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Complete loss of syncytial myotubes upon deletion of MymK gene in human myoblasts. (A) Human Myomaker (MymK) gene structure and the positions of gRNAs and genotyping primers. (B) MymK genotyping results for three KO clones. Arrow points to the position of WT-size amplicon. (C) Sanger sequencing results of MymK genotyping PCR products as shown in (B). The frameshifted codons were highlighted in red. Arrow indicates the position of big deletion. (D) Western blot analysis of myosin heavy chain. Cells are differentiated for 3 days. (E) Myosin immunostaining results of WT and MymK KO myoblasts. Cells were differentiated for 3 days. Arrow points to multinucleated myotube. Nuclei were counterstained with Hoechst and pseudo-colored in green. Scale bar, 100 m. (F) Measurements of fusion for myosin + WT and MymK KO myoblasts. n = 3. The ratio of mononuclear cells was used for statistical analysis. ***P < 0.001. Data are means ± SEM. (G) Myosin immunostaining results to show the rescue of fusion defects of human MymK KO myoblasts by retroviral MymK expression. Cells were differentiated for 3 days. Scale bar, 100 m.
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Myoblast fusion is essential for formations of myofibers, the basic cellular and functional units of skeletal muscles. Recent genetic studies in mice identified two long-sought membrane proteins, Myomaker and Myomixer, which cooperatively drive myoblast fusion. It is unknown whether and how human muscles, with myofibers of tremendously larger size,...
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... were also confirmed by Western blotting analyses (Fig. 1E). Although the expression of myosin varied among MymX KO clones at an early stage of differentiation, their levels were comparable with those from WT cells after full-term differentiation (Fig. 1E). Consistently, MyoG, MYH8, and MymK were expressed at similar levels between genotypes ( fig. S2A), indicating that the differentiation program of human muscle precursor cells was not affected by the absence of MymX ...
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... myotubes that spread over large culture areas for the control group, 63% of MymX KO cells remained mononucleated after full-term differentiation (Fig. 1G). The rest appeared as either binucleated myocytes or small myotubes that contained an average of 4.5 myonuclei (Fig. 1G). Similar results were recapitulated from another three MymX KO clones ( fig. S2, B to D). To verify that the fusion defect was attributed to the exact loss of MymX gene but not to a rare CRISPR off-target effect (if any), we performed rescue experiments. Fusion defects of MymX KO cells can be faithfully rescued by introducing MymX expression construct that harbors silent mutations in the protospacer sequences of ...
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... with mouse MymX KO myoblasts that only rarely fused to generate small myotubes (12)(13)(14), large syncytia that host 6 to 10 myonuclei can be found in human MymX KO culture (Fig. 1G). Although inactivation of MymX gene did not affect MymK expression ( fig. S2A), we tested whether a higher level of MymK could induce a stronger fusion of MymX KO myoblasts. Overexpression of human MymK in MymX KO myoblasts significantly increased the abundance of multinucleations from which even larger syncytia were formed ( fig. S3). This result indicated that human MymX KO myoblasts can fuse in a MymK ...
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... discern these possibilities, we first examined the role of MymK in human myoblasts by CRISPR mutagenesis (Fig. 2A). Genotyping and sequencing revealed biallelic frameshift mutations in all single clones (Fig. 2, B and C) except one allele in clone #G7 that showed in-frame deletions of 30 amino acids (Fig. 2C). Again, the loss of MymK did not affect myogenic differentiation, as normal expression levels for myosin and MyoG were detected (Fig. 2D and ...
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... discern these possibilities, we first examined the role of MymK in human myoblasts by CRISPR mutagenesis (Fig. 2A). Genotyping and sequencing revealed biallelic frameshift mutations in all single clones (Fig. 2, B and C) except one allele in clone #G7 that showed in-frame deletions of 30 amino acids (Fig. 2C). Again, the loss of MymK did not affect myogenic differentiation, as normal expression levels for myosin and MyoG were detected (Fig. 2D and fig. S4A). Human MymK KO myoblasts showed a complete failure of fusion because no muscle ...
