The myogenic electric organ ofSternopygus macrurus: a non-contractile tissue with a skeletal muscle transcriptome

Abstract

In most electric fish species, the electric organ (EO) derives from striated muscle cells that suppress many muscle properties. In the gymnotiform Sternopygus macrurus, mature electrocytes, the current-producing cells of the EO, do not contain sarcomeres, yet they continue to make some cytoskeletal and sarcomeric proteins and the muscle transcription factors (MTFs) that induce their expression. In order to more comprehensively examine the transcriptional regulation of genes associated with the formation and maintenance of the contractile sarcomere complex, results from expression analysis using qRT-PCR were informed by deep RNA sequencing of transcriptomes and miRNA compositions of muscle and EO tissues from adult S. macrurus. Our data show that: (1) components associated with the homeostasis of the sarcomere and sarcomere-sarcolemma linkage were transcribed in EO at levels similar to those in muscle; (2) MTF families associated with activation of the skeletal muscle program were not differentially expressed between these tissues; and (3) a set of microRNAs that are implicated in regulation of the muscle phenotype are enriched in EO. These data support the development of a unique and highly specialized non-contractile electrogenic cell that emerges from a striated phenotype and further differentiates with little modification in its transcript composition. This comprehensive analysis of parallel mRNA and miRNA profiles is not only a foundation for functional studies aimed at identifying mechanisms underlying the transcription-independent myogenic program in S. macrurus EO, but also has important implications to many vertebrate cell types that independently activate or suppress specific features of the skeletal muscle program.

Sternopygus macrurus electric organ transcriptome and cell size exhibit insensitivity to short-term electrical inactivity

“…Previous work has validated gene expression data from our RNA-seq datasets using the control muscle and EO samples (Pinch et al, 2016), as well as electrically silenced muscle samples (Güth et al, 2016). These validations included numerous genes belonging to proteolytic pathways, and the data showed strong similarity in the expression patterns for these genes between qPCR and RNA-seq.…”

“…The methods for extraction of total RNA from samples and use of these samples in quantitative RT-PCR (qPCR) and RNA-seq used in this work were the same as those published previously (Pinch et al, 2016). EO tissues were sampled from a total of three fish – with one fish sampled at each experimental time point (control, 2-day, and 5-days post-ST).…”

“…EO tissues were sampled from a total of three fish – with one fish sampled at each experimental time point (control, 2-day, and 5-days post-ST). All sampling and analysis techniques were performed as described previously (Pinch et al, 2016) with one exception: normalized expression ratios of transcripts between ST and control EO tissues were determined using the DESeq2 package (Love et al, 2014) instead of the EBSeq package.…”

“…cDNA preparation, internal reference gene selection, and data analysis were performed as described previously (Pinch et al, 2016). Primer pair sequences, amplicon sizes, and annealing temperatures are included in Table 1.…”

“…Ultrastructural and immunolabeling studies show that fully differentiated electrocytes in S. macrurus are significantly larger in size than muscle fibers, lack sarcomeres, continue to produce some, but not all, “muscle-specific” proteins including desmin, titin, α-actin, and α-actinin, and, like their myogenic precursors, they are innervated by cholinergic neurons and are multinucleated (Cuellar et al, 2006; Kim et al, 2008, 2004; Patterson and Zakon, 1997; Unguez and Zakon, 1998a). This incomplete muscle-like phenotype is retained even though S. macrurus electrocytes maintain a transcriptome profile that is over ninety percent comparable to that of striated muscle fibers and includes virtually all transcripts associated with the sarcomere and their regulating transcription factors at levels similar to those detected in skeletal muscle cells (Gallant et al, 2014; Pinch et al, 2016). Since electrocytes are electrically activated by electromotoneurons at a continuous rate of 50-200 Hz (Mills et al, 1992), and muscle fibers are activated with tonic and phasic signals of frequencies <20 Hz by a different population of spinal motor neurons (Bellemare et al, 1983; Johnston and Altringham, 1988; Schiaffino and Reggiani, 2011), one question concerns whether impulse activity frequency exerted on the EO regulates properties of the striated muscle program in different ways, and secondly, to what extent the myogenic program is affected by activity-dependent versus activity-independent influences in electrocytes.…”