Molecular regulation of myocyte fusion.

Myocyte fusion is a pivotal process in the development and regeneration of skeletal muscle. Failure during fusion can lead to a range of developmental as well as pathological consequences. This review aims to comprehensively explore the intricate processes underlying myocyte fusion, from the molecular to tissue scale. We shed light on key players, such as the muscle-specific fusogens - Myomaker and Myomixer, in addition to some lesser studied molecules contributing to myocyte fusion. Conserved across vertebrates, Myomaker and Myomixer play a crucial role in driving the merger of plasma membranes of fusing myocytes, ensuring the formation of functional muscle syncytia. Our multiscale approach also delves into broader cell and tissue dynamics that orchestrate the timing and positioning of fusion events. In addition, we explore the relevance of muscle fusogens to human health and disease. Mutations in fusogen genes have been linked to congenital myopathies, providing unique insights into the molecular basis of muscle diseases. We conclude with a discussion on potential therapeutic avenues that may emerge from manipulating the myocyte fusion process to remediate skeletal muscle disorders.

Localized Prox1 Regulates Aortic Valve Endothelial Cell Diversity and Extracellular Matrix Stratification in Mice.

BACKGROUND: Specialized valve endothelial cell (VEC) populations are localized oriented to blood flow in developing aortic and mitral valves, but their roles in valve development and disease are unknown. In the aortic valve (AoV), a population of VECs on the fibrosa side expresses the transcription factor Prox1 together with genes found in lymphatic ECs. In this study, we examine Prox1's role in regulating a lymphatic-like gene network and promoting VEC diversity required for the development of the stratified trilaminar extracellular matrix (ECM) of murine AoV leaflets. METHODS: To determine whether disruption of Prox1 localization affects heart valve development, we generated mice (NFATc1enCre Prox1 gain-of-function) in which Prox1 is overexpressed on the ventricularis side of the AoV beginning in embryonic development. To identify potential targets of Prox1, we performed cleavage under targets and release using nuclease on wild-type and NFATc1enCre Prox1 gain-of-function AoVs with validation by colocalization in vivo using RNA in situ hybridization in NFATc1enCre Prox1 gain-of-function AoVs. Natural induction of Prox1 and target gene expression was evaluated in myxomatous AoVs in a mouse model of Marfan syndrome (Fbn1C1039G/+). RESULTS: The overexpression of Prox1 is sufficient to cause enlargement of AoVs by postnatal day (P)0, as well as a decrease in ventricularis-specific gene expression and disorganized interstitial ECM layers at P7. We identified potential targets of Prox1 known to play roles in lymphatic ECs including Flt1, Efnb2, Egfl7, and Cx37. Ectopic Prox1 colocalized with induced Flt1, Efnb2, and Cx37 expression in NFATc1enCre Prox1 gain-of-function AoVs. Moreover, in Marfan syndrome myxomatous AoVs, endogenous Prox1, and its identified targets, were ectopically induced in ventricularis side VECs. CONCLUSIONS: Our results support a role for Prox1 in localized lymphatic-like gene expression on the fibrosa side of the AoV. Furthermore, localized VEC specialization is required for development of the stratified trilaminar ECM critical for AoV function and is dysregulated in congenitally malformed valves.

Expression of Myomaker and Myomerger in myofibers causes muscle pathology.

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.

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens. We asked if the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver micro-Dystrophin (µDys) to skeletal muscle of a mouse model of Duchenne muscular dystrophy. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Regulation of the myoblast fusion reaction for muscle development, regeneration, and adaptations.

Fusion of plasma membranes is essential for skeletal muscle development, regeneration, exercise-induced adaptations, and results in a cell that contains hundreds to thousands of nuclei within a shared cytoplasm. The differentiation process in myocytes culminates in their fusion to form a new myofiber or fusion to an existing myofiber thereby contributing more synthetic material to the syncytium. The choice for two cells to fuse and become one could be a dangerous event if the two cells are not committed to an allied function. Thus, fusion events are highly regulated with positive and negative factors to fine-tune the process, and requires muscle-specific fusogens (Myomaker and Myomerger) as well as general cellular machinery to achieve the union of membranes. While a unified vertebrate myoblast fusion pathway is not yet established, recent discoveries should make this pursuit attainable. Not only does myocyte fusion impact the normal biology of skeletal muscle, but new evidence indicates dysregulation of the process impacts pathologies of skeletal muscle. Here, I will highlight the molecular players and biochemical mechanisms that drive fusion events in muscle, and discuss how this key myogenic process impacts skeletal muscle diseases.