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... discern these possibilities, we first examined the role of MymK in human myoblasts by CRISPR mutagenesis (Fig. 2A). Genotyping and sequencing revealed biallelic frameshift mutations in all single clones (Fig. 2, B and C) except one allele in clone #G7 that showed in-frame deletions of 30 amino acids (Fig. 2C). Again, the loss of MymK did not affect myogenic differentiation, as normal expression levels for myosin and MyoG were detected (Fig. 2D and fig. S4A). Human MymK KO myoblasts showed a complete failure of fusion because no muscle syncytium (three or more nuclei) was found after the full-term differentiation (Fig. 2, E and F). Same ...
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... mutagenesis (Fig. 2A). Genotyping and sequencing revealed biallelic frameshift mutations in all single clones (Fig. 2, B and C) except one allele in clone #G7 that showed in-frame deletions of 30 amino acids (Fig. 2C). Again, the loss of MymK did not affect myogenic differentiation, as normal expression levels for myosin and MyoG were detected (Fig. 2D and fig. S4A). Human MymK KO myoblasts showed a complete failure of fusion because no muscle syncytium (three or more nuclei) was found after the full-term differentiation (Fig. 2, E and F). Same phenotypes were recapitulated from another three MymK KO clones ( fig. S4, B to D). Validating the specificity of CRISPR targeting, fusion defects of ...
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... deletions of 30 amino acids (Fig. 2C). Again, the loss of MymK did not affect myogenic differentiation, as normal expression levels for myosin and MyoG were detected (Fig. 2D and fig. S4A). Human MymK KO myoblasts showed a complete failure of fusion because no muscle syncytium (three or more nuclei) was found after the full-term differentiation (Fig. 2, E and F). Same phenotypes were recapitulated from another three MymK KO clones ( fig. S4, B to D). Validating the specificity of CRISPR targeting, fusion defects of these human MymK KO cells were rescued by introducing a gRNAinsensitive expression cassette for human MymK (Fig. 2G). Together, MymK is absolutely required for human ...
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... or more nuclei) was found after the full-term differentiation (Fig. 2, E and F). Same phenotypes were recapitulated from another three MymK KO clones ( fig. S4, B to D). Validating the specificity of CRISPR targeting, fusion defects of these human MymK KO cells were rescued by introducing a gRNAinsensitive expression cassette for human MymK (Fig. 2G). Together, MymK is absolutely required for human myoblast ...
Citations
... Human MYOD1-knockout myoblasts were generated by CRISPR-Cas9 mediated gene editing 469 and cultured as described previously (Zhang et al., 2020). Retroviral expression vector pMXs-470 assistance and Shweta Biliya for help with RNAseq library preparation and sequencing. ...
Vertebrates and tunicates are sister groups that share a common fusogenic factor, Myomaker (Mymk), that drives myoblast fusion and muscle multinucleation. Yet they are divergent in when and where they express Mymk. In vertebrates, all developing skeletal muscles express Mymk and are obligately multinucleated. In tunicates, Mymk is only expressed in post-metamorphic multinucleated muscles, but is absent from mononucleated larval muscles. In this study, we demonstrate that cis- regulatory sequence differences in the promoter region of Mymk underlie the different spatiotemporal patterns of its transcriptional activation in tunicates and vertebrates. While in vertebrates Myogenic Regulatory Factors (MRFs) like MyoD1 alone are required and sufficient for Mymk transcription in all skeletal muscles, we show that transcription of Mymk in post-metamorphic muscles of the tunicate Ciona requires the combinatorial activity of MRF/MyoD and Early B-Cell Factor (Ebf). This macroevolutionary difference appears to be encoded in cis, likely due to the presence of a putative Ebf binding site adjacent to predicted MRF binding sites in the Ciona Mymk promoter. We further discuss how Mymk and myoblast fusion might have been regulated in the last common ancestor of tunicates and vertebrates, for which we propose two models.
... Myoblast fusion is a systematic process that involves migration and alignment of membranes of fusion-competent myoblasts, remodelling of cytoskeleton at contact sites followed by opening of fusion pores to allow movement of cytoplasmic content, and eventually amalgamation of two myogenic cells into one (4)(5)(6). Recently, two transmembrane proteins, named Myomaker and Myomerger (also known as Myomixer and Minion) have been identified as major drivers of myoblast fusion in diverse conditions (7)(8)(9)(10). It is also now increasingly clear that myoblast fusion is regulated by multiple signaling pathways that are activated due to interaction of specific membrane proteins between fusion partners or as a part of myogenic differentiation program (11). ...
