Trout myomaker contains 14 minisatellites and two sequence extensions but retains fusogenic function.

The formation of new myofibers in vertebrates occurs by myoblast fusion and requires fusogenic activity of the muscle-specific membrane protein myomaker. Here, using in silico (BLAST) genome analyses, we show that the myomaker gene from trout includes 14 minisatellites, indicating that it has an unusual structure compared with those of other animal species. We found that the trout myomaker gene encodes a 434-amino acid (aa) protein, in accordance with its apparent molecular mass (∼40 kDa) observed by immunoblotting. The first half of the trout myomaker protein (1-220 aa) is similar to the 221-aa mouse myomaker protein, whereas the second half (222-234 aa) does not correspond to any known motifs and arises from two protein extensions. The first extension (∼70 aa) apparently appeared with the radiation of the bony fish clade Euteleostei, whereas the second extension (up to 236 aa) is restricted to the superorder Protacanthopterygii (containing salmonids and pike) and corresponds to the insertion of minisatellites having a length of 30 nucleotides. According to gene expression analyses, trout myomaker expression is consistently associated with the formation of new myofibers during embryonic development, postlarval growth, and muscle regeneration. Using cell-mixing experiments, we observed that trout myomaker has retained the ability to drive the fusion of mouse fibroblasts with C2C12 myoblasts. Our work reveals that trout myomaker has fusogenic function despite containing two protein extensions.

Histological, transcriptomic and in vitro analysis reveal an intrinsic activated state of myogenic precursors in hyperplasic muscle of trout.

BACKGROUND: The dramatic increase in myotomal muscle mass in post-hatching fish is related to their ability to lastingly produce new muscle fibres, a process termed hyperplasia. The molecular and cellular mechanisms underlying fish muscle hyperplasia largely remain unknown. In this study, we aimed to characterize intrinsic properties of myogenic cells originating from hyperplasic fish muscle. For this purpose, we compared in situ proliferation, in vitro cell behavior and transcriptomic profile of myogenic precursors originating from hyperplasic muscle of juvenile trout (JT) and from non-hyperplasic muscle of fasted juvenile trout (FJT) and adult trout (AT). RESULTS: For the first time, we showed that myogenic precursors proliferate in hyperplasic muscle from JT as shown by in vivo BrdU labeling. This proliferative rate was very low in AT and FJT muscle. Transcriptiomic analysis revealed that myogenic cells from FJT and AT displayed close expression profiles with only 64 differentially expressed genes (BH corrected p-val < 0.001). In contrast, 2623 differentially expressed genes were found between myogenic cells from JT and from both FJT and AT. Functional categories related to translation, mitochondrial activity, cell cycle, and myogenic differentiation were inferred from genes up regulated in JT compared to AT and FJT myogenic cells. Conversely, Notch signaling pathway, that signs cell quiescence, was inferred from genes down regulated in JT compared to FJT and AT. In line with our transcriptomic data, in vitro JT myogenic precursors displayed higher proliferation and differentiation capacities than FJT and AT myogenic precursors. CONCLUSIONS: The transcriptomic analysis and examination of cell behavior converge to support the view that myogenic cells extracted from hyperplastic muscle of juvenile trout are intrinsically more potent to form myofibres than myogenic cells extracted from non-hyperplasic muscle. The generation of gene expression profiles in myogenic cell extracted from muscle of juvenile trout may yield insights into the molecular and cellular mechanisms controlling hyperplasia and provides a useful list of potential molecular markers of hyperplasia.

miR-210 expression is associated with methionine-induced differentiation of trout satellite cells.

