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Simple Summary: Myomaker has been reported to play an important role in regulating myoblast fusion. However, the role of Myomaker gene in skeletal muscle growth in economic fish during post-hatching stage is unclear. This study showed that the growth of Chinese perch was significantly decreased when Myomaker was inhibited by Myomaker-siRNA. Furthermore, both the diameter of muscle fibers and the number of nuclei in single muscle fibers were significantly reduced in the Myomaker-siRNA group, whereas, there was no significant difference in the number of proliferating cells between the control and Myomaker-siRNA groups. Together, these findings indicate that Myomaker may promote the hypertrophy of muscle fibers and growth of fast muscle in Chinese perch by promoting myoblast fusion. The fusion of myoblasts is a crucial stage in the growth and development of skeletal muscle. Myomaker is an important myoblast fusion factor that plays a crucial role in regulating myoblast fusion. However, the function of Myomaker in economic fish during posthatching has been poorly studied. In this study, we found that the expression of Myomaker in the fast muscle of Chinese perch (Siniperca chuatsi) was higher than that in other tissues. To determine the function of Myomaker in fast muscle, Myomaker-siRNA was used to knockdown Myomaker in Chinese perch and the effect on muscle growth was determined. The results showed that the growth of Chinese perch was significantly decreased in the Myomaker-siRNA group. Furthermore, both the diameter of muscle fibers and the number of nuclei in single muscle fibers were significantly reduced in the Myomaker-siRNA group, whereas there was no significant difference in the number of BrdU-positive cells (proliferating cells) between the control and the Myomaker-siRNA groups. Together, these findings indicate that Myomaker may regulate growth of fast muscle in Chinese perch juveniles by promoting myoblast fusion rather than proliferation. [ABSTRACT FROM AUTHOR]

Carey Fineman Ziter Syndrome (CFZS) is a rare autosomal recessive disease caused by mutations in the MYMK locus which encodes the protein, myomaker. Myomaker is essential for fusion and concurrent myonuclei donation of muscle progenitors during growth and development. Strikingly, in humans, MYMK mutations appear to prompt myofiber hypertrophy but paradoxically, induce generalised muscle weakness. As the underlying cellular mechanisms remain unexplored, the present study aimed to gain insights by combining myofiber deep-phenotyping and proteomic profiling. Hence, we isolated individual muscle fibers from CFZS patients and performed mechanical, 3D morphological and proteomic analyses. Myofibers from CFZS patients were ~ 4x larger than controls and possessed ~ 2x more myonuclei than those from healthy subjects, leading to disproportionally larger myonuclear domain volumes. These greater myonuclear domain sizes were accompanied by smaller intrinsic cellular force generating-capacities in myofibers from CFZS patients than in control muscle cells. Our complementary proteomic analyses indicated remodelling in 233 proteins particularly those associated with cellular respiration. Overall, our findings suggest that myomaker is somewhat functional in CFZS patients, but the associated nuclear accretion may ultimately lead to non-functional hypertrophy and altered energy-related mechanisms in CFZS patients. All of these are likely contributors of the muscle weakness experienced by CFZS patients. [ABSTRACT FROM AUTHOR]

Myoblast fusion is essential for skeletal muscle development, growth, and regeneration. However, the molecular mechanisms underlying myoblast fusion and differentiation are not fully understood. Previously, we reported that interleukin-4 (IL-4) promotes myoblast fusion; therefore, we hypothesized that IL-4 signaling might regulate the expression of the molecules involved in myoblast fusion. In this study, we showed that in addition to fusion, IL-4 promoted the differentiation of C2C12 myoblast cells by inducing myoblast determination protein 1 (MyoD) and myogenin, both of which regulate the expression of myomerger and myomaker, the membrane proteins essential for myoblast fusion. Unexpectedly, IL-4 treatment increased the expression of myomerger, but not myomaker, in C2C12 cells. Knockdown of IL-4 receptor alpha (IL-4Rα) in C2C12 cells by small interfering RNA impaired myoblast fusion and differentiation. We also demonstrated a reduction in the expression of MyoD, myogenin, and myomerger by knockdown of IL-4Rα in C2C12 cells, while the expression level of myomaker remained unchanged. Finally, cell mixing assays and the restoration of myomerger expression partially rescued the impaired fusion in the IL-4Rα-knockdown C2C12 cells. Collectively, these results suggest that the IL-4/IL-4Rα axis promotes myoblast fusion and differentiation via the induction of myogenic regulatory factors, MyoD and myogenin, and myomerger. [ABSTRACT FROM AUTHOR]

