Myoblast fusion is a critical process in the formation of multinucleated muscle fibers, essential for both skeletal muscle development and regeneration. In adults, satellite cells, muscle-specific progenitors, are responsible for muscle growth and repair. Upon activation, these cells proliferate, differentiate, and fuse to mend damaged myofibers. However, the complete understanding of the proteins and signaling pathways governing this fusion process remains incomplete. Proteins such as MYOMAKER (MYMK) and MYOMIXER (MYMX) exhibit fusogenic activity, and mutations in their genes lead to Carey-Fineman-Ziter syndrome, a myopathy. Similarly, limb-girdle muscular dystrophy type 2B (LGMD2B), caused by Dysferlin (DYSF) mutations, is linked to impaired membrane repair and myoblast fusion. Other Ferlin-related proteins, like MYOFERLIN, also regulate membrane resealing and cell fusion. Unlike MYMK and MYMX, DYSF isn't strictly essential, but its absence results in smaller and damaged muscle fibers. Given the significant impact of neuromuscular disorders affecting millions globally, a thorough understanding of myoblast fusion regulation is crucial for developing targeted therapeutics. Previous studies have utilized MYMK to direct heterologous reprogrammed cells to fuse with dystrophic muscles for Dystrophin delivery. The RAC1-DOCK1 pathway is evolutionarily conserved and essential for myoblast fusion; DOCK1 activates RAC signaling with ELMO proteins, and this complex's activity is tightly regulated by auto-inhibitory interactions. Although the ELMO proteins' role in vertebrate myogenesis was unclear, *Drosophila* studies hinted at their involvement. This research aims to demonstrate the essential role of mammalian ELMO proteins in myoblast fusion and show how modulating their conformation can enhance muscle regeneration and improve outcomes in limb-girdle muscular dystrophy.

The literature extensively documents the importance of myoblast fusion in muscle development and regeneration. Studies have identified key fusogenic proteins like MYMK and MYMX, whose mutations cause Carey-Fineman-Ziter syndrome. Dysferlin's role in membrane repair and fusion has also been established, with its deficiency resulting in LGMD2B. The RAC1-DOCK1 pathway's crucial role in regulating myoblast fusion is well-documented, with DOCK1 acting as an activator of RAC signaling in conjunction with ELMO proteins. The auto-inhibitory regulation of the ELMO/DOCK complex is a key area of research, influencing the overall signaling output. Previous work in *Drosophila* suggested a possible role for ELMO proteins in myogenesis, providing a foundation for this study exploring their mammalian counterparts. The use of MYMK in delivering Dystrophin to dystrophic muscles highlights the potential of manipulating myoblast fusion for therapeutic purposes. The review also touches upon the clinical significance of myoblast fusion deficiencies, with neuromuscular disorders affecting millions worldwide, emphasizing the urgent need for therapeutic advancements.

The study employed a multi-faceted approach combining in vivo and in vitro techniques. Initially, whole-mount RNA in situ hybridization was used to map the expression profiles of Elmo1, Elmo2, and Elmo3 genes in mouse embryos and adult muscles. To investigate the role of ELMO proteins, the researchers generated various mouse models. An Elmo2 knockout model was created using a LacZ reporter gene to track Elmo2 expression. Conditional knockout (cKO) models were generated using Myf5CRE and Pax3CRE mice for muscle-specific Elmo2 deletion. To manipulate ELMO2 conformation, knock-in mouse lines were created with mutations in the ELMO2 RBD (to decrease signaling) and ELMO2 EID (to increase signaling). The functionality of these mutations was verified using methods like NMR spectroscopy and isothermal titration calorimetry. Muscle regeneration was assessed using cardiotoxin (CTX)-induced injury in the tibialis anterior (TA) muscle, followed by analysis of muscle fiber cross-sectional area (CSA), number of nuclei per fiber, and the presence of centrally located nuclei. Primary myoblasts were isolated from various mouse models to assess fusion in vitro, including fusion assays and mixed cell population assays with live cell tracking dyes. RNA-Seq was employed to analyze transcriptomes of differentiated myoblasts. Immunohistochemistry was used extensively for staining of various proteins (e.g., MHC, DESMIN, PAX7, DYSTROPHIN). Statistical analyses, including Student's t-tests, were used to compare different groups. Structural models were generated using available PDB coordinates. Finally, the study also included experiments using Dysferlin-null mice to evaluate the impact of ELMO2 conformational changes on a disease model.

The study revealed that ELMO1 and ELMO2 have redundant functions and are essential for myoblast fusion during embryonic development. Mice lacking both ELMO1 and ELMO2 (muscle-specific) exhibited severe myoblast fusion defects and died at birth, likely due to respiratory failure from underdeveloped muscles. Manipulating ELMO2 conformation significantly impacted myoblast fusion. The Elmo2RBD mutation (decreased signaling) resulted in smaller muscle fibers and reduced nuclei per fiber, both during development and regeneration. Conversely, the Elmo2EID mutation (increased signaling) led to larger muscle fibers and increased nuclei per fiber. The Elmo2EID mutation improved muscle regeneration following CTX-induced injury, characterized by larger regenerated fibers and increased fusion events. In vitro experiments confirmed that these effects on fusion were cell-intrinsic, with Elmo2EID myoblasts demonstrating increased fusion capacity compared to controls. Furthermore, the study demonstrated that even after multiple cycles of degeneration/regeneration, the Elmo2EID mutation maintained larger muscle fibers, without depleting satellite cell pools. Finally, in Dysferlin-null mice (LGMD2B model), expressing ELMO2 in an open conformation (Elmo2EID) rescued the myoblast fusion defects, improving several dystrophic features, including centrally located nuclei and necrotic myofibers.

The findings demonstrate the critical and evolutionarily conserved roles of ELMO1 and ELMO2 in myoblast fusion. The ability to genetically modulate ELMO2 conformation provides a powerful tool for manipulating myoblast fusion efficiency. The results highlight the mechanistic importance of ELMO2's RBD and EID domains in regulating the interaction with downstream signaling molecules, such as RHOG and ARL4A. The improved muscle regeneration in Elmo2EID mice and the rescue of dystrophic features in Dysferlin-null mice strongly suggest that this approach holds therapeutic potential for muscle diseases. The lack of effect on satellite cell pools in the Elmo2EID mice further supports the therapeutic implications. Future studies should explore the full repertoire of ELMO/DOCK interacting proteins in myoblasts, as well as the potential involvement of ELMO proteins in cell-cell adhesion mechanisms. The study's success in manipulating ELMO2 conformation provides a strong foundation for future research aimed at developing novel therapies for muscle diseases.

This research demonstrates the indispensable role of ELMO1 and ELMO2 in myoblast fusion and reveals a novel therapeutic strategy by manipulating ELMO2 conformation to enhance muscle regeneration. The Elmo2EID mutation, promoting an open conformation, significantly improved myoblast fusion, muscle regeneration, and ameliorated dystrophic phenotypes in a LGMD2B model. This approach offers a promising therapeutic avenue for muscle diseases and warrants further investigation into its application in various conditions affecting muscle regeneration.

While the study provides compelling evidence for the role of ELMO2 conformation in myoblast fusion, some limitations exist. The use of mouse models may not perfectly reflect human physiology, and further validation in human systems is necessary. The study primarily focused on skeletal muscle; the implications for other muscle types require further investigation. The mechanism by which ELMO2 conformation affects myoblast fusion needs additional elucidation, specifically regarding the involvement of other cell-surface proteins and signaling pathways. The long-term effects of manipulating ELMO2 conformation and the potential for compensatory mechanisms require further investigation.