... Myomaker, a membrane protein, is critical for myoblast fusion. A recent study demonstrated that promoter of Mymk contains E-Box motifs to which MyoD transcription factor binds to induce the gene expression of Myomaker for myotube formation (9). The results of the present study suggest the IRE1α-XBP1 signaling axis promotes myoblast fusion through transcriptional upregulation of Mymk gene (Fig. 6). ...
Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. Inositol requiring enzyme 1 alpha (IRE1α) is one of the arms of the unfolded protein response (UPR) that regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in adult muscle stem cells (i.e. satellite cells) of mice impairs skeletal muscle regeneration primarily through inhibiting myoblast fusion step. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits fusion of cultured myoblasts during myogenesis. Genome-wide transcriptome analysis revealed that knockdown of IRE1α or XBP1 deregulates the gene expression of molecules involved in the regulation of myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including Myomaker (Mymk) during myogenic differentiation. Our study demonstrates that spliced XBP1 (sXBP1) transcription factor binds to the promoter region of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Finally, our results show that inducible deletion of IRE1α in satellite cells inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation in the gene expression of profusion molecules. Significance Statement: Myoblast fusion is an essential step for regeneration and post-natal growth of skeletal muscle. We demonstrate that the activation of the IRE1α/XBP1 arm of the unfolded protein response induces myoblast fusion through augmenting the gene expression of multiple profusion molecules, including myomaker. This study has identified a novel signaling axis that link ER stress-induced non-myogenic signaling pathway to myoblast fusion. Augmenting the activity of IRE1α/XBP1 pathway could be a potential therapeutic strategy for various muscle degenerative diseases.
... initial cell types, including adipocytes, neuroblastomas, and liver cells, into myogenic cells 29 . MYMK, MYMX, MYOG, and MYHC are regulated under the control of MyoD1 28,30 . Regarding UDCs, MyoD1 needs other epigenetic factors to convert them to the skeletal muscle lineage. ...
Human urine-derived cells (UDCs) are primary cultured cells originating from the upper urinary tract and are known to be multipotent. We previously developed MYOD1 -transduced UDCs (MYOD1-UDCs) as a model recapitulating the pathogenesis of Duchenne muscular dystrophy (DMD) caused by a lack of dystrophin. MYOD1-UDCs also allow evaluation of the efficacy of exon skipping with antisense oligonucleotides. However, despite the introduction of MYOD1 , some MYOD1-UDCs failed to form myotubes, possibly because of heterogeneity among UDCs. Here, we carried out single-cell RNA-sequencing analyses and revealed that CD90/Thy-1 was highly expressed in a limited subpopulation of UDCs with high myogenic potency. Furthermore, CD90-positive MYOD1-UDCs, but not CD90-negative cells, could form myotubes expressing high levels of myosin heavy chain and dystrophin. Notably, overexpression of CD90 in CD90-negative MYOD1-UDCs did not enhance myogenic differentiation, whereas CD90 suppression in CD90-positive UDCs led to decreased myotube formation and decreased myosin heavy chain expression. CD90 may thus contribute to the fusion of single-nucleated MYOD1-UDCs into myotubes but is not crucial for promoting the expression of late muscle regulatory factors. Finally, we confirmed that CD90-positive MYOD1-UDCs derived from patients with DMD were a valuable tool for obtaining a highly reproducible and stable evaluation of exon skipping using antisense oligonucleotide.
... Thereafter MyoD of the myogenic regulatory factor (MRF) family is a pioneer transcription factor that initiates muscle-specific gene expression and can then bind to MyoG and exert transcriptional activity (Londhe and Davie, 2011). Moreover, MyoG and Myomaker play important roles in myoblast fusion as key factors in terminal myoblast differentiation (Hasty et al., 1993;Nabeshima et al., 1993;Chen et al., 2020;Zhang et al., 2020a). In addition, MyHC expression also indicates that myoblasts begin to fuse into multinucleated myotubes and form myofibers (Francis-West et al., 2003). ...
