A novel in vitro model of sarcopenia using BubR1 hypomorphic C2C12 myoblasts

Abstract

Sarcopenia is the age-related loss of skeletal muscle mass and function with adverse outcomes that include physical disability, poor quality of life, and death. The detailed molecular mechanisms remain unknown. An in vitro muscle atrophy model is needed to enable mechanistic studies. To create such a model, we employed BubR1 insufficiency which causes premature ageing in mice. Using C2C12 cells, a recognized in vitro model of the skeletal muscle cell, we obtained the BubR1 hypomorphic C2C12 (C2C12BKD) cells by using shRNA. The resulting C2C12BKD cells displayed several characteristics of the sarcopenic muscle cell. In C2C12BKD cells, formation of myotubes, assessed by analysis of fusion index, was markedly reduced as was the expression of myogenin and MyoD, two marker genes for myogenesis. Moreover, the cells showed increased expression of the muscle-specific ubiquitin ligases Atrogin-1 and MuRF-1, indicating increased protein degradation through the ubiquitin-proteasome dependent proteolytic pathway. These results suggest that C2C12BKD cells are potentially useful as a novel in vitro model of sarcopenia.

Functional skeletal muscle model derived from SOD1-mutant ALS patient iPSCs recapitulates hallmarks of disease progression

“…Fusion index was quantified for myotubes stained with MyHC and nuclear stain, 4′,6-diamidino-2-phenylindole, (DAPI). The number of nuclei within myotubes was divided by the amount of total nuclei to obtain a number between 0 and 1, where 0 means no fusion and 1 is fusion of all myoblasts [53][54][55][56] . Simply put, Myotube contraction assessment.…”

Muscle wasting and aging: Experimental models, fatty infiltrations, and prevention

“…Comment citer ce document : Brioche, T. (Auteur de correspondance), Pagano, A., Py, G., Chopard Mendias et al, 2012). More recently, Nozaki et al (2015) developed the C2C12 cell line from the BubR1 hypomorphic mouse model of accelerated-aging (Baker et al, 2008); these cells are characterized by a subpopulation of muscle stem cells expressing p16Ink4a (Nozaki et al, 2015). This modified cell line shows increased expression of the muscle-specific ubiquitin ligases MAFbx and MuRF-1, reduced expression of MyoD and myogenin, and a decreased fusion index (Nozaki et al, 2015).…”

Degradation of ribosomal and chaperone proteins is attenuated during the differentiation of replicatively aged C2C12 myoblasts

“…Similarly, murine C 2 C 12 myoblasts that have undergone replicative ageing in vitro exhibit lesser expression of the myogenic regulator factors (MRF), myogenin and myoblast determination protein 1 (MyoD) [9,10] and impairments in myoblast differentiation and fusion [9][10][11]. These hallmarks of muscle ageing in vitro also occur in muscle cells upon silencing of the progeroid gene, mitotic checkpoint serine/threonine-protein kinase (BubR1) [12]. The mechanisms that link the loss of differentiation and lack of fusion of replicatively aged myoblasts with differences in the expression of MRF are unknown but could include deficits in proteostasis.…”

“… 9 , 10 , 11 These hallmarks of muscle ageing in vitro also occur in muscle cells upon silencing of the progeroid gene, mitotic checkpoint serine/threonine‐protein kinase (BubR1). 12 The mechanisms that link the loss of differentiation and lack of fusion of replicatively aged myoblasts with differences in the expression of MRF are unknown but could include deficits in proteostasis. C2C12 myoblasts exhibit high levels of protein turnover and are enriched with chaperone proteins and ribosomal protein subunits during early differentiation.…”