In fish, data on microRNAs (miRNAs) involved in myogenesis are scarce. In order to identify miRNAs involved in satellite cell differentiation, we used a methionine depletion/replenishment protocol to synchronize myogenic cell differentiation. Our results validated that methionine removal (72 h) from the medium strongly decreased myoD1 and myogenin expression, indicating differentiation arrest. In contrast, methionine replenishment rescued expression of myoD1 and myogenin, showing a resumption of differentiation. We performed a miRNA array analysis of myogenic cells under three conditions: presence of methionine for 72 h (control), absence of methionine for 72 h (Meth-) and absence of methionine for 48 h followed by 24 h of methionine replenishment (Meth-/+). A clustering analysis identified three clusters: cluster I corresponds to miRNA upregulated only in Meth-/+ conditions; cluster II corresponds to miRNA downregulated only in Meth-/+ conditions; cluster III corresponds to miRNAs with high expression in control, low expression in Meth- conditions and intermediate expression after methionine replenishment (Meth-/+). Cluster III was very interesting because it fitted with the data obtained for myoD1 and myogenin (supporting an involvement in differentiation) and contained seven miRNAs with muscle-related function (e.g. miR-133a) and one (miR-210) with unknown function. Based on our previously published miRNA repertoire ( Juanchich et al., 2016), we confirmed miR-133a was expressed only in white muscle and showed that miR-210 had strong expression in white muscle. We also showed that miR-210 expression was upregulated during differentiation of satellite cells, suggesting that miR-210 was potentially involved in the differentiation of satellite cells.

Dynamic expression of tgf-ß2, tgf-ß3 and inhibin ßA during muscle growth resumption and satellite cell differentiation in rainbow trout (Oncorhynchus mykiss).

Members of the TGF-ß superfamily are involved in numerous cell functions; however, except for myostatin, their roles in the regulation of muscle growth in fish are completely unknown. We measured tgf-ß1, tgf-ß2, tgf-ß3, inhibin ßA (inh) and follistatin (fst) gene expression during muscle growth recovery following a fasting period. We observed that tgf-ß1a and tgf-ß2 expression were quickly down-regulated after refeeding and that tgf-ß3 reached its highest level of expression 7days post-refeeding, mirroring myogenin expression. Inh ßA1 mRNA levels decreased sharply after refeeding, in contrast to fst b2 expression, which peaked at day 2. No significant modification of expression was observed for tgf-ß1a, tgf-ß1b, tgf-ß1c and tgf-ß6 during refeeding. In vitro, tgf-ß2 and inh ßA1 expression decreased during the differentiation of satellite cells, whereas tgf-ß3 expression increased following the same pattern as myogenin. Surprisingly, fst b1 and fst b2 expression decreased during differentiation, whereas no variation was observed in fst a1 and fst a2 expression levels. In vitro analyses also indicated that IGF1 treatment up-regulated tgf-ß3, inh ßA1 and myogenin expression, and that MSTN treatment increased fst b1 and fst b2 expression. In conclusion, we showed that the expression of tgf-ß2, tgf-ß3 and inh ßA1 is dynamically regulated during muscle growth resumption and satellite cell differentiation, strongly suggesting that these genes have a role in the regulation of muscle growth.

Dynamics of pax7 expression during development, muscle regeneration, and in vitro differentiation of satellite cells in rainbow trout (Oncorhynchus mykiss).

Essential for muscle fiber formation and hypertrophy, muscle stem cells, also called satellite cells, reside beneath the basal lamina of the muscle fiber. Satellite cells have been commonly identified by the expression of the Paired box 7 (Pax7) due to its specificity and the availability of antibodies in tetrapods. In fish, the identification of satellite cells remains difficult due to the lack of specific antibodies in most species. Based on the development of a highly sensitive in situ hybridization (RNAScope®) for pax7, we showed that pax7+ cells were detected in the undifferentiated myogenic epithelium corresponding to the dermomyotome at day 14 post-fertilization in rainbow trout. Then, from day 24, pax7+ cells gradually migrated into the deep myotome and were localized along the muscle fibers and reach their niche in satellite position of the fibres after hatching. Our results showed that 18 days after muscle injury, a large number of pax7+ cells accumulated at the wound site compared to the uninjured area. During the in vitro differentiation of satellite cells, the percentage of pax7+ cells decreased from 44% to 18% on day 7, and some differentiated cells still expressed pax7. Taken together, these results show the dynamic expression of pax7 genes and the follow-up of these muscle stem cells during the different situations of muscle fiber formation in trout.