... In addition, Myomaker, a muscle-specific fusion factor, is one of the key factors in the differentiation of myoblasts in humans and mice. Zhang et al., 2020a). Our study found that the mRNA expression levels of MyoG, MyoD, MyHC, and Myomaker were significantly downregulated after the overexpression of RRM2, which also significantly reduced the protein expression levels of MyHC; the MyHC immunofluorescence results were consistent with this finding. ...
During myogenesis and regeneration, the proliferation and differentiation of myoblasts play key regulatory roles and may be regulated by many genes. In this study, we analyzed the transcriptomic data of chicken primary myoblasts at different periods of proliferation and differentiation with protein‒protein interaction network, and the results indicated that there was an interaction between cyclin-dependent kinase 1 (CDK1) and ribonucleotide reductase regulatory subunit M2 (RRM2). Previous studies in mammals have a role for RRM2 in skeletal muscle development as well as cell growth, but the role of RRM2 in chicken is unclear. In this study, we investigated the effects of RRM2 on skeletal muscle development and regeneration in chickens in vitro and in vivo. The interaction between RRM2 and CDK1 was initially identified by co-immunoprecipitation and mass spectrometry. Through a dual luciferase reporter assay and quantitative real-time PCR, we identified the core promoter region of RRM2, which is regulated by the SP1 transcription factor. In this study, through cell counting kit-8 assays, 5-ethynyl-2′-deoxyuridine incorporation assays, flow cytometry, immunofluorescence staining, and Western blot analysis, we demonstrated that RRM2 promoted the proliferation and inhibited the differentiation of myoblasts. In vivo studies showed that RRM2 reduced the diameter of muscle fibers and slowed skeletal muscle regeneration. In conclusion, these data provide preliminary insights into the biological functions of RRM2 in chicken muscle development and skeletal muscle regeneration.
... Muscle differentiation involves two distinct but related processes -the expression of MRFs and muscle structural proteins, and the fusion between mononucleated myoblasts to generate multinucleated myofibers 12 . Inhibition of MRF expression could result in a defect and/or delay in myoblast fusion 43 . Since N-WASP, WIP2 and WAVE2 are expressed in muscle cells before and during differentiation, it is possible that they may affect both muscle-specific gene expression and myoblast fusion. ...
Invasive membrane protrusions play a central role in a variety of cellular processes. Unlike filopodia, invasive protrusions are mechanically stiff and propelled by branched actin polymerization. However, how branched actin filaments are organized to create finger-like invasive protrusions remains a longstanding question in cell biology. Here, by examining the mammalian fusogenic synapse, where invasive protrusions are generated to promote cell membrane juxtaposition and fusion, we have uncovered the mechanism underlying invasive protrusion formation. We show that two Arp2/3 nucleation promoting factors (NPFs), WAVE and N-WASP, exhibit distinct and complementary localization patterns in the protrusions. While WAVE is at the leading edge, N-WASP is recruited by its interacting protein, WIP, to the shaft of the protrusion. During protrusion growth, new branched actin filaments are polymerized at the periphery of the shaft and crosslinked to preexisting actin bundles by the “pioneer” actin-bundling protein dynamin. The thickened actin bundles are further stabilized by WIP, which functions as a WH2 domain-mediated actin-bundling protein. Disrupting any of these components results in defective protrusions and myoblast fusion in cultured cells and/or in mouse embryos. Thus, our study has revealed the intricate spatiotemporal coordination between two NPFs and two actin-bundling proteins in creating invasive protrusions and has general implications in understanding protrusion formation in many cellular processes beyond cell-cell fusion.
... These cells, called myoblasts, can proliferate and, upon extracellular cues, exit the cell cycle, downregulate PAX7, and express myogenin (MYOG) [2], another regulator factor involved in myoblast engagement into myogenic differentiation. Differentiated cells, called myocytes, fuse to form multinucleated myofibers [3]. ...
... The necessity of myoblasts to fuse to each other to form myotubes has been substantiated and extended through the genetic manipulation of genes that are uniquely expressed by myoblasts. Specifically, deletion of myomaker or myomixer (also called myomerger and minion) prevented myotube formation during in vitro and embryonic myogenesis [56][57][58][59]. Myomaker and myomixer-mediated myoblast-myoblast fusion is also required for muscle regeneration. ...
