OTUB2 coordinates myogenic differentiation and mitochondrial function Yeongmin Kim College of Life Sciences and Medical Engineering

Abstract

Sarcopenia is characterized by the progressive decline in skeletal muscle mass, strength, and function, largely associated with aging and reduced physical activity. As global life expectancy continues to increase, the growing prevalence of sarcopenia has drawn increasing attention as a major age-related health issue. However, therapeutic options remain limited, with exercise being the only established intervention, and no approved drug therapy is currently available. The maintenance of skeletal muscle mass depends on the dynamic balance between muscle formation and muscle degeneration. In this context, promoting myogenesis by activating quiescent muscle stem cells in adult muscle may provide a promising approach for treating sarcopenia. Myogenesis is a highly coordinated process in which proliferating myoblasts differentiate into multinucleated myotubes. During this process, precise protein turnover is required to eliminate proliferation- associated proteins while stabilizing myogenic proteins. Thus, selective protein turnover mediated by the ubiquitin-proteasome system (UPS) may be essential for proper myogenic progression. Despite the potential importance of UPS-mediated protein regulation, the functional relationship between deubiquitinating enzymes (DUBs) and myogenesis remains poorly understood. Thus, we investigated how DUBs contribute to the regulation of myogenesis. To identify potential DUB candidates, we analyzed the intersection between DUB-encoding genes and common differentially expressed genes identified during C2C12 myogenic differentiation and skeletal muscle regeneration. Among the candidate DUBs, OTUB2 was selected for further investigation, and its expression gradually increased during C2C12 myogenic differentiation. Treatment with an OTUB2 inhibitor impaired C2C12 myogenic differentiation and reduced both the protein and mRNA levels of MyoD, MyoG, and MHC. Consistently, OTUB2 knockdown suppressed myogenesis, whereas OTUB2 overexpression enhanced myogenic differentiation, indicating a positive regulatory role of OTUB2 in myogenesis. We next examined whether OTUB2 regulates cellular metabolism using Seahorse analysis. Basal respiration remained largely unchanged, whereas maximal respiration was significantly reduced upon OTUB2 inhibition. In addition, OTUB2 inhibition led to reductions in PGC1α expression, mtDNA levels, and OXPHOS-related genes, indicating that OTUB2 may be involved in maintaining mitochondrial biogenesis and respiratory capacity. The role of OTUB2 inhibition was further examined in vivo using a CTX-induced muscle regeneration model. Treatment with an OTUB2 inhibitor suppressed muscle regeneration after CTX injury, and CSA quantification revealed significantly smaller regenerating muscle fibers in the OTUB2 inhibitor-treated group. Overall, these findings highlight OTUB2 as a potential regulator of myogenesis and muscle regeneration, suggesting that further mechanistic investigation of OTUB2 may advance our understanding of muscle biology and support future therapeutic development for sarcopenia.