Background: Skeletal muscle development and regeneration depend on cellular fusion of myogenic progenitors to generate multinucleated myofibers. These progenitors utilize two muscle-specific fusogens, Myomaker and Myomerger, which function by remodeling cell membranes to fuse to each other or to existing myofibers. Myomaker and Myomerger expression is restricted to differentiating progenitor cells as they are not detected in adult myofibers. However, Myomaker remains expressed in myofibers from mice with muscular dystrophy. Ablation of Myomaker from dystrophic myofibers results in reduced membrane damage, leading to a model where persistent fusogen expression in myofibers, in contrast to myoblasts, is harmful. Methods: Dox-inducible transgenic mice were developed to ectopically express Myomaker or Myomerger in the myofiber compartment of skeletal muscle. We quantified indices of myofiber membrane damage, such as serum creatine kinase and IgM+ myofibers, and assessed general muscle histology, including central nucleation, myofiber size, and fibrosis. Results: Myomaker or Myomerger expression in myofibers independently caused membrane damage at acute time points. This damage led to muscle pathology, manifesting with centrally nucleated myofibers and muscle atrophy. Dual expression of both Myomaker and Myomerger in myofibers exacerbated several aspects of muscle pathology compared to expression of either fusogen by itself. Conclusions: These data reveal that while myofibers can tolerate some level of Myomaker and Myomerger, expression of a single fusogen above a threshold or co-expression of both fusogens is damaging to myofibers. These results explain the paradigm that their expression in myofibers can have deleterious consequences in muscle pathologies and highlight the need for their highly restricted expression during myogenesis and fusion. [ABSTRACT FROM AUTHOR]

Skeletal muscle formation is an extremely important step in animal growth and development. Recent studies have found that TMEM8c (also known as Myomaker, MYMK), a muscle-specific transmembrane protein, can promote myoblast fusion and plays a key role in the normal development of skeletal muscle. However, the effect of Myomaker on porcine (Sus scrofa) myoblast fusion and the underlying regulatory mechanisms remain largely unknown. Therefore, in this study, we focused on the role and corresponding regulatory mechanism of the Myomaker gene during skeletal muscle development, cell differentiation, and muscle injury repair in pigs. We obtained the entire 3′ UTR sequence of porcine Myomaker using the 3′ RACE approach and found that miR-205 inhibited porcine myoblast fusion by targeting the 3′ UTR of Myomaker. In addition, based on a constructed porcine acute muscle injury model, we discovered that both the mRNA and protein expression of Myomaker were activated in the injured muscle, while miR-205 expression was significantly inhibited during skeletal muscle regeneration. The negative regulatory relationship between miR-205 and Myomaker was further confirmed in vivo. Taken together, the present study reveals that Myomaker plays a role during porcine myoblast fusion and skeletal muscle regeneration and demonstrates that miR-205 inhibits myoblast fusion through targeted regulation of the expression of Myomaker. [ABSTRACT FROM AUTHOR]

Human antigen R (HuR) is an RNA-binding protein that contributes to a wide variety of biological processes and diseases. HuR has been demonstrated to regulate muscle growth and development, but its regulatory mechanisms are not well understood, especially in goats. In this study, we found that HuR was highly expressed in the skeletal muscle of goats, and its expression levels changed during longissimus dorsi muscle development in goats. The effects of HuR on goat skeletal muscle development were explored using skeletal muscle satellite cells (MuSCs) as a model. The overexpression of HuR accelerated the expression of myogenic differentiation 1 (MyoD), Myogenin (MyoG), myosin heavy chain (MyHC), and the formation of myotubes, while the knockdown of HuR showed opposite effects in MuSCs. In addition, the inhibition of HuR expression significantly reduced the mRNA stability of MyoD and MyoG. To determine the downstream genes affected by HuR at the differentiation stage, we conducted RNA-Seq using MuSCs treated with small interfering RNA, targeting HuR. The RNA-Seq screened 31 upregulated and 113 downregulated differentially expressed genes (DEGs) in which 11 DEGs related to muscle differentiation were screened for quantitative real-time PCR (qRT-PCR) detection. Compared to the control group, the expression of three DEGs (Myomaker, CHRNA1, and CAPN6) was significantly reduced in the siRNA-HuR group (p < 0.01). In this mechanism, HuR bound to Myomaker and increased the mRNA stability of Myomaker. It then positively regulated the expression of Myomaker. Moreover, the rescue experiments indicated that the overexpression of HuR may reverse the inhibitory impact of Myomaker on myoblast differentiation. Together, our findings reveal a novel role for HuR in promoting muscle differentiation in goats by increasing the stability of Myomaker mRNA. [ABSTRACT FROM AUTHOR]

