Proceedings of The Physiological Society

University of Central Lancashire (2002) J Physiol 543P, S237


Effect of calcineurin inhibition and MEK1/2 inhibition upon metabolic enzyme activities in primary cultures of rat skeletal muscle

J. Higginson*, H. Wackerhage*, N. Woods*, P. Schjerling†, A. Ratkevicius‡, N. Grunnet‡ and B. Quistorff‡

*Department of Biological Sciences, University of Central Lancashire, Preston PR1 2HE, UK, †Copenhagen Muscle Research Centre, Rigshospitalet, University of Copenhagen and ‡Department of Medical Biochemistry and Genetics, University of Copenhagen, Denmark

Little is known of the signal transduction pathways that control differential gene expression in fast and slow skeletal muscle fibres. Earlier studies indicated that both the calcineurin pathway (Chin et al. 1998) and the ERK1/2 pathway (Murgia et al. 2000) can increase the percentage of slow fibres in vivo. Here we investigated the effect of calcineurin blockade with cyclosporin A and ERK1/2 blockade with U0126 upon the enzyme activities in primary rat skeletal muscle culture. Donor animals were killed humanely under approval of the ethical committee of the University of Copenhagen. Cells were either not treated (control) or incubated with either 0.25 mM cyclosporin A or 10 mM U0126 (n = 4 for each group). The enzymes measured were creatine kinase (CK), lactate dehydrogenase (LDH), hexokinase (HEX), malate dehydrogenase (MDH), citrate synthase (CS), pyruvate dehydrogenase (PDH total and percentage active form) and β-hydroxyacyl-CoA dehydrogenase (HAD). Differences as a result of treatment (control, cyclosporin A, U0126, n = 4 for each group) were analysed using a dependent ANOVA (P < 0.05 was considered significant). Both CK and LDH activity were significantly increased following cyclosporin A treatment compared with control. Activities of CK, LDH, HEX, MDH and HAD were all significantly increased above control following U0126 treatment. The percentage of PDH in its active form showed a significant decrease with U0126 treatment. These results suggest that both the calcineurin/NFAT and ERK1/2 pathway control some but not all enzymes that are expressed in a fibre type-specific manner. We conclude that it is an oversimplification to assume that a single signal transduction pathway is capable of controlling each gene that is expressed in a fibre type-specific manner. Bioinformatic approaches or microarray studies will be needed to characterize the sets of genes that are regulated by signal transduction pathways and to understand the complexity of fibre type-specific gene regulation.

All procedures accord with current national guidelines.

Where applicable, experiments conform with Society ethical requirements