The dihydropyridine receptor (DHPR), normally a voltage-dependent calcium channel, functions in skeletal muscle essentially as a voltage sensor, triggering intracellular calcium release for excitation-contraction coupling. In addition to this fast calcium release, via ryanodine receptor (RYR) channels, depolarization of skeletal myotubes evokes slow calcium waves, unrelated to contraction, that involve the cell nucleus (Jaimovich, E., R. Reyes, J.L. Liberona, and J.A. Powell. 2000. Am. J. Physiol. Cell Physiol. 278:C998–C1010). We tested the hypothesis that DHPR may also be the voltage sensor for these slow calcium signals. In cultures of primary rat myotubes, 10 μM nifedipine (a DHPR inhibitor) completely blocked the slow calcium (fluo-3-fluorescence) transient after 47 mM K+ depolarization and only partially reduced the fast Ca2+ signal. Dysgenic myotubes from the GLT cell line, which do not express the α1 subunit of the DHPR, did not show either type of calcium transient following depolarization. After transfection of the α1 DNA into the GLT cells, K+ depolarization induced slow calcium transients that were similar to those present in normal C2C12 and normal NLT cell lines. Slow calcium transients in transfected cells were blocked by nifedipine as well as by the G protein inhibitor, pertussis toxin, but not by ryanodine, the RYR inhibitor. Since slow Ca2+ transients appear to be mediated by IP3, we measured the increase of IP3 mass after K+ depolarization. The IP3 transient seen in control cells was inhibited by nifedipine and was absent in nontransfected dysgenic cells, but α1-transfected cells recovered the depolarization-induced IP3 transient. In normal myotubes, 10 μM nifedipine, but not ryanodine, inhibited c-jun and c-fos mRNA increase after K+ depolarization. These results suggest a role for DHPR-mediated calcium signals in regulation of early gene expression. A model of excitation-transcription coupling is presented in which both G proteins and IP3 appear as important downstream mediators after sensing of depolarization by DHPR.

The signaling mechanisms by which skeletal muscle electrical activity leads to changes in gene expression remain largely undefined. We have reported that myotube depolarization induces calcium signals in the cytosol and nucleus via inositol 1,4,5-trisphosphate (IP(3)) and phosphorylation of both ERK1/2 and cAMP-response element-binding protein (CREB). We now describe the calcium dependence of P-CREB and P-ERK induction and of the increases in mRNA of the early genes c-fos, c-jun, and egr-1. Increased phosphorylation and early gene activation were maintained in the absence of extracellular calcium, while the increase in intracellular calcium induced by caffeine could mimic the depolarization stimulus. Depolarization performed either in the presence of the IP(3) inhibitors 2-aminoethoxydiphenyl borate or xestospongin C or on cells loaded with BAPTA-AM, in which slow calcium signals were abolished, resulted in decreased activation of the early genes examined. Both early gene activation and CREB phosphorylation were inhibited by ERK phosphorylation blockade. These data suggest a role for calcium in the transcription-related events that follow membrane depolarization in muscle cells.

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Skeletal muscle is a highly plastic tissue with a remarkable capacity to adapt itself to challenges imposed by contractile activity. Adaptive response, that include hypertrophy and activation of oxidative mechanisms have been associated with transient changes in transcriptional activity of specific genes. To define the set of genes regulated by a depolarizing stimulus, we used 22 K mouse oligonucleotide microarrays. Total RNA from C2C12 myotubes was obtained at 2, 4, 18, and 24 h after high K+ stimulation. cDNA from control and depolarized samples was labeled with cyanine 3 or 5 dyes prior to microarray hybridization. Loess normalization followed by statistical analysis resulted in 423 differentially expressed genes using an unadjusted P-value < or = 0.01 as cut off. Depolarization affects transcriptional activity of a limited number of genes, mainly associated with metabolism, cell communication and response to stress. A number of genes related to Ca2+ signaling pathways are induced at 4 h, reinforcing the potential role of Ca2+ in early steps of signal transduction that leads to gene expression. Significant changes in the expression of molecules involved in muscle cell structure were observed; K+-depolarization increased Tnni1 and Acta1 mRNA levels in both differentiated C2C12 and rat skeletal muscle cells in primary culture. Of these two, depolarization induced slow Ca2+ transients appear to have a role only in the regulation of Tnni1 transcriptional activity. We suggest that depolarization induced expression of a small set of genes may underlie Ca2+ dependent plasticity of skeletal muscle cells.

Contracting skeletal muscle produces and releases interleukin-6 (IL-6) in high amounts. Nevertheless, the mechanisms underlying IL-6 expression are not understood. Because inositol-1,4,5-trisphosphate (IP(3))-mediated slow Ca(2+) signals evoked by depolarization of skeletal myotubes appears to play a role in the regulation of gene expression, we examined its involvement on IL-6 transcription. With the use of semiquantitative RT-PCR, we have shown that K(+) depolarization of myotubes induces a transient increase in IL-6 mRNA level, which peaks at 3-4 h and is independent of extracellular Ca(2+). Inhibitors of IP(3)-dependent Ca(2+) signals, like 2-aminoethoxydiphenyl borate (2-APB) and U-73122, decreased activation of IL-6 gene expression as did Ca(2+) signals inhibitor BAPTA-AM, whereas ryanodine, a fast Ca(2+) transient inhibitor, had no effect on IL-6 induction. Depolarization of myotubes transiently transfected with a reporter gene construct, containing 651 bp of IL-6 promoter, induced a twofold increase in promoter activity, which was abolished by either 2-APB or U-73122 and remained unaffected after ryanodine treatment. Site-directed mutagenesis of parental construct allowed us to identify activator protein-1 and NF-kappaB sequences as regulatory elements involved in IL-6 upregulation. Our results provide evidence for involvement of IP(3)-mediated Ca(2+) signals on IL-6 transcription in skeletal muscle cells.

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Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) drive calcium signals involved in skeletal muscle excitation-transcription coupling and plasticity; IP(3)R subtype distribution and downstream events evoked by their activation have not been studied in human muscle nor has their possible alteration in Duchenne muscular dystrophy (DMD). We studied the expression and localization of IP(3)R subtypes in normal and DMD human muscle and in normal (RCMH) and dystrophic (RCDMD) human muscle cell lines. In normal muscle, both type 1 IP(3)Rs (IP(3)R1) and type 2 IP(3)Rs (IP(3)R2) show a higher expression in type II fibers, whereas type 3 IP(3)Rs (IP(3)R3) show uniform distribution. In DMD biopsies, all fibers display a homogeneous IP(3)R2 label, whereas 24 +/- 7% of type II fibers have lost the IP(3)R1 label. RCDMD cells show 5-fold overexpression of IP(3)R2 and down-regulation of IP(3)R3 compared with RCMH cells. A tetanic stimulus induces IP(3)-dependent slow Ca(2+) transients significantly larger and faster in RCDMD cells than in RCMH cells as well as significant ERK1/2 phosphorylation in normal but not in dystrophic cells. Excitation-driven gene expression was different among cell lines; 44 common genes were repressed in RCMH cells and expressed in RCDMD cells or vice versa. IP(3)-dependent Ca(2+) release may play a significant role in DMD pathophysiology.