Skeletal muscle is formed by multinucleated myofibers originated by waves of hyperplasia and hypertrophy during myogenesis. Tissue damage triggers a regeneration process including new myogenesis and muscular remodeling. During myogenesis, the fusion of myoblasts is a key step that requires different genes' expression, including the fusogens myomaker and myomixer. The present work aimed to characterize these proteins in gilthead sea bream and their possible role in in vitro myogenesis, at different fish ages and during muscle regeneration after induced tissue injury. Myomaker is a transmembrane protein highly conserved among vertebrates, whereas Myomixer is a micropeptide that is moderately conserved. myomaker expression is restricted to skeletal muscle, while the expression of myomixer is more ubiquitous. In primary myocytes culture, myomaker and myomixer expression peaked at day 6 and day 8, respectively. During regeneration, the expression of both fusogens and all the myogenic regulatory factors showed a peak after 16 days post-injury. Moreover, myomaker and myomixer were present at different ages, but in fingerlings there were significantly higher transcript levels than in juveniles or adult fish. Overall, Myomaker and Myomixer are valuable markers of muscle growth that together with other regulatory molecules can provide a deeper understanding of myogenesis regulation in fish. [ABSTRACT FROM AUTHOR]

The whole-genome sequence of the olive flounder (Paralichthys olivaceus) provides a basis for gene functional analyses, which is important for the aquaculture industry. Understanding gene function will help us to select better economic traits such as fast growth and better culture conditions, which further will increase the aquaculture output. Gene knockout is an important reverse genetics approach for in vivo studies of gene function. In this study, the CRISPR/Cas9 genome editing method with a microinjection system using a simple braked needle was employed in olive flounder. After injection in embryos, green fluorescent protein expression was detected in 40% of larvae. The proportion of normal-hatched larvae was approximately 50%. Different mutations, including short indels and fragment deletions, were found in our test genes gsdf and myomaker. Additionally, we detected more than one mutation in a single larva. In summary, our microinjection technique and CRISPR/Cas9 can be applied to study gene functions in olive flounder. [ABSTRACT FROM AUTHOR]

Background: Spinal muscular atrophy is an inherited neurodegenerative disease caused by insufficient levels of the survival motor neuron (SMN) protein. Recently approved treatments aimed at increasing SMN protein levels have dramatically improved patient survival and have altered the disease landscape. While restoring SMN levels slows motor neuron loss, many patients continue to have smaller muscles and do not achieve normal motor milestones. While timing of treatment is important, it remains unclear why SMN restoration is insufficient to fully restore muscle size and function. We and others have shown that SMN‐deficient muscle precursor cells fail to efficiently fuse into myotubes. However, the role of SMN in myoblast fusion is not known. Methods: In this study, we show that SMN‐deficient myoblasts readily fuse with wild‐type myoblasts, demonstrating fusion competency. Conditioned media from wild type differentiating myoblasts do not rescue the fusion deficit of SMN‐deficient cells, suggesting that compromised fusion may primarily be a result of altered membrane dynamics at the cell surface. Transcriptome profiling of skeletal muscle from SMN‐deficient mice revealed altered expression of cell surface fusion molecules. Finally, using cell and mouse models, we investigate if myoblast fusion can be rescued in SMN‐deficient myoblast and improve the muscle pathology in SMA mice. Results: We found reduced expression of the muscle fusion proteins myomaker (P = 0.0060) and myomixer (P = 0.0051) in the muscle of SMA mice. Suppressing SMN expression in C2C12 myoblast cells reduces expression of myomaker (35% reduction; P < 0.0001) and myomixer, also known as myomerger and minion, (30% reduction; P < 0.0001) and restoring SMN levels only partially restores myomaker and myomixer expression. Ectopic expression of myomixer improves myofibre number (55% increase; P = 0.0006) and motor function (35% decrease in righting time; P = 0.0089) in SMA model mice and enhances motor function (82% decrease in righting time; P < 0.0001) and extends survival (28% increase; P < 0.01) when administered in combination with an antisense oligonucleotide that increases SMN protein levels. Conclusions: Here, we identified reduced expression of muscle fusion proteins as a key factor in the fusion deficits of SMN‐deficient myoblasts. This discovery provides a novel target to improve SMA muscle pathology and motor function, which in combination with SMN increasing therapy could enhance clinical outcomes for SMA patients. [ABSTRACT FROM AUTHOR]

