A New Muscle Disuse Model Using A Removable Tissue-engineering Muscle

Abstract

PURPOSE: Physical inactivity and subsequent skeletal muscle disuse may cause muscle atrophy, which is associated with a reduction in muscle function, physical performance, quality of life, and loss of independence, particularly in elderly. Traditionally, 2D muscle cell cultures (e.g. C2C12) have been used to study biological processes and identify and validate pharmacological and/or nutritional compounds to treat skeletal muscle disease including muscle atrophy. However, cell culture studies basically use a pharmacological method to induce muscle atrophy, and not an actual muscle disuse induced by a reduction of mechanical stress (i.e., mechanical unloading). Previously Nakamura et al. (2017) developed 3D engineered muscle (OITem: Osaka institute technology engineered muscle) with artificial tendons at both ends of the muscle, which is removable, thereby allowing us to mechanically release muscle tension by taking off (removing) one side of the muscle. Therefore, the purpose of the present study was to investigate whether removable tissue-engineering muscle effectively induce muscle atrophy, and thus is useful for muscle disuse model. METHODS: OITem was made from C2C12 skeletal muscle cells and a cold type-I collagen gel and placed between two artificial tendons (Nakamura et al., 2017). OITem was differentiated for 2-weeks and then divided into two groups: 1-week stretched on control group (CON: n = 6) in which the both ends of the muscle fixed with tendons, and 1-week stretched off group (OFF: n = 6) in which the one side of the muscle was removed. We analyzed expressions of muscle atrophy-related proteins MuRF-1 and Atrogin-1, and myogenesis-related protein myogenin using Western blotting. As well, we analyzed expression of MHC-fast or -slow (fast- or slow-twitch muscle fiber specific myosin heavy chain (MHC), respectively). RESULTS: The protein expression of MuRF-1 and myogenin was significantly lower in the OFF than those in the CON (P < 0.05). Moreover, the protein expression of MHC-fast and -slow was significantly lower in the OFF than those in the CON (P < 0.01). However, the protein expression of Atrogin-1 did not differ between two groups. CONCLUSIONS: The present findings suggest that OITem may be useful model for muscle disuse.