Initially we characterised growth responses to altered nutritional input at the transcriptional and tissue levels in the fast skeletal muscle of juvenile gilthead sea bream. Fish reared at 21-22°C (range) were fed a commercial diet at 3% body mass d(-1) (non-satiation feeding, NSF) for 4 weeks, fasted for 4d (F) and then fed to satiation (SF) for 21d. 13 out of 34 genes investigated showed consistent patterns of regulation between nutritional states. Fasting was associated with a 20-fold increase in MAFbx, and a 5-fold increase in Six1 and WASp expression, which returned to NSF levels within 16h of SF. Refeeding to satiation was associated with a rapid (<24 h) 12 to 17-fold increase in UNC45, Hsp70 and Hsp90α transcripts coding for molecular chaperones associated with unfolded protein response pathways. The growth factors FGF6 and IGF1 increased 6.0 and 4.5-fold within 16 h and 24 h of refeeding respectively. The average growth in diameter of fast muscle fibres was checked with fasting and significant fibre hypertrophy was only observed after 13d and 21d SF. To investigate developmental plasticity in growth responses we used the same experimental protocol with fish reared at either 17.5-18.5°C (range) (LT) or 21-22°C (range) (HT) to metamorphosis and then transferred to 21-22°C. There were persistent effects of development temperature on muscle growth patterns with 20% more fibres of lower average diameter in LT than HT group of similar body size. Altering the nutritional input to the muscle to stimulate growth revealed cryptic changes in the expression of UNC45 and Hsp90α with higher transcript abundance in the LT than HT groups, whereas there were no differences in the expression of MAFbx and Six1. It was concluded that myogenesis and gene expression patterns during growth are not fixed, but can be modified by temperature during the early stages of the life cycle.

Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (Gh) express Gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. Restricting Gh-transgenics to the same lower ration (TR) as wild-type fish (WT) results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. In order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of Gh and nutritional signals affects gene regulation we used RNA-seq to compare the fast skeletal muscle transcriptome in TR and WT coho salmon.

Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (GH) show substantially faster growth than wild-type (WT) fish. We fed GH-transgenic salmon either to satiation (1 year; TF) or the same smaller ration of wild-type fish (2 years; TR), resulting in groups matched for body size to WT salmon. The myotomes of TF and WT fish had the same number and size distribution of muscle fibres, indicating a twofold higher rate of fibre recruitment in the GH transgenics. Unexpectedly, calorie restriction was found to decrease the rate of fibre production in transgenics, resulting in a 20% increase in average fibre size and reduced costs of ionic homeostasis. Genes for myotube formation were downregulated in TR relative to TF and WT fish. We suggest that muscle fibre size optimisation allows the reallocation of energy from maintenance to locomotion, explaining the observation that calorie-restricted transgenics grow at the same rate as WT fish whilst exhibiting markedly higher foraging activity.

Heat shock proteins 90 (Hsp90) have an essential role in sarcomere formation and differentiation in skeletal muscle and also act as molecular chaperones during protein folding impacting a wide range of physiological processes. We characterised and provided a phylogenetically consistent nomenclature for the complete repertoire of six Hsp90 paralogues present in duplicated salmonid fish genomes (Hsp90α1a, Hsp90α1b, Hsp90α2a, Hsp90α2b, Hsp90ß1a and Hsp90ß1b). The expression of paralogues in fast skeletal muscle was investigated using in vivo fasting-feeding experiments and primary myogenic cultures. Fasted juvenile Atlantic salmon (Salmo salar) showed a transient 2 to 8-fold increase in the expression of all 4 Hsp90α paralogues within 24h of satiation feeding. Hsp90α1a and hsp90α1b also showed a pronounced secondary increase in expression after 10 days, concomitant with muscle differentiation and the expression of myogenin and sarcomeric proteins (mlc2, myhc). Hsp90ß1b was constitutively expressed whereas Hsp90ß1a expression was downregulated 10-fold between fasted and fed individuals. Hsp90α1a and Hsp90α1b were upregulated 10 to 15-fold concomitant with myotube formation and muscle differentiation in vitro whereas other Hsp90 paralogues showed no change in expression. In cells starved of amino acid (AA) and serum for 72h the addition of AA, but not insulin-like growth factor 1, increased phosphorylation of mTor and expression of all 4 hsp90α paralogues and associated co-chaperones including hsp30, tbcb, pdia4, pdia6, stga and fk504bp1, indicating a general activation of the protein folding response. In contrast, Hsp90ß1a expression in vitro was unresponsive to AA treatment indicating that some other as yet uncharacterised signal(s) regulate its expression in response to altered nutritional state.