Skeletal muscle fibers count on nuclear numbers for growth.

Skeletal muscle cells are noteworthy for their syncytial nature, with each myofiber accumulating hundreds or thousands of nuclei derived from resident muscle stem cells (MuSCs). These nuclei are accrued through cell fusion, which is controlled by the two essential fusogens Myomaker and Myomerger that are transiently expressed within the myogenic lineage. While the absolute requirement of fusion for muscle development has been known for decades, the underlying need for the magnitude of multinucleation in muscle remains mysterious. Possible advantages of multinucleation include the potential it affords for transcriptional diversity within these massive cells, and as a means of increasing DNA content to support optimal cell size and function. In this article, we review recent advances that elucidate the relationship between myonuclear numbers and establishment of myofiber size, and discuss how this new information refines our understanding of the concept of myonuclear domains (MND), the cytoplasmic volumes that each resident myonucleus can support. Finally, we explore the potential consequences and costs of multinucleation and its impacts on myonuclear transcriptional reserve capacity, growth potential, myofiber size regulation, and muscle adaptability. We anticipate this report will not only serve to highlight the latest advances in the basic biology of syncytial muscle cells but also provide information to help design the next generation of therapeutic strategies to maintain muscle mass and function.

FOXF1 is required for the oncogenic properties of PAX3-FOXO1 in rhabdomyosarcoma.

The PAX3-FOXO1 fusion protein is the key oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue malignancy with a particularly poor prognosis. Identifying key downstream targets of PAX3-FOXO1 will provide new therapeutic opportunities for treatment of FP-RMS. Herein, we demonstrate that Forkhead Box F1 (FOXF1) transcription factor is uniquely expressed in FP-RMS and is required for FP-RMS tumorigenesis. The PAX3-FOXO1 directly binds to FOXF1 enhancers and induces FOXF1 gene expression. CRISPR/Cas9 mediated inactivation of either FOXF1 coding sequence or FOXF1 enhancers suppresses FP-RMS tumorigenesis even in the presence of PAX3-FOXO1 oncogene. Knockdown or genetic knockout of FOXF1 induces myogenic differentiation in PAX3-FOXO1-positive FP-RMS. Over-expression of FOXF1 decreases myogenic differentiation in primary human myoblasts. In FP-RMS tumor cells, FOXF1 protein binds chromatin near enhancers associated with FP-RMS gene signature. FOXF1 cooperates with PAX3-FOXO1 and E-box transcription factors MYOD1 and MYOG to regulate FP-RMS-specific gene expression. Altogether, FOXF1 functions downstream of PAX3-FOXO1 to promote FP-RMS tumorigenesis.

Nuclear numbers in syncytial muscle fibers promote size but limit the development of larger myonuclear domains.

Mammalian cells exhibit remarkable diversity in cell size, but the factors that regulate establishment and maintenance of these sizes remain poorly understood. This is especially true for skeletal muscle, comprised of syncytial myofibers that each accrue hundreds of nuclei during development. Here, we directly explore the assumed causal relationship between multinucleation and establishment of normal size through titration of myonuclear numbers during mouse neonatal development. Three independent mouse models, where myonuclear numbers were reduced by 75, 55, or 25%, led to the discovery that myonuclei possess a reserve capacity to support larger functional cytoplasmic volumes in developing myofibers. Surprisingly, the results revealed an inverse relationship between nuclei numbers and reserve capacity. We propose that as myonuclear numbers increase, the range of transcriptional return on a per nuclear basis in myofibers diminishes, which accounts for both the absolute reliance developing myofibers have on nuclear accrual to establish size, and the limits of adaptability in adult skeletal muscle.

Myonuclear content regulates cell size with similar scaling properties in mice and humans.

