SummaryFusion of myoblasts is essential for the formation of multi-nucleated muscle fibers. However, the identity of myogenic proteins that directly govern this fusion process has remained elusive. Here, we discovered a muscle-specific membrane protein, named Myomaker, that controls myoblast fusion. Myomaker is expressed on the cell surface of myoblasts during fusion and is down-regulated thereafter. Over-expression of Myomaker in myoblasts dramatically enhances fusion and genetic disruption of Myomaker in mice causes perinatal death due to an absence of multi-nucleated muscle fibers. Remarkably, forced expression of Myomaker in fibroblasts promotes fusion with myoblasts, demonstrating the direct participation of this protein in the fusion process. Pharmacologic perturbation of the actin cytoskeleton abolishes the activity of Myomaker, consistent with prior studies implicating actin dynamics in myoblast fusion. These findings reveal a long-sought myogenic fusion protein both necessary and sufficient for mammalian myoblast fusion and provide new insights into the molecular underpinnings of muscle formation.

Muscular dystrophies comprise a diverse group of genetic disorders that lead to muscle wasting and, in many instances, premature death 1 . Many mutations that cause muscular dystrophy compromise the support network that connects myofilament proteins within the cell to the basal lamina outside the cell, rendering the sarcolemma more permeable or leaky. Here we show that deletion of the gene encoding cyclophilin D (Ppif) rendered mitochondria largely insensitive to the calcium overloadinduced swelling associated with a defective sarcolemma, thus reducing myofiber necrosis in two distinct models of muscular dystrophy. Mice lacking δ-sarcoglycan (Scgd −/− mice) showed markedly less dystrophic disease in both skeletal muscle and heart in the absence of Ppif. Moreover, the premature lethality associated with deletion of Lama2, encoding the α-2 chain of laminin-2, was rescued, as were other indices of dystrophic disease. Treatment with the cyclophilin inhibitor Debio-025 similarly reduced mitochondrial swelling and necrotic disease manifestations in mdx mice, a model of Duchenne muscular dystrophy, and in Scgd −/− mice. Thus, mitochondrial-dependent necrosis represents a prominent disease mechanism in muscular dystrophy, suggesting that inhibition of cyclophilin D could provide a new pharmacologic treatment strategy for these diseases.The muscular dystrophies are inherited disorders that mostly affect striated muscle tissue, resulting in progressive muscle weakness, wasting and, in many instances, premature death 1 . Many characterized mutations in humans that result in muscular dystrophy cause alterations either in structural proteins that attach the underlying contractile proteins to the basal lamina, which provides rigidity to the skeletal muscle cell membrane (sarcolemma), or in proteins that HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript directly stabilize or repair the cell membrane 1-3 . For example, loss of dystrophin or other components of the dystrophin-glycoprotein complex leads to a fundamental alteration in the physical properties of the sarcolemma, permitting contraction-induced microtears and the unregulated exchange of ions, increasing the influx of calcium 2-4 . Moreover, total intracellular calcium or subsarcolemmal calcium levels have been found to be elevated in skeletal muscle cells or fibers from dystrophic mice 5,6 .In response to sustained increases in intracellular calcium concentrations, such as after ischemic injury, mitochondria undergo a so-called 'permeability transition' 7 . This process results in the regulated formation of a large pore complex that spans the outer and inner mitochondrial membranes, leading to an irreversible loss of matrix and intermembrane contents and swelling of the mitochondria. If this transition state is not reversed in a timely manner, the mitochondria can rupture, causing necrotic and/or apoptotic cell death. Cyclophilin D is a mitochondrial matrix prolyl cis-trans isomerase that directly regulates calcium-and reactive oxygen specie...

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.

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.

Muscular dystrophy is a general term encompassing muscle disorders that cause weakness and wasting, typically leading to premature death. Membrane instability, as a result of a genetic disruption within the dystrophin-glycoprotein complex (DGC), is thought to induce myofiber degeneration, although the downstream mechanism whereby membrane fragility leads to disease remains controversial. One potential mechanism that has yet to be definitively proven in vivo is that unregulated calcium influx initiates disease in dystrophic myofibers. Here we demonstrate that calcium itself is sufficient to cause a dystrophic phenotype in skeletal muscle independent of membrane fragility. For example, overexpression of transient receptor potential canonical 3 (TRPC3) and the associated increase in calcium influx resulted in a phenotype of muscular dystrophy nearly identical to that observed in DGC-lacking dystrophic disease models, including a highly similar molecular signature of gene expression changes. Furthermore, transgene-mediated inhibition of TRPC channels in mice dramatically reduced calcium influx and dystrophic disease manifestations associated with the mdx mutation (dystrophin gene) and deletion of the ␦-sarcoglycan (Scgd) gene. These results demonstrate that calcium itself is sufficient to induce muscular dystrophy in vivo, and that TRPC channels are key disease initiators downstream of the unstable membrane that characterizes many types of muscular dystrophy.fibrosis ͉ necrosis ͉ degeneration ͉ skeletal muscle

