The muscle specific Myomaker protein that controls myoblast fusion was initially found in mice as a 221 aa protein, and it was described to have a similar transcription profile as those of myod and myogenin 10. In fish, Myomaker was first described in zebrafish 7, and recently it has been characterized in rainbow trout 8 and yellowfin seabream (Acanthopagrus latus) 23. In all the three species, the gene is structured in 6 exons, but differences in the length of the protein are marked, being as long as 434 aa in rainbow trout 8, while just 285 aa in non-salmonid species such as yellowfin seabream 23 and gilthead seabream. The protein sequence alignment between mice, rainbow trout and gilthead seabream, pointed out that the N-terminal half of the rainbow trout Myomaker was similar to the mice and gilthead seabream sequences, while the C-terminal half did not have homology with any known motifs 8. The phylogenetic analysis showed that Myomaker is a well conserved protein across vertebrate organisms, from fish to mammals 8,10. The gilthead sea bream Myomaker protein presented a homology of 89%, 81%and 71% with zebrafish, rainbow trout and mouse, respectively. Moreover, a clear evolution among fish species was observed. and the gilthead sea bream Myomaker sequence resulted more closely related to other perciform species, such as the European sea bass (D. labrax), the beloniform species, such as O. latipes, or the salmonids.
The fusogenic gene myomixer was also firstly identified in mice, contained a single exon and encoded an 84 aa protein. Additionally, the mice myomixer gene had another transcript form, less conserved, which had 3 exons and yielded a protein of 108 aa 9,12, but until today, a single myomixer transcript has been described in the few fish species where this gene has been studied. Indeed, the only described gilthead sea bream myomixer transcript (ENSSAUT00010028952.1) that was found in the gilthead sea bream genome deposited in Ensembl contained only one exon that encoded a 75 aa protein, similarly to the rainbow trout myomixer (77 aa), although in this salmonid species, the gene is structured in two exons 13.
The Myomixer protein sequence showed weak cross-species conservation, with mammals and fish sharing only 36% identity 13,15. Among fish, the Myomixer gilthead sea bream showed an homology of 70.67% and 69.33% with the zebrafish and rainbow trout Myomixer protein sequences, respectively (unpublished data). Nevertheless, the crucial AxLyCxL motif of the Myomixer micropeptide presented a high conservation across vertebrates. As in the case of Myomaker, the gilthead sea bream Myomixer sequence was more related to that from other perciformes, such as the European sea bass, beloniformes or salmoniformes. Other phylogenetic analysis performed in gilthead sea bream, such as those for Myogenin and Preproghrelin proteins also showed that these molecules evolved in the same way, being closer to other perciforms, while being more distant to salmoniformes or cypriniformes 24.
Regarding the tissue screening, on one side, the gene expression of myomaker in gilthead sea bream showed a narrow distribution among tissues, being expressed mainly in white and red skeletal muscles. Such expression distribution was also observed in rainbow trout 8 and yellowfin seabream 23. On the other hand, the myomixer pattern of expression in adult gilthead sea bream was not restricted to white and red muscles, thus differing from the findings in rainbow trout and mice 12,13.
While in mammals, regenerative myogenesis is a well-known process, in fish, some aspects remain unclear. Regeneration in gilthead sea bream after muscle injury was first studied by Rowlerson and coworkers 25. The histological analysis showed a high cellular proliferation with a greater deposition of connective tissue and new small myofibers formation around the lesion site by 7–11 days after the injury was made. In rainbow trout muscle, after 20 and 30 days of a mechanical injury, an alteration of the muscle fiber organization was found at the site of damage due to a high deposit of connective tissue with small muscle fibers 8,26. The regenerative process is not complete at that time and coincides with the onset of a peak of myogenin, myomaker and myomixer expression 8,27. The differences observed between the two species regarding the moment when the new myofibers were formed during myogenesis could be due to the distinct metabolic rates, being higher in the gilthead sea bream, reared at 21-23ºC, compared to the rainbow trout, reared at 10-15ºC 8,13,28. At day 1, the injury was easily observed with the naked eye, while on days 8 and 16, the muscle damage was no longer visually noticeable (pictures shown in Supplementary Material). This could mean that new myofibers would already be forming to repair the muscle injury. At a transcriptional level, there were appreciable differences between rainbow trout and gilthead sea bream indicating also a faster regenerative process in the latter. In the current study, the expression of MRFs, along with the myomaker and myomixer fusogens was evaluated through the regenerative process. In mice, both myomaker and myomixer expression was strongly detected in regenerating muscle 3 days after the injury and then rapidly decreased in less than 2 days when the new myofibers were formed, which indicated that both proteins are essential for muscle regeneration 9,10. The regulation of the expression of both genes is mediated by two E-boxes in the promoter, which are described as targets of MyoD and Myogenin 14. Furthermore, specifically knocking out myomaker in the mice satellite cells in vivo completely impaired myoblast fusion, thus resulting in a complete blocking of muscle regeneration 11.
