Proceedings of The Physiological Society
Kings College London (2005) J Physiol 565P, PC158
Differential gene expression upon depolarization of skeletal muscle cells
Juretic, Nevenka ; Jaimovich, Enrique ; Munroe, David ; Riveros, Nora ;
1. Centro de Estudios Moleculares de la Celula, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile. 2. Laboratory of Molecular Technology, National Cancer Institute (NCI), Frederick, MD, USA.
It has been extensively reported that adaptive response of skeletal muscle to exercise or to electrical stimuli involves changes in specific gene expression, nevertheless the cellular mechanisms underlying muscle adaptation remain unknown (Pette, 1998). The slow calcium transient that follows membrane depolarization in skeletal muscle cells (Jaimovich et al. 2000; Araya et al. 2003) is involved in initial steps of signaling pathways leading to transcriptional activation of some early genes (Carrasco et al. 2003). To further explore on the subject we analyzed Interleukin-6 (IL-6) expression in K+ depolarized myotubes, since IL-6 has been reported as one of the genes differentially expressed when skeletal muscle is submitted to work overload (Carson et al. 2002). Myotubes in primary culture were obtained from neonatal rats (humanely killed; Jaimovich et al. 2000). Using semi quantitative RT-PCR, we have shown that K+-evoked depolarization induces a transient increase (357±19%, mean±SEM, n=3) in IL-6 mRNA levels, with a maximum at 3 h. Depolarization performed in the presence of slow calcium signals suppressors like U73122 (138±17%, n=3) and 2-aminoethoxydiphenyl borate (94±6%, n=3), resulted in decreased activation of IL-6 gene expression. These results suggest a regulatory role for the slow calcium signal on IL-6 gene transcription. To identify other genes that could be regulated by the same depolarizing stimuli we made use of oligonucleotide microarrays analysis (Compugen 22K oligomouse, 21.920 genes). Total RNA from C2C12 myotubes was obtained at different times after 5 min of high K+ stimulation. cDNA prepared from control and depolarized samples was labelled with cyanine 3 or 5 prior to microarray hybridization. Analysis (6 arrays for each time) was performed using NCI— Center for Information Technology of NIH Microarray Database tools. We focused our interest on 58 genes whose expression changed at least 2-fold up or down related to non-depolarized conditions. The main differences in the transcriptional response were observed for genes involved in metabolism, signal transduction and stress response. Evidence involving slow calcium signals in the expression of some of these selected genes could contribute to clarify the mechanisms that link changes in membrane potential and the adaptive response in skeletal muscle cells.
Where applicable, experiments conform with Society ethical requirements