... That is, few, if any, regenerating myofibers were observed in injured muscles of mice conditionally depleted of myomaker or myomixer [56,60]. These seminal findings demonstrate that the fusogenic properties of myomaker and myomixer are required for nascent myotube formation during in vitro myogenesis, embryonic myogenesis, and muscle regeneration [24,[56][57][58][59]. ...
... The necessity of myoblasts to fuse to each other to form myotubes has been substantiated and extended through the genetic manipulation of genes that are uniquely expressed by myoblasts. Specifically, deletion of myomaker or myomixer (also called myomerger and minion) prevented myotube formation during in vitro and embryonic myogenesis [56][57][58][59]. Myomaker and myomixer-mediated myoblast-myoblast fusion is also required for muscle regeneration. ...
... That is, few, if any, regenerating myofibers were observed in injured muscles of mice conditionally depleted of myomaker or myomixer [56,60]. These seminal findings demonstrate that the fusogenic properties of myomaker and myomixer are required for nascent myotube formation during in vitro myogenesis, embryonic myogenesis, and muscle regeneration [24,[56][57][58][59]. ...
Injury to skeletal muscle through trauma, physical activity, or disease initiates a process called muscle regeneration. When injured myofibers undergo necrosis, muscle regeneration gives rise to myofibers that have myonuclei in a central position, which contrasts the normal, peripheral position of myonuclei. Myofibers with central myonuclei are called regenerating myofibers and are the hallmark feature of muscle regeneration. An important and underappreciated aspect of muscle regeneration is the maturation of regenerating myofibers into a normal sized myofiber with peripheral myonuclei. Strikingly, very little is known about processes that govern regenerating myofiber maturation after muscle injury. As knowledge of myofiber formation and maturation during embryonic, fetal, and postnatal development has served as a foundation for understanding muscle regeneration, this narrative review discusses similarities and differences in myofiber maturation during muscle development and regeneration. Specifically, we compare and contrast myonuclear positioning, myonuclear accretion, myofiber hypertrophy, and myofiber morphology during muscle development and regeneration. We also discuss regenerating myofibers in the context of different types of myofiber necrosis (complete and segmental) after muscle trauma and injurious contractions. The overall goal of the review is to provide a framework for identifying cellular and molecular processes of myofiber maturation that are unique to muscle regeneration.
... These cells, called myoblasts, can proliferate and, upon extracellular cues, exit the cell cycle, downregulate PAX7, and express myogenin (MYOG) [2], another regulator factor involved in myoblast engagement into myogenic differentiation. Differentiated cells, called myocytes, fuse to form multinucleated myofibers [3]. ...
The ability to recapitulate muscle differentiation in vitro enables the exploration of mechanisms underlying myogenesis and muscle diseases. However, obtaining myoblasts from patients with neuromuscular diseases or from healthy subjects poses ethical and procedural challenges that limit such investigations. An alternative consists in converting skin fibroblasts into myogenic cells by forcing the expression of the myogenic regulator MYOD. Here, we directly compared cellular phenotype, transcriptome, and nuclear lamina-associated domains (LADs) in myo-converted human fibroblasts and myotubes differentiated from myoblasts. We used isogenic cells from a 16-year-old donor, ruling out, for the first time to our knowledge, genetic factors as a source of variations between the two myogenic models. We show that myo-conversion of fibroblasts upregulates genes controlling myogenic pathways leading to multinucleated cells expressing muscle cell markers. However, myotubes are more advanced in myogenesis than myo-converted fibroblasts at the phenotypic and transcriptomic levels. While most LADs are shared between the two cell types, each also displays unique domains of lamin A/C interactions. Furthermore, myotube-specific LADs are more gene-rich and less heterochromatic than shared LADs or LADs unique to myo-converted fibroblasts, and they uniquely sequester developmental genes. Thus, myo-converted fibroblasts and myotubes retain cell type-specific features of radial and functional genome organization. Our results favor a view of myo-converted fibroblasts as a practical model to investigate the phenotypic and genomic properties of muscle cell differentiation in normal and pathological contexts, but also highlight current limitations in using fibroblasts as a source of myogenic cells.
... Our results show that MYOG and MYOD bind to Myh7-Sol-E1 and Myh7-Sol-E4. MYOG and MYOD are critical transcription factors in skeletal muscle differentiation [53][54][55][56]. Myogenesis is orchestrated through a series of transcriptional controls governed by MRFs. ...