Myomaker is a muscle-specific membrane protein that is essential for myoblast fusion. Myomaker is regulated by myoblast determination protein (MyoD), a muscle-specific basic helix-loop-helix (bHLH) transcription factor in higher vertebrates. However, the transcriptional regulatory mechanism of the myomaker gene has not been explored in marine fishes. In the present study, molecular cloning, bioinformatic analysis and transcriptional analysis of Acanthopagrus latus myomaker (Almyomaker) were performed. The open reading frame (ORF) sequence of Almyomaker is 858 bp, which encodes a polypeptide of 285 amino acids. Moreover, phylogenetic and gene structure analysis indicates that Almyomaker is highly conserved among vertebrates. The tissue distribution pattern shows that Almyomaker is more highly expressed in white muscle than in other tissues. Furthermore, to explore whether two MyoDs are modulators of Almyomaker, a promoter analysis was performed using progressive deletion mutations of Almyomaker. The results of promoter activity assays show that Almyomaker expression is notably activated by two MyoDs. Transcriptional activity of the Almyomaker promoter was observed to dramatically decrease after targeted mutation of the MyoD1 M1 and MyoD2 M2 binding sites. In summary, MyoD1 and MyoD2 play an important role in the regulation of Almyomaker expression and may promote myoblast fusion during muscle development and growth by modulating Almyomaker expression. [ABSTRACT FROM AUTHOR]

Skeletal muscle plays essential roles in motor function, energy, and glucose metabolism. Skeletal muscle formation occurs through a process called myogenesis, in which a crucial step is the fusion of mononucleated myoblasts to form multinucleated myofibers. The myoblast/myocyte fusion is triggered and coordinated in a muscle-specific way that is essential for muscle development and post-natal muscle regeneration. Many molecules and proteins have been found and demonstrated to have the capacity to regulate the fusion of myoblast/myocytes. Interestingly, two newly discovered muscle-specific membrane proteins, Myomaker and Myomixer (also called Myomerger and Minion), have been identified as fusogenic regulators in vertebrates. Both Myomaker and Myomixer–Myomerger–Minion have the capacity to directly control the myogenic fusion process. Here, we review and discuss the latest studies related to these two proteins, including the discovery, structure, expression pattern, functions, and regulation of Myomaker and Myomixer–Myomerger–Minion. We also emphasize and discuss the interaction between Myomaker and Myomixer–Myomerger−Minion, as well as their cooperative regulatory roles in cell–cell fusion. Moreover, we highlight the areas for exploration of Myomaker and Myomixer–Myomerger–Minion in future studies and consider their potential application to control cell fusion for cell-therapy purposes. [ABSTRACT FROM AUTHOR]

Myoblast fusion is a vital step for skeletal muscle development, growth, and regeneration. Loss of Jamb, Jamc, or Myomaker (Mymk) function impaired myoblast fusion in zebrafish embryos. In addition, mymk mutation hampered fish muscle growth. However, the effect of Jamb and Jamc deficiency on fish muscle growth is not clear. Moreover, whether jamb;jamc and jamb;mymk double mutations have stronger effects on myoblast fusion and muscle growth remains to be investigated. Here, we characterized the muscle development and growth in jamb, jamc, and mymk single and double mutants in zebrafish. We found that although myoblast fusion was compromised in jamb and jamc single or jamb;jamc double mutants, these mutant fish showed no defect in muscle cell fusion during muscle growth. The mutant fish were able to grow into adults that were indistinguishable from the wild-type sibling. In contrast, the jamb;mymk double mutants exhibited a stronger muscle phenotype compared to the jamb and jamc single and double mutants. The jamb;mymk double mutant showed reduced growth and partial lethality, similar to a mymk single mutant. Single fiber analysis of adult skeletal myofibers revealed that jamb, jamc, or jamb;jamc mutants contained mainly multinucleated myofibers, whereas jamb;mymk double mutants contained mostly mononucleated fibers. Significant intramuscular adipocyte infiltration was found in skeletal muscles of the jamb;mymk mutant. Collectively, these studies demonstrate that although Jamb, Jamc, and Mymk are all involved in myoblast fusion during early myogenesis, they have distinct roles in myoblast fusion during muscle growth. While Mymk is essential for myoblast fusion during both muscle development and growth, Jamb and Jamc are dispensable for myoblast fusion during muscle growth. [ABSTRACT FROM AUTHOR]

Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood. [ABSTRACT FROM AUTHOR]

