Lineage tracing of newly accrued nuclei in skeletal myofibers uncovers distinct transcripts and interplay between nuclear populations.

Multinucleated skeletal muscle cells have an obligatory need to acquire additional nuclei through fusion with activated skeletal muscle stem cells when responding to both developmental and adaptive growth stimuli. A fundamental question in skeletal muscle biology has been the reason underlying this need for new nuclei in syncytial cells that already harbor hundreds of nuclei. To begin to answer this long-standing question, we utilized nuclear RNA-sequencing approaches and developed a lineage tracing strategy capable of defining the transcriptional state of recently fused nuclei and distinguishing this state from that of pre-existing nuclei. Our findings reveal the presence of conserved markers of newly fused nuclei both during development and after a hypertrophic stimulus in the adult. However, newly fused nuclei also exhibit divergent gene expression that is determined by the myogenic environment to which they fuse. Moreover, accrual of new nuclei through fusion is required for nuclei already resident in adult myofibers to mount a normal transcriptional response to a load-inducing stimulus. We propose a model of mutual regulation in the control of skeletal muscle development and adaptations, where newly fused and pre-existing myonuclear populations influence each other to maintain optimal functional growth.

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.

Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers.

While the majority of cells contain a single nucleus, cell types such as trophoblasts, osteoclasts, and skeletal myofibers require multinucleation. One advantage of multinucleation can be the assignment of distinct functions to different nuclei, but comprehensive interrogation of transcriptional heterogeneity within multinucleated tissues has been challenging due to the presence of a shared cytoplasm. Here, we utilized single-nucleus RNA-sequencing (snRNA-seq) to determine the extent of transcriptional diversity within multinucleated skeletal myofibers. Nuclei from mouse skeletal muscle were profiled across the lifespan, which revealed the presence of distinct myonuclear populations emerging in postnatal development as well as aging muscle. Our datasets also provided a platform for discovery of genes associated with rare specialized regions of the muscle cell, including markers of the myotendinous junction and functionally validated factors expressed at the neuromuscular junction. These findings reveal that myonuclei within syncytial muscle fibers possess distinct transcriptional profiles that regulate muscle biology.

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.

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.

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.