Vitamin D elicits tissue-specific isoform expression in Atlantic salmon muscles

Background: Accounting for isoforms is likely key to understanding muscle transcriptomic divergence. Muscles offer classic examples of tissues where changes in isoforms alter function and structure in response to stimuli like increased exercise or novel nutrient regimes. To determine how an essential micronutrient alters muscle isoform production, we examined how vitamin D supplementation influences transcription at multiple hierarchical levels across four Atlantic salmon ( Salmo salar ) muscle tissues. Specifically, we investigated whether analyses of differential transcript expression (DTE), differential transcript usage (DTU), and alternative splicing (DAS) recovered different responses to vitamin D compared to differential gene expression (DGE) alone. Results: Vitamin D modulates salmon muscle transcription at the level of the gene, transcript, and splice junctions in all four muscle tissues. However, the strongest effects at all levels were found in the heart. There was little overlap among significant genes found at the gene, transcript, and splicing levels and the distribution of isoforms per significant gene varied across DGE, DTE, and DTU, indicating that each method tends to identify unique sets of genes. For example, we found that several myosin light chain kinase isoforms were particularly impacted by vitamin D in the heart, but many of the genes exhibited isoforms that were differentially expressed in opposing directions and were thus masked in DGE analysis. We also identified myofibrillar genes that are impacted by vitamin D at all levels of gene regulation, demonstrating that vitamin D impacts many structural proteins that are directly involved in striated muscle contractions. Conclusions: Vitamin D influences several muscle tissue types across multiple levels of transcriptomic divergence. Further, the limited overlap among significant genes found at the gene, transcript, and splicing levels suggests examining only DGE can mask important genes that show differential effects at other levels. Myofibrillar gene isoforms that directly influence muscle contractions in critical organs like the heart could provide an especially fruitful avenue of additional investigation into the transcriptional impacts of vitamin D. Together, these findings clarify how vitamin D influences muscle differentiation, health, and function.