Muscle fibers are the largest cells in the body, and one of its few syncytia. Individual cell sizes are variable and adaptable, but what governs cell size has been unclear. We find that muscle fibers are DNA scarce compared to other cells, and that the nuclear number (N) adheres to the relationship N = aVb where V is the cytoplasmic volume. N invariably scales sublinearly to V (b < 1), making larger cells even more DNA scarce. N scales linearly to cell surface in adult humans, in adult and developing mice, and in mice with genetically reduced N, but in the latter the relationship eventually fails when they reach adulthood with extremely large myonuclear domains. Another exception is denervation-atrophy where nuclei are not eliminated. In conclusion, scaling exponents are remarkably similar across species, developmental stages and experimental conditions, suggesting an underlying scaling law where DNA-content functions as a limiter of muscle cell size.

Myocyte-derived Myomaker expression is required for regenerative fusion but exacerbates membrane instability in dystrophic myofibers.

Muscle progenitor cell fusion is required for the formation and regeneration of multinucleated skeletal muscle fibers. Chronic muscle regeneration in Duchenne muscular dystrophy (DMD) is characterized by ongoing fusion of satellite cell (SC) progeny, but the effects of fusion on disease and the mechanisms by which fusion is accomplished in this setting are not fully understood. Using the mdx mouse model of DMD, we deleted the fusogenic protein Myomaker in SCs or myofibers. Following deletion in SCs, mice displayed a complete lack of myocyte fusion, resulting in severe muscle loss, enhanced fibrosis, and significant functional decline. Reduction of Myomaker in mature myofibers in mdx mice, however, led to minimal alterations in fusion dynamics. Unexpectedly, myofiber-specific deletion of Myomaker resulted in improvement of disease phenotype, with enhanced function and decreased muscle damage. Our data indicate that Myomaker has divergent effects on dystrophic disease severity depending upon its compartment of expression. These findings show that myocyte fusion is absolutely required for effective regeneration in DMD, but persistent Myomaker expression in myofibers due to ongoing fusion may have unintended deleterious consequences for muscle integrity. Thus, sustained activation of a component of the myogenic program in dystrophic myofibers exacerbates disease.

Lipid Mixing Assay for Murine Myoblast Fusion and Other Slow Cell-cell Fusion Processes.

Lipid mixing (redistribution of lipid probes between fusing membranes) has been widely used to study early stages of relatively fast viral and intracellular fusion processes that take seconds to minutes. Lipid mixing assays are especially important for identification of hemifusion intermediates operationally defined as lipid mixing without content mixing. Due to unsynchronized character and the slow rate of the differentiation processes that prime the cells for cell-cell fusion processes in myogenesis, osteoclastogenesis and placentogenesis, these fusions take days. Application of lipid mixing assays to detect early fusion intermediates in these very slow fusion processes must consider the continuous turnover of plasma membrane components and potential fusion-unrelated exchange of the lipid probes between the membranes. Here we describe the application of lipid mixing assay in our work on myoblast fusion stage in development and regeneration of skeletal muscle cells. Our approach utilizes conventional in vitro model of myogenic differentiation and fusion based on murine C2C12 cells. When we observe the appearance of first multinucleated cells, we lift the cells and label them with either fluorescent lipid DiI as a membrane probe or CellTrackerTM Green as a content probe. Redistribution of the probes between the cells is scored by fluorescence microscopy. Hemifused cells are identified as mononucleated cells labeled with both content- and membrane probes. The interpretation must be supported by a system of negative controls with fusion-incompetent cells to account for and minimize contributions of fusion-unrelated exchange of the lipid probes. This approach with minor modifications has been used for investigating fusion of primary murine myoblasts, osteoclast precursors and fusion mediated by a gamete fusogen HAP2, and likely can be adopted for other slow cell-cell fusion processes.

Cell Fusion: Merging Membranes and Making Muscle.

Cell fusion is essential for the development of multicellular organisms, and plays a key role in the formation of various cell types and tissues. Recent findings have highlighted the varied protein machinery that drives plasma-membrane merger in different systems, which is characterized by diverse structural and functional elements. We highlight the discovery and activities of several key sets of fusion proteins that together offer an evolving perspective on cell membrane fusion. We also emphasize recent discoveries in vertebrate myoblast fusion in skeletal muscle, which is composed of numerous multinucleated myofibers formed by the fusion of progenitor cells during development.

Myonuclear accretion is a determinant of exercise-induced remodeling in skeletal muscle.

