Skeletal Muscle Stromal Cells Derived From MuRF1 Knockout Mice Are Resistant to Adipogenesis

Abstract

Background: Intramuscular adipose tissue has been found to contribute to muscle dysfunction and is associated with a sedentary lifestyle, aging, and glucocorticoid treatment. Muscle ring finger 1 knockout (MuRF1 KO) mice have been shown to be protected from muscle loss following disuse, aging, and glucocorticoid treatment. In this study we used in vitro techniques to determine if MuRF1 KO muscle stromal cells are resistant to adipogenesis. Methods: Stromal cells were isolated from skeletal muscle tissue of MuRF1 KO and wild type mice. These cells were expanded in culture until 70–90% confluent, then differentiated in either a myogenic media formulation (myogenic cultures) or an adipogenic media formulation (adipogenic cultures). Gene expression was analyzed by qRT-PCR, protein content was measured by bicinchoninic acid assay, and triglyceride content was analyzed by colorimetric assay. We also analyzed isolated stromal cells, which had not been expanded in culture, by flow cytometry to identify myogenic satellite cells (SCs) and fibro-adipogenic progenitors (FAPs). Results: Wild type adipogenic cultures had higher expression of Trim63, the gene encoding MuRF1, when compared to wild type myogenic cultures. Adipogenic cultures had higher expression of adipogenic programing and lipid handling genes than myogenic cultures. These adipogenic cultures also had higher triglyceride content than myogenic cultures. The expression of adipogenic programing genes and lipid handling genes were lower in cultures derived from MuRF1 KO mice compared to wild type derived cultures; however, there was no statistically significant difference in the triglyceride content between the two genotypes. Analysis of stromal cell populations by flow cytometry indicated no difference in the FAP:SC ratio between wild type and MuRF1 KO mice. Conclusions: These results indicate that although there are no differences in the ratio of FAPs to SCs in wild type and MuRF1 KO mice, the MuRF1 KO cultures appear to be resistant to adipogenesis. The mechanism behind this resistance to adipogenesis remains to be elucidated.