RT-PCR was used to measure c-fo.7 and c-jim mRNA levels as previously described [ 51. ... Osbaldeston, N.J., Lee, D.M., Cox, V.M , Eaves, L., Morrison,. 6.
Biochemical Society Transactions (1995) 23 3273 The effect of various stretch and electrical stimulation regimes on proto-oncogene induction in skeletal muscle NICOLA J. DAWES, DAVID M. LEE, VALERIE M. COX, HUIEN NGA and DAVID F. GOLDSPINK Muscle Research Unit, Dept. of Clinical Medicine, University of Leeds, Leeds LS2 9JT.
In intact, striated muscle, a variety of mechanical stimuli have been shown to lead to rapid and transient increases in the expression of c-fos, c-jun and other early response genes; examples include pressure overloading of the heart [I] and tenotomy of a hnctional synergistic muscle [2]. The c-Fos and c-Jun proteins are components of the heterodimeric mammalian AP- 1 transcription factor [3]. The cellular role of this complex is in transcriptional regulation as an adaptive response to external stimuli transduced via second messengers [4]. In order to investigate the roles of fos and jrin cellular oncogenes in muscle cell signalling we have studied their expression under conditions known to induce either hypertrophy or alterations in gene expression. We have previously demonstrated that c-fos and c-jzin mRNA are transiently induced in rabbit latissimus dorsi (LD) in response to stretch and/or lOHz electrical stimulation [5]. In these initial studies, the LD was stretched to approximately 15% above its resting length using an inflatable tissue expander placed beneath the muscle. Here we show that the degree of stretch is correlated to the level of induction of the c-fos and c-jun message. In addition, we have compared the expression of c-fo.~and c-jiin mRNA in the LD in response to 2Hz and lOHz electrical stimulation. Either stretch or electrical stimulation was imposed upon the LD as described previously [5]. For the differential stretch regime, increasing volumes of saline were used to impose increasing degrees of stretch to the muscle, i.e., from about 8 to 20% above the resting level. In all cases, stretch was carried out for a period of 1 hour, since previous studies [5] had shown peak c-fos and c+in mRNA expression at this time point. Electrical stimulation (using 2Hz stimulators) was carried out for varying time periods up to 12 hours. Contralateral muscles served as internal controls whilst external control muscles were provided by non-operated or sham-operated muscles. Total RNA was extracted according to the method of Chomczynski and Sacchi [6]. RT-PCR was used to measure c-fo.7 and c-jim mRNA levels as previously described [51. Figure 1 shows the expression of c-fos (A) and c-jun (B) mRNA in response to differing degrees of stretch (i.e. varying volumes of saline in the expander). Both c-fos and c-jun mRNA levels increased from approximately 2- to 35-fold (when compared to external control muscles) as the degree of stretch was varied from around 8% (20ml saline) to around 20% (70mI saline). As with the temporal expression profiles seen previously [5], the pattern of c-fos and c-jun mRNA expression seen here illustrates simultaneous induction of the two genes. This is consistent with the formation of the AP-I transcription factor. The temporal expression of c-fos and c-jun mRNA in response to 2Hz electrical stimulation showed a very similar pattern to that previously demonstrated with 1OHz electrical stimulation [5]. Peak expression of both proto-oncogenes was seen after around 4.5 to 6 hours of stimulation, with fold increases of approximately 15- to 20-fold compared to external controls. It appears that increasing the degree of stretch imposed on the LD muscle leads to increased induction of c-fos and c-jzin mRNA. However, the patterns of c-fos and c-jun induction following electrical stimulation at two different frequencies ( 2 H z and 1OHz) showed identical expression profiles.
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Figure I . ExDression of c:fo.s (A) and c j u n (B) mRNA in LD subiected to differing degrees of stretch Hatched and open bars represent experimental and contralateral muscles respectively. This work was supported by the British Heart Foundation
1. Schunkert, H., Jahn, L., Izumo, S., Apstein, C.S. & Lorell, B.H. (1991) PNAS 88, 11480-11484 2. Whitelaw, P.F. & Hesketh, J.E. (1992) Biochem. J 281, 143-
I47 3. Curran, T. & Franza, B.R. (1988) Cell 55, 395-397 4. Kahn, P. & Graf, T. (eds.) Oncogenes and Growth Control. Springer, Berlin 5. Osbaldeston, N.J., Lee, D.M., Cox, V.M , Eaves, L., Morrison, J.F.J. & Goldspink, D.F (1993) J. Physiol. 473, 132P 6. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem 162, 156-159
