The ERG1A Potassium Channel Induces Expression of Genes Related to Skeletal Muscle Atrophy in C2C12 Myotubes

Abstract

The ERG1A potassium channel alternative splice variant is detected at low abundance in normal skeletal muscle; however, it is up-regulated in atrophying skeletal muscle, where it has been shown to modulate both intracellular calcium levels and ubiquitin proteasome proteolysis (UPP). The pathways by which this modulation occurs are not known. Therefore, we transduced C2C12 myotubes with either an adenovirus encoding HERG or an appropriate control virus (n=6). At 48 hours after viral treatments, we extracted total RNA from these cells and reverse transcribed them into cDNA, selecting for coding sequences (i.e., mRNA) by using poly(T) oligomers. The cDNA libraries were sequenced on Illumina's NovaSeq platform and sequence quality was assessed using FastQC (v 0.11.7) for all samples. Quality trimming was performed with the FASTX-Toolkit (v 0.0.14) to remove bases with a Phred33 score of less than 30. The resulting reads of at least 50 bases were mapped against the reference genome using STAR. The mapping results and the annotation file for the reference genome were used as input for HTSeq7 (v 0.7.0) to obtain read counts. Counts from all replicates were merged together to produce a read count matrix for all samples and this count matrix was used for downstream differential gene expression analysis (DGEA). DGEA between treatment and control was carried out using ‘R’ (v 3.5.1). The data demonstrate that HERG expression does produce numerous changes in the gene expression profile of C2C12 myotubes. Indeed, we find that HERG potentially modulates expression of numerous genes (see Table) connected with skeletal muscle atrophy, specifically ubiquitin proteasome proteolysis and with the cytokine interferon, which has been connected with muscle atrophy. The data suggest that HERG does play a role in modulation of protein degradation in skeletal muscle.