Mature adipocytes inhibit differentiation of myogenic cells but stimulate proliferation of fibro-adipogenic precursors derived from trout muscle in vitro.

Interactions between tissues and cell types, mediated by cytokines or direct cell-cell exchanges, regulate growth. To determine whether mature adipocytes influence the in vitro growth of trout mononucleated muscle cells, we developed an indirect coculture system, and showed that adipocytes (5 × 106 cells/well) derived from perivisceral adipose tissue increased the proliferation (BrdU-positive cells) of the mononucleated muscle cells (26% vs. 39%; p < 0.001) while inhibiting myogenic differentiation (myosin+) (25% vs. 15%; p < 0.001). Similar effects were obtained with subcutaneous adipose tissue-derived adipocytes, although requiring more adipocytes (3 × 107 cells/well vs. 5 × 106 cells/well). Conditioned media recapitulated these effects, stimulating proliferation (31% vs. 39%; p < 0.001) and inhibiting myogenic differentiation (32 vs. 23%; p < 0.001). Adipocytes began to reduce differentiation after 24 h, whereas proliferation stimulation was observed after 48 h. While adipocytes did not change pax7+ and myoD1/2+ percentages, they reduced myogenin+ cells showing inhibition from early differentiation stage. Finally, adipocytes increased BrdU+ cells in the Pdgfrα+ population but not in the myoD+ one. Collectively, our results demonstrate that trout adipocytes promote fibro-adipocyte precursor proliferation while inhibiting myogenic cells differentiation in vitro, suggesting the key role of adipose tissue in regulating fish muscle growth.

Myostatin induces atrophy of trout myotubes through inhibiting the TORC1 signaling and promoting Ubiquitin-Proteasome and Autophagy-Lysosome degradative pathways.

Myostatin (MSTN) is well known as a potent inhibitor of muscle growth in mammals and has been shown to both inhibit the growth promoting TORC1 signaling pathway and promote Ubiquitin-Proteasomal and Autophagy-Lysosomal degradative routes. In contrast, in non-mammalian species, despite high structural conservation of MSTN sequence, functional conservation is only assumed. Here, we show that treatment of cultured trout myotubes with human recombinant MSTN (huMSTN) resulted in a significant decrease of their diameter by up to 20%, validating the use of heterologous huMSTN in our in vitro model to monitor the processes by which this growth factor promotes muscle wasting in fish. Accordingly, huMSTN stimulation prevented the full activation by IGF1 of the TORC1 signaling pathway, as revealed by the analysis of the phosphorylation status of 4E-BP1. Moreover, the levels of the proteasome-dependent protein Atrogin1 exhibited an increase in huMSTN treated cells. Likewise, we observed a stimulatory effect of huMSTN treatment on the levels of LC3-II, the more reliable marker of the Autophagy-Lysosomal degradative system. Overall, these results show for the first time in a piscine species the effect of MSTN on several atrophic and hypertrophic pathways and support a functional conservation of this growth factor between lower and higher vertebrates.

FoxO1 is not a key transcription factor in the regulation of myostatin (mstn-1a and mstn-1b) gene expression in trout myotubes.

