MON-713 Nuclear Corepressor; SMRT Acts as an Important Regulator for Both Beta-Oxidation and the Maturation of Myogenesis in Mouse C2C12 Cell

Abstract

Background: Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT; NCoR2) is a transcriptional corepressor which has been recognized as an important player in the regulation of hepatic lipogenesis and the somatic development in mouse embryo. SMRT protein is also widely expressed in the mouse connective tissues, for example adipocyte and skeletal muscle, and we recently reported that the mouse of global deletion of SMRT causes significant obesity which is independent from thyroid hormone signaling and thermogenesis. However, the tissue specific role of SMRT in skeletal muscle is still unelucidated. Methods: To clarify this, we took the gene targeting strategy for SMRT using CRISPR Cas9, and made the myogenic C2C12 clone which lacks SMRT protein (C2C12-SMRTKO; SKO). For this study, wild type C2C12 cell (WT) and SKO cell were cultured in differentiation medium (DMEM+2% horse serum) for 5-6 days, and analyses for gene expression compared two cell types were performed. Results: We found the significant up-regulations of muscle specific beta-oxidation related genes (ex. Ppar delta, Ampk2), and higher protein level of PGC-1A in the SKO cell, suggesting that SKO cell had similar gene profile to that of rodent skeletal muscle in the exercise test. On the other hand, confocal microscopic analysis showed SKO cell had decreased cell-fusion and loss of myotube, indicating that the morphology was similar to immature mouse myoblasts. Further gene analyses compared between WT and SKO cell demonstrated that SKO cell had higher expressions of myogenic markers; MyoD and Myogenin. However, interestingly, the lower expressions of muscle constitutive genes; MHC, Actin, and Alpha-dystrobrevin were found in the SKO cell. These data indicate that the SKO cell has incomplete muscle fiber formation. Conclusion: Taken together, we demonstrate that SMRT works as a pivotal transcriptional mediator for both beta-oxidation and the process of myotube formation in C2C12 cell. Further inquiry for the cause of sarcopenia-like phenotype manifested in the SKO cell will be needed.