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

Abstract

The formation of new myofibers in vertebrates occurs by myoblast fusion and requires fusogenic activity of the musclespecific 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 (ϳ40 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 (ϳ70 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.

Myomixer is expressed during embryonic and post-larval hyperplasia, muscle regeneration and fusion of myoblats in rainbow trout (Oncorhynchus mykiss)

“…in fry, juvenile and to a lesser extend in mature fish. Our results clearly indicate that the expression pattern of myomixer is similar to that of the myomaker in trout (Landemaine et al, 2019) during embryonic and post-larval stages. In addition, we did not observe myomixer and myomaker expression in myofibers, but only in small cells that should be fusing muscle precursors.…”

“…To determine whether myomixer is up-regulated during the muscle regeneration, we measured its expression in muscle following mechanical injury. In our previous study, we observed that the formation of new fibers and the increase of myogenin expression occurred 30 days following injury (Landemaine et al, 2019). Consistently, myomixer expression remained stable up to 16 days and was sharply up-regulated on day 30 with 6-fold higher expression in injured muscle than in the control one ( Figure 5).…”

“…Our qPCR analyses showed that myomixer expression was strongly stimulated in white muscle 30 days after injury, in parallel with the appearance of newly formed myofibers (Landemaine et al, 2019;Montfort et al, 2016). This kinetic of myomixer expression during muscle regeneration, is comparable to that one of myomaker and myogenin (Landemaine et al, 2019).…”

“…As described in Landemaine et al, (2019), this experiment was carried out at the INRAE facility PEIMA (Sizun, Britany, France). Briefly, 1530 ± 279 g rainbow trout ( O. mykiss ) were anesthetized with MS-222 (50 ml/l) and using a sterile 1.2-mm needle, the left side of each fish was injured by a puncture behind to the dorsal fin and above the lateral line.…”

“…Recently, we identified the unique myomaker ortholog in rainbow trout and revealed its unusual sequence. Indeed, the trout myomaker protein contains 14 minisatellites and two sequence extensions leading to a protein of 434 aa instead of 221 in zebrafish (Landemaine et al, 2019). In vitro , ectopic expression of trout myomaker in mouse fibroblasts promotes fusion with C2C12 myoblasts.…”