The fusion of muscle precursor cells is a required event for proper skeletal muscle development and regeneration. Numerous proteins have been implicated to function in myoblast fusion; however, the majority are expressed in diverse tissues and regulate numerous cellular processes. How myoblast fusion is triggered and coordinated in a muscle-specific manner has remained a mystery for decades. Through the discovery of two muscle-specific fusion proteins, Myomaker and Myomerger-Minion, we are now primed to make significant advances in our knowledge of myoblast fusion. This article reviews the latest findings regarding the biology of Myomaker and Minion-Myomerger, places these findings in the context of known pathways in mammalian myoblast fusion, and highlights areas that require further investigation. As our understanding of myoblast fusion matures so does our potential ability to manipulate cell fusion for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Cell-cell fusion is essential for fertilization and organ development. Dedicated proteins known as fusogens are responsible for mediating membrane fusion. However, until recently, these proteins either remained unidentified or were poorly understood at the mechanistic level. Here, we review how fusogens surmount multiple energy barriers to mediate cell-cell fusion. We describe how early preparatory steps bring membranes to a distance of ~10 nm, while fusogens act in the final approach between membranes. The mechanical force exerted by cell fusogens and the accompanying lipidic rearrangements constitute the hallmarks of cell-cell fusion. Finally, we discuss the relationship between viral and eukaryotic fusogens, highlight a classification scheme regrouping a superfamily of fusogens called Fusexins, and propose new questions and avenues of enquiry. [ABSTRACT FROM AUTHOR]

Skeletal muscle formation requires fusion of mononucleated myoblasts to form multinucleated myofibers. The muscle-specific membrane proteins myomaker and myomixer cooperate to drive mammalian myoblast fusion. Whereas myomaker is highly conserved across diverse vertebrate species, myomixer is a micropeptide that shows relatively weak cross-species conservation. To explore the functional conservation of myomixer, we investigated the expression and function of the zebrafish myomixer ortholog. Here we show that myomixer expression during zebrafish embryogenesis coincides with myoblast fusion, and genetic deletion of myomixer using CRISPR/Cas9 mutagenesis abolishes myoblast fusion in vivo. We also identify myomixer orthologs in other species of fish and reptiles, which can cooperate with myomaker and substitute for the fusogenic activity of mammalian myomixer. Sequence comparison of these diverse myomixer orthologs reveals key amino acid residues and a minimal fusogenic peptide motif that is necessary for promoting cell-cell fusion with myomaker. Our findings highlight the evolutionary conservation of the myomaker-myomixer partnership and provide insights into the molecular basis of myoblast fusion. [ABSTRACT FROM AUTHOR]

The fusion of myoblasts is an important step during skeletal muscle differentiation. A recent study in mice found that a transmembrane protein called Myomaker, which is specifically expressed in muscle, is critical for myoblast fusion. However, the cellular mechanism of its roles and the regulatory mechanism of its expression remain unclear. Chicken not only plays an importantrole in meat production but is also an ideal model organism for muscle development research. Here, we report that Myomaker is also essential for chicken myoblast fusion. Forced expression of Myomaker in chicken primary myoblasts promotes myoblast fusion, whereas knockdown of Myomaker by siRNA inhibits myoblast fusion. MYOD and MYOG, which belong to the family of myogenic regulatory factors, can bind to a conserved E-box located proximal to the Myomaker transcription start site and induce Myomaker transcription. Additionally, mi-140-3p can inhibit Myomaker expression and myoblast fusion, at least in part, by binding to the 3' UTR of Myomaker in vitro. These findings confirm the essential roles of Myomaker in avian myoblast fusion and show that MYOD, MYOG and mi-140-3p can regulate Myomaker expression. [ABSTRACT FROM AUTHOR]

From lifeless viral particles to complex multicellular organisms, membrane fusion is inarguably the important fundamental biological phenomena. Sitting at the heart of membrane fusion are protein mediators known as fusogens. Despite the extensive functional and structural characterization of these proteins in recent years, scientists are still grappling with the fundamental mechanisms underlying membrane fusion. From an evolutionary perspective, fusogens follow divergent evolutionary principles in that they are functionally independent and do not share any sequence identity; however, they possess structural similarity, raising the possibility that membrane fusion is mediated by essential motifs ubiquitous to all. In this review, we particularly emphasize structural characteristics of small-molecular-weight fusogens in the hope of uncovering the most fundamental aspects mediating membrane–membrane interactions. By identifying and elucidating fusion-dependent functional domains, this review paves the way for future research exploring novel fusogens in health and disease. [ABSTRACT FROM AUTHOR]