), a typical fast-twitch muscle, of adult Dutch rabbits by immobilising one of the hind-limbs in plantar flexion using a plaster cast. Electrical stimulation of the EDL was achieved via the peronealnerw. Conhuous impulses of lo& t'recIuency welt delivered to the muscle using an external stimulator. A cambination of these two regimes was used in a third experimental p u p . These stimuli were maintained for varying time periods up to 24 hours. Contralakral muscles served as internal controls, whilst external controls were provided by shamoperated QT non-operated animals. The animals were killed and the muscle samples immediately dissected out and fiozen in liquid nitrogen. Total RNA was extracted from the muscle according to the methcd of Chomczynski and Sacchi (6). 2pg m p l e s of total RNA were used for reverse transcription. The multmg cDNA samples were subsequently amphfkd over 24 or 28 cycles of FCR using cdun or c-fos primers mpctively. Quantitation of the target cDNAs was achieved by using a serial dilution of either rat c-fos or vdun cDNA amplified alongside the samples.PCR products were run on a 2% agarose gel and the negative of a photograph of the gel was scanned using an LKB laser denaitometer. Reaction parameters were predetermined to emure that ampMation was within the exponential phase and that the absorbance values measured by the densitometer were within the linear range of the film. Stretching of the EDL induced peak expression of c-fos and cjun mRNAs after approximately 1 how, whilst electrical stimulation induced peak expression around 4.5 to 6 hours. A combination of the two regimes led to peak expression of these cellular oncogenes after 1 how, but with sigdicant increases in mRNA levels as early as 15 minutes following initiation of the stimuli (see Table 1.). Comparisons between the three trammg regimes clearly show that stretch alone inducea earlier and greater increases in ctfos and c-jun mRNA levels than electrical sthulation alone. are combined, peak c-fos mRNA However, when the re&% levels are further doubled compand to str:tch (Table 1.)

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Table 1. Peak expression of c-fos and cgun mRNA levels (expressed as fold inmasea compand to zeo-time controls) in re;.lponse to different training regimes. TlYUUllgreaUne E1ec.s25 fold?/6h cyos mRNA 25 fold?/6h c-pn mRNA

stretch 50 fold?/lh

35 fold?/lh

T&bd 140 fold?/lh Not done

Indeed, a combmation of stretch and low-electncal shulahon has been show to n b t e the transformahon of fasttwstch skeletal muscle mto slow twstch more rapxdly than eledncal &mulahon alone (7).

D.P., Sheng, M., Lester, F.L. & Gmnberg, M.E. (1989) Genes & De~el.3,304-3 13. 2. Kabn, P.& Graf, T. (eds.) (1986) Oncogenes, Growth & Control. Springer,Berh. 3. -0, S.,Nadal-Ginard, B. & Mahdavi (1988) PNAS U.S.A. 85,339-343. 4. Bauters, C., Moalic, J.M., Bercovici, J., Moms, C., EmanoilRaviers, R., schiffino, S., Swynghedauw, B. (1988) J. Mol. Cell. Cardiol. 20,97-101. 5. Whitelaw, P.F.& Hesketh, J.E. (1992) Biochem. J. 281, 143147. 6.Chomczydci, P. & sacchi,N. (1987) Anal.Biochem. 162, 156-159. 7. Goldspink, D.F., Eastem, J., Winterbum, S.K., Williams, P.E. & Goldspink, G.E. (1991) J. Cad. Surg. 6,218-224. 1. B-I,