In mammals, much evidence has demonstrated the important role of myostatin (MSTN) in regulating muscle mass and identified the transcription factor forkhead box O (FoxO) 1 as a key regulator of its gene expression during atrophy. However, in trout, food deprivation leads to muscle atrophy without an increase of the expression of mstn genes in the muscle. We therefore studied the relationship between FoxO1 activity and the expression of both mstn genes (mstn1a and mstn1b) in primary culture of trout myotubes. To this aim, two complementary studies were undertaken. In the former, FoxO1 protein activity was modified with insulin-like growth factor-I (IGF-I) treatment, and the consequences on the expression of both mstn genes were monitored. In the second experiment, the expression of both studied genes was modified with growth hormone (GH) treatment, and the activation of FoxO1 protein was investigated. We found that IGF-I induced the phosphorylation of FoxO1 and FoxO4. Moreover, under IGF-I stimulation, FoxO1 was no longer localized in the nucleus, indicating that this growth factor inhibited FoxO1 activity. However, IGF-I treatment had no effect on mstn1a and mstn1b expression, suggesting that FoxO1 would not regulate the expression of mstn genes in trout myotubes. Furthermore, the treatment of myotubes with GH decreased the expression of both mstn genes but has no effect on the phosphorylation of FoxO1, FoxO3, and FoxO4 nor on the nuclear translocation of FoxO1. Altogether, our results showed that mstn1a and mstn1b expressions were not associated with FoxO activity, indicating that FoxO1 is likely not a key regulator of mstn genes in trout myotubes.

The production of fluorescent transgenic trout to study in vitro myogenic cell differentiation.

BACKGROUND: Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (Oncorhynchus mykiss) carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter, and cultivated genetically modified myogenic cells derived from these fish. RESULTS: In transgenic trout, green fluorescence appeared in fast muscle fibers as early as the somitogenesis stage and persisted throughout life. Using an in vitro myogenesis system we observed that satellite cells isolated from the myotomal muscle of transgenic trout expressed GFP about 5 days post-plating as they started to fuse. GFP fluorescence persisted subsequently in myosatellite cell-derived myotubes. Using this in vitro myogenesis system, we showed that the rate of muscle cell differentiation was strongly dependent on temperature, one of the most important environmental factors in the muscle growth of poikilotherms. CONCLUSIONS: We produced MLC2f-gfp transgenic trout that exhibited fluorescence in their fast muscle fibers. The culture of muscle cells extracted from these trout enabled the real-time monitoring of myogenic differentiation. This in vitro myogenesis system could have numerous applications in fish physiology to evaluate the myogenic activity of circulating growth factors, to test interfering RNA and to assess the myogenic potential of fish mesenchymal stem cells. In ecotoxicology, this system could be useful to assess the impact of environmental factors and marine pollutants on fish muscle growth.

In vitro characterization of proliferation and differentiation of trout satellite cells.

Fish satellite cells have been extracted from various species, but the myogenic characteristics of these cells in culture remain largely unknown. We show here that 60%-70% of the adherent cells are myogenic based on their immunoreactivity for the myogenic regulatory factor MyoD. In DMEM containing 10% fetal calf serum (FCS), trout myoblasts display rapid expression of myogenin (18% of myogenin-positive cells at day 2) combined with rapid fusion into myotubes (50% of myogenin-positive nuclei and 30% nuclei in myosin heavy chain [MyHC]-positive cells at day 7). These kinetics of differentiation are reminiscent of the behavior of fetal myoblasts in mammals. However, not all the myogenic cells differentiate; this subpopulation of cells might correspond to the previously named "reserve" cells. More than 90% of the BrdU-positive cells are also positive for MyoD, indicating that myogenic cells proliferate in vitro. By contrast, less than 1% of myogenin-positive cells are positive for BrdU suggesting that myogenin expression occurs only in post-mitotic cells. In order to maximize either the proliferation or the differentiation of cells, we have defined new culture conditions based on the use of a proliferation medium (F10+10%FCS) and a differentiation medium (DMEM+2%FCS). Three days after switching the medium, the differentiation index (% MyHC-positive nuclei) is 40-fold higher than that in proliferation medium, whereas the proliferation index (% BrdU-positive nuclei) is three-fold lower. Stimulation of cell proliferation by insulin-like growth factor 1 (IGF1), IGF2, and FGF2 is greater in F10 medium. The characterization of these extracted muscle cells thus validates the use of this in vitro system of myogenesis in further studies of the myogenic activity of growth factors in trout.

Identification of TGF-ß, inhibin ßA and follistatin paralogs in the rainbow trout genome.