In fish, the implication of Myomaker and Myomixer in muscle regeneration has only been studied recently in rainbow trout, where the expression of both genes, drastically increased only at 30 days post injury, along with myogenin 8,13. Such response coincides with the observations in injured mice models where, although the regeneration process occurs in a much shorter time, the expression of both myomaker and myomixer peaked at 3 days post injury5. In the present study, the muscle regeneration experiment in gilthead sea bream showed that myomaker, myomixer and all the MRFs were strongly upregulated at day 16, while most of the genes were decreasing at day 30, thus presenting the gilthead sea bream a faster response than that observed in trout, where the analyzed genes were upregulated only after 30 days of recovery 8. Moreover, another aspect that could affect the metabolic rate, in addition to the temperature at which the fish are reared, is the influence of fish size. In the regeneration experiment with rainbow trout performed by Landemaine and coworkers 8 the authors used 1 kg fish, in comparison with the 15 g gilthead sea bream that were utilized in the present study. In general, smaller fish shows higher metabolic rates and therefore, faster muscle regeneration. Moreover, the present findings are in agreement with those of Rowlerson and coworkers 25 by histological studies in gilthead sea bream, as the new small myofibers were deposited in the lesion site around day 8 post-injury, at the same moment that it was observed a significant increase in MRFs, myomaker and myomixer gene expression. Overall, these data support the importance of both, Myomaker and Myomixer in the regenerative process of skeletal muscle in gilthead sea bream.
The in vitro study performed showed that myomaker presented a peak of expression levels at day 6 decreasing later progressively, while for myomixer, the highest expression was found at day 8 This temporal distribution is in agreement with the previously described role of both molecules 5. Thus, the action of Myomaker inducing the hemifusion of the membranes would happen slightly earlier than that of Myomixer, which is the formation and expansion of the pore between the plasma membranes 5. The current results coincide with those obtained in rainbow trout in vitro myoblasts, where the expression of both myomaker and myomixer was increasing progressively during differentiation stages 8,13.
The MRF expression during in vitro gilthead sea bream myogenesis was well described by our group 17 and the results obtained here are consistent with that first report. The myod1 early peak of expression agrees with the function of this transcription factor at the onset of the myogenesis in conjunction with the myod2, which classically appears more delayed than the myod1 29. The upregulation of myogenin at day 6 and the maintenance of high levels still at day 8 coincide with its role regulating the myoblast differentiation progress, as well as with the maximum levels of mrf4 at day 8, a factor more involved in the finalization and maturation of myotubes. Thus, the high parallelism between myogenin, myomaker and myomixer expression is quite clear and it is consequent with the role of these three factors on the later stages of myogenesis. In fact, the interaction of both fusogens with Myogenin could be explained by the presence of E-boxes in their promoters as pointed out in rainbow trout 8.
Finally, the comparison of myomaker and myomixer transcript levels in fish at different ages suggests that both factors play a more active function at the stage of fingerlings. These results are in agreement with the findings described in rainbow trout 8,13 where the expression of myomixer and myomaker were maximum at the stage of embryo, decreasing progressively at 15, 150 and 1500 grams. All this information supports the role of both factors in somitogenesis or strong growing stages such us in fingerlings to decrease in juveniles or adults where the level of hyperplasia is less important. Thus, in mouse and zebrafish, the expression of myomixer declines soon after somitogenesis 9,15, whereas in trout its expression is maintained throughout post-larval growth, i.e., in fry, juvenile and to a lesser extend in mature fish.
Overall, the present results support that myomaker and myomixer play in gilthead seabream an important role not only during developmental myogenesis, especially at the second part of the process, when the myocytes differentiation takes place, but also during regenerative myogenesis where their upregulation takes place only after 16 days of recovery, pointing out their role during the later differentiation stages. Therefore, our results contribute to understand the role of myomaker and myomixer in a fish species of undetermined growth, normally living at high temperature waters and with high interest for aquaculture.