Title

 

Authors

F. A. Iannotti¹; V. Barrese²; L. Formisano³; and M. Taglialatela<sup>1,2

1</sup>Department of Health Sciences, University of Molise, Campobasso, Italy; ²Department of Neuroscience, University of Naples Federico II, Naples, Italy; ³University of Sannio, Benevento, Italy.

 

Abstract

Drastic changes in the transcriptional regulation of potassium channels is a pivotal event during skeletal muscle differentiation (1). Recently, using murine C2C12 cells as experimental in vitro model, we found that voltage-dependent potassium channels belonging to the Kv7 (KCNQ) subfamily are expressed at the myoblast (proliferating) stage, and their transcripts and proteins exhibit robust increases during the myotube formation, with Kv7.4 showing the highest degree of regulation. Moreover, the pharmacological activation of Kv7 channels with retigabine influenced cell proliferation, differentiation, and survival in response to myotoxic stimuli  (2). In the present study, we have further investigated the potential contribution of Kv7.4 subunits in skeletal muscle differentiation and the transcriptional mechanisms controlling Kv7.4 expression in skeletal muscle cells. Using real-time PCR experiments and Western blot analysis,we found that the reduction of  Kv7.4 mRNA using by RNA interference drastically decreased the expression of canonical skeletal muscle differentiation markers such as myogenin, troponin and PAX3. Moreover, in Kv7.4-silenced C2C12 cells, retigabine was effects on myotube formation during myogenesis were completely abrogated, suggesting that Kv7.4 expression plays a crucial role for the induction and/or maintenance of the differentiated state in skeletal myotubes. In order toidentify the molecular mechanisms responsible for the changes in Kv7.4 expression during C2C12 differentiation, we concentrated on the potential role of the RE1-silencing transcription factor (REST), a well-knowntranscriptional repressor of several neuronal differentiation genes both in non-neuronal cells and in neural progenitor cells. REST regulates gene expression by recruiting histone modifying and chromatin remodeling proteins upon binding to a specific DNA sequence (RE1-sequence) (3). Intriguingly, in C2C12 cells, REST expression levels inversely correlated to those of Kv7.4; in fact, REST transcripts and protein were markedly reduced during myotube formation, and REST over-expression drastically reduced the transcript levels of Kv7.4, as well as of the skeletal differentiation markers myogenin, troponin and PAX3. Moreover, REST suppression by RNA interference increased the expression levels of the mRNAs for both skeletal muscle differentiation markers and Kv7.4. Bioinformatic analysis revealed the presence of potential REST binding sites (RE1-sites) in the 5’UTR region and in the first intron of Kv7.4 gene conserved across different species. Chromatin immunoprecipitation assays (ChiPs) showed that REST effectively binds to RE1-sites in the Kv7.4 gene, exhibiting a higher binding efficiency in undifferentiated myoblasts when compared to differentiated myotubes. In conclusion, these data suggest a specific and pivotal role for K_v7.4 during skeletal muscle differentiation in vitro, and highlight a novel transcriptional regulation mechanism for this potassium channel subunit.
 
References:

1. Cooper E.; A new role for ion channels in myoblast fusion.J Cell Biol. 2001 May 14;153(4):F9-12.
2. Iannotti et al; Expression, localization, and pharmacological role of Kv7 potassium channels in skeletal muscle proliferation, differentiation, and survival after myotoxic insults. 2010 Mar; 332 (3):811-20
3. Majumder S.; REST in good times and bad: roles in tumor suppressor and oncogenic activities. Cell Cycle. 2006 2006 Sep;5(17):1929-35