Since their initial discovery, TGF-ß superfamily members have been considered multifunctional growth and differentiation factors in many cell types. Various studies have clearly demonstrated the key roles of specific TGF-ß members in muscle growth, including myostatin and inhibin as well as genes, such as follistatin. By binding to TGF-ß members, follistatin prevents TGF-ß from binding to its receptors and thus neutralizes its activity. Here, we report the identification of the gene sequences of four TGF-ß isoforms and three paralogs of TGF-ß1, which we called TGF-ß1a, TGF-ß1b and TGF-ß1c, four sequences of inhibin ßA paralogs; and two sequences of follistatin paralogs from rainbow trout. A phylogenetic analysis clearly indicated the existence of four monophyletic clades, corresponding to TGF-ß1, -ß2, -ß3 and -ß6. Based on their sequence identity TGF-ß1a and -ß1c are grouped together, whereas TGF-ß1b appears more divergent even though it is grouped within the TGF-ß1 clade. Alignments and phylogenetic analyses showed that the protein sequences of TGF-ß, inhibin ßA and follistatin are extremely well conserved (>90%) relative to each other; however, their regulation and expression patterns are different. TGF-ß2 and -ß3 showed the most abundant expression in muscle and were the main TGF-ß members expressed in this tissue. Follistatin and inhibin ßA paralogs were expressed in all tissues examined but with different patterns. Our identification of multiple copies of TGF-ß, inhibin ßA and follistatin with different expression patterns suggests non-redundant functions for these paralogs in rainbow trout.

Myostatin inhibits proliferation but not differentiation of trout myoblasts.

The muscle growth in mammals is regulated by several growth factors including myostatin (MSTN), a member of the transforming growth factor-beta (TGF-beta) superfamily. To date, it is unknown in fish whether MSTN could have any effect on proliferation or differentiation of myogenic cells. Using culture of trout satellite cells, we showed that mstn1a and mstn1b mRNA are expressed in myoblasts and that their expression decreased in differentiating myoblasts. We also demonstrated that a treatment with huMSTN decreased the proliferation of IGF1-stimulated myoblasts in a dose-dependent manner. By contrast, treatment of myoblasts with 100 nM of huMSTN for three days, did not affect the percentage of positive cells for myogenin neither the percentage of nuclei in myosin positive cells. Moreover, our results clearly indicated that huMSTN treatment had no effect on MyoD and myogenin protein levels, which suggests that huMSTN did not strongly affect MyoD activity. In conclusion, we showed that huMSTN inhibited proliferation but not differentiation of trout myoblasts, probably resulting from a lack of huMSTN effect on MyoD activity. Altogether, these results show high interspecies differences in the function of MSTN.

Differentially expressed proteins in rainbow trout adipocytes isolated from visceral and subcutaneous tissues.

In rainbow trout, subcutaneous (in dorsal and ventral positions) and visceral fat deposits are known to influence the yield of edible flesh, whilst their respective roles in metabolism, storage and release of fatty acids have not, so far, been directly studied. The present work aimed to identify, by using 2D electrophoresis, proteins differentially expressed in isolated mature adipocytes originating from these various localizations in prepubescent females. A total of nine proteins were estimated to be differentially expressed according to the localisation of the adipocytes. Seven protein spots were considered to be present in the three fat deposits at differing abundances, and among them only six were estimated as being specific to fat tissues. Among these, five were more abundant in subcutaneous adipocytes of both sites compared to perivisceral adipocytes. Four were identified: three as H-FABP, ATP synthase, serum deprivation-response protein, indicating higher metabolic activity in subcutaneous adipocytes, while the latter, annexin, indicative of a higher proportion of less mature adipocytes, as also suggested by their smaller mean diameter. The more abundant protein in visceral isolated adipocytes is actin, known to be involved in cytoskeleton structure and to increase during adipogenesis. This allows us to suggest their more mature stage of development, in relation with their higher mean diameter.