Exploring the Integrated Role of miRNAs and lncRNAs in Regulating the Transcriptional Response to Amino Acids and Insulin-like Growth Factor 1 in Gilthead Sea Bream (Sparus aurata) Myoblasts.

In this study, gilthead sea bream (Sparus aurata) fast muscle myoblasts were stimulated with two pro-growth treatments, amino acids (AA) and insulin-like growth factor 1 (Igf-1), to analyze the transcriptional response of mRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and to explore their possible regulatory network using bioinformatic approaches. AA had a higher impact on transcription (1795 mRNAs changed) compared to Igf-1 (385 mRNAs changed). Both treatments stimulated the transcription of mRNAs related to muscle differentiation (GO:0042692) and sarcomere (GO:0030017), while AA strongly stimulated DNA replication and cell division (GO:0007049). Both pro-growth treatments altered the transcription of over 100 miRNAs, including muscle-specific miRNAs (myomiRs), such as miR-133a/b, miR-206, miR-499, miR-1, and miR-27a. Among 111 detected lncRNAs (>1 FPKM), only 30 were significantly changed by AA and 11 by Igf-1. Eight lncRNAs exhibited strong negative correlations with several mRNAs, suggesting a possible regulation, while 30 lncRNAs showed strong correlations and interactions with several miRNAs, suggesting a role as sponges. This work is the first step in the identification of the ncRNAs network controlling muscle development and growth in gilthead sea bream, pointing out potential regulatory mechanisms in response to pro-growth signals.

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.

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.

Ascorbic Acid Supplementation Improves Skeletal Muscle Growth in Pacu (Piaractus mesopotamicus) Juveniles: In Vivo and In Vitro Studies.

In fish, fasting leads to loss of muscle mass. This condition triggers oxidative stress, and therefore, antioxidants can be an alternative to muscle recovery. We investigated the effects of antioxidant ascorbic acid (AA) on the morphology, antioxidant enzyme activity, and gene expression in the skeletal muscle of pacu (Piaractus mesopotamicus) following fasting, using in vitro and in vivo strategies. Isolated muscle cells of the pacu were subjected to 72 h of nutrient restriction, followed by 24 h of incubation with nutrients or nutrients and AA (200 µM). Fish were fasted for 15 days, followed by 6 h and 15 and 30 days of refeeding with 100, 200, and 400 mg/kg of AA supplementation. AA addition increased cell diameter and the expression of anabolic and cell proliferation genes in vitro. In vivo, 400 mg/kg of AA increased anabolic and proliferative genes expression at 6 h of refeeding, the fiber diameter and the expression of genes related to cell proliferation at 15 days, and the expression of catabolic and oxidative metabolism genes at 30 days. Catalase activity remained low in the higher supplementation group. In conclusion, AA directly affected the isolated muscle cells, and the higher AA supplementation positively influenced muscle growth after fasting.

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.

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.

Exposure to Bacteriophages T4 and M13 Increases Integrin Gene Expression and Impairs Migration of Human PC-3 Prostate Cancer Cells.

The interaction between bacteriophages and integrins has been reported in different cancer cell lines, and efforts have been undertaken to understand these interactions in tumor cells along with their possible role in gene alterations, with the aim to develop new cancer therapies. Here, we report that the non-specific interaction of T4 and M13 bacteriophages with human PC-3 cells results in differential migration and varied expression of different integrins. PC-3 tumor cells (at 70% confluence) were exposed to 1 × 107 pfu/mL of either lytic T4 bacteriophage or filamentous M13 bacteriophage. After 24 h of exposure, cells were processed for a histochemical analysis, wound-healing migration assay, and gene expression profile using quantitative real-time PCR (qPCR). qPCR was performed to analyze the expression profiles of integrins ITGAV, ITGA5, ITGB1, ITGB3, and ITGB5. Our findings revealed that PC-3 cells interacted with T4 and M13 bacteriophages, with significant upregulation of ITGAV, ITGA5, ITGB3, ITGB5 genes after phage exposure. PC-3 cells also exhibited reduced migration activity when exposed to either T4 or M13 phages. These results suggest that wildtype bacteriophages interact non-specifically with PC-3 cells, thereby modulating the expression of integrin genes and affecting cell migration. Therefore, bacteriophages have future potential applications in anticancer therapies.

Ascorbic acid stimulates the in vitro myoblast proliferation and migration of pacu (Piaractus mesopotamicus).

The postembryonic growth of skeletal muscle in teleost fish involves myoblast proliferation, migration and differentiation, encompassing the main events of embryonic myogenesis. Ascorbic acid plays important cellular and biochemical roles as an antioxidant and contributes to the proper collagen biosynthesis necessary for the structure of connective and bone tissues. However, whether ascorbic acid can directly influence the mechanisms of fish myogenesis and skeletal muscle growth remains unclear. The aim of our work was to evaluate the effects of ascorbic acid supplementation on the in vitro myoblast proliferation and migration of pacu (Piaractus mesopotamicus). To provide insight into the potential antioxidant role of ascorbic acid, we also treated myoblasts in vitro with menadione, which is a powerful oxidant. Our results show that ascorbic acid-supplemented myoblasts exhibit increased proliferation and migration and are protected against the oxidative stress caused by menadione. In addition, ascorbic acid increased the activity of the antioxidant enzyme superoxide dismutase and the expression of myog and mtor, which are molecular markers related to skeletal muscle myogenesis and protein synthesis, respectively. This work reveals a direct influence of ascorbic acid on the mechanisms of pacu myogenesis and highlights the potential use of ascorbic acid for stimulating fish skeletal muscle growth.