Review: Understanding fish muscle biology in the indeterminate growth species pacu (Piaractus mesopotamicus).

The muscle phenotype of fish is regulated by numerous factors that, although widely explored, still need to be fully understood. In this context, several studies aimed to unravel how internal and external stimuli affect the muscle growth of these vertebrates. The pacu (Piaractus mesopotamicus) is a species of indeterminate muscular growth that quickly reaches high body weight. For this reason, it adds great importance to the productive sector, along with other round fish. In this context, we aimed to compile studies on fish biology and skeletal muscle growth, focusing on studies by our research group that used pacu as an experimental model along with other species. Based on these studies, new muscle phenotype regulators were identified and explored in vivo, in vitro, and in silico studies, which strongly contribute to advances in understanding muscle growth mechanisms with future applications in the productive sector.

Identification of Novel Genes Associated with Fish Skeletal Muscle Adaptation during Fasting and Refeeding Based on a Meta-Analysis.

The regulation of the fish phenotype and muscle growth is influenced by fasting and refeeding periods, which occur in nature and are commonly applied in fish farming. However, the regulators associated with the muscle responses to these manipulations of food availability have not been fully characterized. We aimed to identify novel genes associated with fish skeletal muscle adaptation during fasting and refeeding based on a meta-analysis. Genes related to translational and proliferative machinery were investigated in pacus (Piaractus mesopotamicus) subjected to fasting (four and fifteen days) and refeeding (six hours, three and fifteen days). Our results showed that different fasting and refeeding periods modulate the expression of the genes mtor, rps27a, eef1a2, and cdkn1a. These alterations can indicate the possible protection of the muscle phenotype, in addition to adaptive responses that prioritize energy and substrate savings over cell division, a process regulated by ccnd1. Our study reveals the potential of meta-analysis for the identification of muscle growth regulators and provides new information on muscle responses to fasting and refeeding in fish that are of economic importance to aquaculture.

Amino Acids and IGF1 Regulation of Fish Muscle Growth Revealed by Transcriptome and microRNAome Integrative Analyses of Pacu (Piaractus mesopotamicus) Myotubes.

Amino acids (AA) and IGF1 have been demonstrated to play essential roles in protein synthesis and fish muscle growth. The myoblast cell culture is useful for studying muscle regulation, and omics data have contributed enormously to understanding its molecular biology. However, to our knowledge, no study has performed the large-scale sequencing of fish-cultured muscle cells stimulated with pro-growth signals. In this work, we obtained the transcriptome and microRNAome of pacu (Piaractus mesopotamicus)-cultured myotubes treated with AA or IGF1. We identified 1228 and 534 genes differentially expressed by AA and IGF1. An enrichment analysis showed that AA treatment induced chromosomal changes, mitosis, and muscle differentiation, while IGF1 modulated IGF/PI3K signaling, metabolic alteration, and matrix structure. In addition, potential molecular markers were similarly modulated by both treatments. Muscle-miRNAs (miR-1, -133, -206 and -499) were up-regulated, especially in AA samples, and we identified molecular networks with omics integration. Two pairs of genes and miRNAs demonstrated a high-level relationship, and involvement in myogenesis and muscle growth: marcksb and miR-29b in AA, and mmp14b and miR-338-5p in IGF1. Our work helps to elucidate fish muscle physiology and metabolism, highlights potential molecular markers, and creates a perspective for improvements in aquaculture and in in vitro meat production.

An insight on the impact of teleost whole genome duplication on the regulation of the molecular networks controlling skeletal muscle growth.

Fish muscle growth is a complex process regulated by multiple pathways, resulting on the net accumulation of proteins and the activation of myogenic progenitor cells. Around 350-320 million years ago, teleost fish went through a specific whole genome duplication (WGD) that expanded the existent gene repertoire. Duplicated genes can be retained by different molecular mechanisms such as subfunctionalization, neofunctionalization or redundancy, each one with different functional implications. While the great majority of ohnolog genes have been identified in the teleost genomes, the effect of gene duplication in the fish physiology is still not well characterized. In the present study we studied the effect of WGD on the transcription of the duplicated components controlling muscle growth. We compared the expression of lineage-specific ohnologs related to myogenesis and protein balance in the fast-skeletal muscle of pacus (Piaractus mesopotamicus-Ostariophysi) and Nile tilapias (Oreochromis niloticus-Acanthopterygii) fasted for 4 days and refed for 3 days. We studied the expression of 20 ohnologs and found that in the great majority of cases, duplicated genes had similar expression profiles in response to fasting and refeeding, indicating that their functions during growth have been conserved during the period after the WGD. Our results suggest that redundancy might play a more important role in the retention of ohnologs of regulatory pathways than initially thought. Also, comparison to non-duplicated orthologs showed that it might not be uncommon for the duplicated genes to gain or loss new regulatory elements simultaneously. Overall, several of duplicated ohnologs have similar transcription profiles in response to pro-growth signals suggesting that evolution tends to conserve ohnolog regulation during muscle development and that in the majority of ohnologs related to muscle growth their functions might be very similar.

Maternal protein restriction changes structural and metabolic gene expression in the skeletal muscle of aging offspring rats.

Maternal protein restriction affects postnatal skeletal muscle physiology with impacts that last through senility. To investigate the morphological and molecular characteristics of skeletal muscle in aging rats subjected to maternal protein restriction, we used aged male rats (540 days old) born of dams fed a protein restricted diet (6% protein) during pregnancy and lactation. Using morphological, immunohistochemical and molecular analyses, we evaluated the soleus (SOL) and extensor digitorum longus (EDL) muscles, muscle fiber cross-sectional area (CSA) (n=8), muscle fiber frequency (n=5) and the gene expression (n=8) of the oxidative markers (succinate dehydrogenase-Sdha and citrate synthase-CS) and the glycolytic marker (lactate dehydrogenase-Ldha). Global transcriptome analysis (n=3) was also performed to identify differentially regulated genes, followed by gene expression validation (n=8). The oxidative SOL muscle displayed a decrease in muscle fiber CSA (*p<0.05) and in the expression of oxidative metabolism marker Sdha (***p<0.001), upregulation of the anabolic Igf-1 (**p<0.01), structural Chad (**p<0.01), and Fmod (*p<0.05) genes, and downregulation of the Hspb7 (**p<0.01) gene. The glycolytic EDL muscle exhibited decreased IIA (*p<0.05) and increased IIB (*p<0.05) fiber frequency, and no changes in muscle fiber CSA or in the expression of oxidative metabolism genes. In contrast, the gene expression of Chad (**p<0.01) was upregulated and the Myog (**p<0.01) gene was downregulated. Collectively, our morphological, immunohistochemical and molecular analyses showed that maternal protein restriction induced changes in the expression of metabolic, anabolic, myogenic, and structural genes, mainly in the oxidative SOL muscle, in aged offspring rats.