Properties of skeletal muscle in the teleost Sternopygus macrurus are unaffected by short-term electrical inactivity

Abstract

Skeletal muscle is distinguished from other tissues on the basis of its shape, biochemistry, and physiological function. Based on mammalian studies, fiber size, fiber types, and gene expression profiles are regulated, in part, by the electrical activity exerted by the nervous system. To address whether similar adaptations to changes in electrical activity in skeletal muscle occur in teleosts, we studied these phenotypic properties of ventral muscle in the electric fish Sternopygus macrurus following 2 and 5 days of electrical inactivation by spinal transection. Our data show that morphological and biochemical properties of skeletal muscle remained largely unchanged after these treatments. Specifically, the distribution of type I and type II muscle fibers and the cross-sectional areas of these fiber types observed in control fish remained unaltered after each spinal transection survival period. This response to electrical inactivation was generally reflected at the transcript level in real-time PCR and RNA-seq data by showing little effect on the transcript levels of genes associated with muscle fiber type differentiation and plasticity, the sarcomere complex, and pathways implicated in the regulation of muscle fiber size. Data from this first study characterizing the acute influence of neural activity on muscle mass and sarcomere gene expression in a teleost are discussed in the context of comparative studies in mammalian model systems and vertebrate species from different lineages.

Keywords: fiber types in teleost fish; muscle inactivation; muscle transcriptome; skeletal muscle atrophy; spinal cord transection.

Fig. 1.

Schematic of the electromotor circuitry of Sternopygus macrurus and site of spinal transection (ST). Skeletal muscle fibers in the ventral muscle allow movement of the fin and are innervated by a population of spinal motoneurons called somatomotoneurons (SMNs) that receive electrical signals from motor areas within the brain. Spinal transection surgery was performed as indicated to electrically silence posteriorly located SMNs and innervated muscle fibers. Concurrently, the ST surgery also silenced electromotoneurons innervating electrocytes in the electric organ (EO). Only the most distal portion of ventral muscle (dashed outline, dark gray) and the caudal portion of the electric organ (solid outline, dark gray) were harvested for analysis. While EO samples were included in the assembly of the transcriptomic dataset, the current study examined only the effects on ventral muscle.

Fig. 2.

Fiber types in the ventral muscle of S. macrurus. Tissue cross section showing the ventral musculature of a tail from a control fish immunolabeled with an antibody against type I sarcomeric myosin heavy chain (green). Left (L) and right (R) myomeres are numbered in increasing order along the ventral-to-dorsal axis. Morphological and fiber type distribution analyses were performed bilaterally on myomeres 3 and 4 from tails of control and ST fish (n = 3 each). Scale bar, 1,000 μm.

Fig. 3.

Effect of ST on cross-sectional area of muscle fiber types in S. macrurus. Minimum Feret diameter measurements were obtained for type I (A) and type II (B) muscle fibers from control and 5-day ST fish (n = 3 each) and used to calculate cross-sectional area, which was then normalized against total tail area to account for small differences in fish size. The number of fibers analyzed for each sample is indicated in parentheses. Data are represented as box plots with each open circle representing a single fiber measurement and “X” indicating the mean cross-sectional area for each fish.

Fig. 4.

Effect of ST on muscle fiber type distribution in S. macrurus. Average type I muscle fiber distribution in myomeres 3 and 4 from control and spinal transected fish ventral muscle (n = 3 each) was determined as the average ratio of total cross-sectional area from type I muscle fibers to the total area of all muscle cells from four myomeres (i.e., L3, R3, L4, R4). Bars represent means ± SD for each sample.

Fig. 5.

Heat map of transcript abundance ratios of muscle transcription factor and sarcomeric genes between control and ST fish. Normalized transcript abundance ratios were determined for 2-day ST/control and 5-day ST/control muscle samples and the log₂-transformed ratios were visualized as heat maps using the “heatmap.2” function in the “gplots” package in R for muscle transcription factors (A) and sarcomere genes (B). Transcription factors were ordered by the fiber type specificity of their target genes, whereas sarcomere genes were sorted by expression pattern using the default row clustering method in the “heatmap.2” function. Genes flagged as differentially expressed by DESeq2 are indicated by including the unadjusted P value overlaying the appropriate color cell.

Fig. 6.

Quantification of transcript abundance ratios of selected genes of interest between spinally transected and control ventral muscle using real-time PCR (qPCR). Primers listed in Table 1 were used to amplify transcripts of interest. Abundance ratios determined by qPCR (n ≥ 4) for 2-day ST/control and 5-day ST/control were normalized using 3 internal references genes (rps11, snrpb, cct5) as described in materials and methods. Results are shown with means ± SE. *Statistically significant results with P < 0.05 after multiple testing adjustment. +Genes with statistical significance (P < 0.05) before, but not after, multiple testing adjustment.

Fig. 7.

Heat map of transcript abundance ratios of genes involved in cell size regulation between ST and control ventral muscle. Analysis and representation of data are the same as in Fig. 5. Genes within each pathway were sorted by expression pattern using the default row clustering method in the “heatmap.2” function.