Skeletal muscle adapts to external stimuli such as increased work. Muscle progenitors (MPs) control muscle repair due to severe damage, but the role of MP fusion and associated myonuclear accretion during exercise are unclear. While we previously demonstrated that MP fusion is required for growth using a supra-physiological model (Goh and Millay, 2017), questions remained about the need for myonuclear accrual during muscle adaptation in a physiological setting. Here, we developed an 8 week high-intensity interval training (HIIT) protocol and assessed the importance of MP fusion. In 8 month-old mice, HIIT led to progressive myonuclear accretion throughout the protocol, and functional muscle hypertrophy. Abrogation of MP fusion at the onset of HIIT resulted in exercise intolerance and fibrosis. In contrast, ablation of MP fusion 4 weeks into HIIT, preserved exercise tolerance but attenuated hypertrophy. We conclude that myonuclear accretion is required for different facets of exercise-induced adaptive responses, impacting both muscle repair and hypertrophic growth.

Myomaker and Myomerger Work Independently to Control Distinct Steps of Membrane Remodeling during Myoblast Fusion.

Classic mechanisms for membrane fusion involve transmembrane proteins that assemble into complexes and dynamically alter their conformation to bend membranes, leading to mixing of membrane lipids (hemifusion) and fusion pore formation. Myomaker and Myomerger govern myoblast fusion and muscle formation but are structurally divergent from traditional fusogenic proteins. Here, we show that Myomaker and Myomerger independently mediate distinct steps in the fusion pathway, where Myomaker is involved in membrane hemifusion and Myomerger is necessary for fusion pore formation. Mechanistically, we demonstrate that Myomerger is required on the cell surface where its ectodomains stress membranes. Moreover, we show that Myomerger drives fusion completion in a heterologous system independent of Myomaker and that a Myomaker-Myomerger physical interaction is not required for function. Collectively, our data identify a stepwise cell fusion mechanism in myoblasts where different proteins are delegated to perform unique membrane functions essential for membrane coalescence.

Myoblast fusion confusion: the resolution begins.

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.

Insights into the localization and function of myomaker during myoblast fusion.

Multinucleated skeletal muscle fibers form through the fusion of myoblasts during development and regeneration. Previous studies identified myomaker (Tmem8c) as a muscle-specific membrane protein essential for fusion. However, the specific function of myomaker and how its function is regulated are unknown. To explore these questions, we first examined the cellular localization of endogenous myomaker. Two independent antibodies showed that whereas myomaker does localize to the plasma membrane in cultured myoblasts, the protein also resides in the Golgi and post-Golgi vesicles. These results raised questions regarding the precise cellular location of myomaker function and mechanisms that govern myomaker trafficking between these cellular compartments. Using a synchronized fusion assay, we demonstrated that myomaker functions at the plasma membrane to drive fusion. Trafficking of myomaker is regulated by palmitoylation of C-terminal cysteine residues that allows Golgi localization. Moreover, dissection of the C terminus revealed that palmitoylation was not sufficient for complete fusogenic activity suggesting a function for other amino acids within this C-terminal region. Indeed, C-terminal mutagenesis analysis highlighted the importance of a C-terminal leucine for function. These data reveal that myoblast fusion requires myomaker activity at the plasma membrane and is potentially regulated by proper myomaker trafficking.

Myomerger induces fusion of non-fusogenic cells and is required for skeletal muscle development.

Despite the importance of cell fusion for mammalian development and physiology, the factors critical for this process remain to be fully defined, which has severely limited our ability to reconstitute cell fusion. Myomaker (Tmem8c) is a muscle-specific protein required for myoblast fusion. Expression of myomaker in fibroblasts drives their fusion with myoblasts, but not with other myomaker-expressing fibroblasts, highlighting the requirement of additional myoblast-derived factors for fusion. Here we show that Gm7325, which we name myomerger, induces the fusion of myomaker-expressing fibroblasts. Thus, myomaker and myomerger together confer fusogenic activity to otherwise non-fusogenic cells. Myomerger is skeletal muscle-specific and genetic deletion in mice results in a paucity of muscle fibres demonstrating its requirement for normal muscle formation. Myomerger deficient myocytes differentiate and harbour organized sarcomeres but are fusion-incompetent. Our findings identify myomerger as a fundamental myoblast fusion protein and establish a system that begins to reconstitute mammalian cell fusion.

Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy.

Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy.

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.