Muscular dystrophies (MDs) comprise a group of degenerative muscle disorders characterized by progressive muscle wasting and often premature death. The primary defect common to most MDs involves disruption of the dystrophin-glycoprotein complex (DGC). This leads to sarcolemmal instability and Ca 2+ influx, inducing cellular necrosis. Here we have shown that the dystrophic phenotype observed in δ-sarcoglycan-null (Sgcd -/-) mice and dystrophin mutant mdx mice is dramatically improved by skeletal muscle-specific overexpression of sarcoplasmic reticulum Ca 2+ ATPase 1 (SERCA1). Rates of myofiber central nucleation, tissue fibrosis, and serum creatine kinase levels were dramatically reduced in Sgcd -/-and mdx mice with the SERCA1 transgene, which also rescued the loss of exercise capacity in Sgcd -/-mice. Adeno-associated virus-SERCA2a (AAV-SERCA2a) gene therapy in the gastrocnemius muscle of Sgcd -/-mice mitigated dystrophic disease. SERCA1 overexpression reversed a defect in sarcoplasmic reticulum Ca 2+ reuptake that characterizes dystrophic myofibers and reduced total cytosolic Ca 2+ . Further, SERCA1 overexpression almost completely rescued the dystrophic phenotype in a mouse model of MD driven solely by Ca 2+ influx. Mitochondria isolated from the muscle of SERCA1-Sgcd -/-mice were no longer swollen and calpain activation was reduced, suggesting protection from Ca 2+ -driven necrosis. Our results suggest a novel therapeutic approach using SERCA1 to abrogate the altered intracellular Ca 2+ levels that underlie most forms of MD.

The serine/threonine phosphatase calcineurin is an important regulator of calcium-activated intracellular responses in eukaryotic cells. In higher eukaryotes, calcium/ calmodulin-mediated activation of calcineurin facilitates direct dephosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT). Recently, controversy has surrounded the role of calcineurin in mediating skeletal muscle cell hypertrophy. Here we examined the ability of calcineurin-deficient mice to undergo skeletal muscle hypertrophic growth following mechanical overload (MOV) stimulation or insulin-like growth factor-1 (IGF-1) stimulation. Two distinct models of calcineurin deficiency were employed: calcineurin A␤ gene-targeted mice, which show a Ϸ50% reduction in total calcineurin, and calcineurin B1-LoxP-targeted mice crossed with a myosin light chain 1f cre knock-in allele, which show a greater than 80% loss of total calcineurin only in skeletal muscle. Calcineurin A␤؊/؊ and calcineurin B1-LoxP(fl/fl)-MLC-cre mice show essentially no defects in muscle growth in response to IGF-1 treatment or MOV stimulation, although calcineurin A␤؊/؊ mice show a basal defect in total fiber number in the plantaris and a mild secondary reduction in growth, consistent with a developmental defect in myogenesis. Both groups of genetargeted mice show normal increases in Akt activation following MOV or IGF-1 stimulation. However, overloadmediated fiber-type switching was dramatically impaired in calcineurin B1-LoxP(fl/fl)-MLC-cre mice. NFAT-luciferase reporter transgenic mice failed to show a correlation between IGF-1-or MOV-induced hypertrophy and calcineurin-NFAT-dependent signaling in vivo. We conclude that calcineurin expression is important during myogenesis and fiber-type switching, but not for muscle growth in response to hypertrophic stimuli.

Regeneration of injured adult skeletal muscle involves fusion of activated satellite cells to form new myofibers. Myomaker is a muscle-specific membrane protein required for fusion of embryonic myoblasts, but its potential involvement in adult muscle regeneration has not been explored. We show that myogenic basic helixloop-helix (bHLH) transcription factors induce myomaker expression in satellite cells during acute and chronic muscle regeneration. Moreover, genetic deletion of myomaker in adult satellite cells completely abolishes muscle regeneration, resulting in severe muscle destruction after injury. Myomaker is the only muscle-specific protein known to be absolutely essential for fusion of embryonic and adult myoblasts.