MuRF1 and MuRF2 are key players in skeletal muscle regeneration involving myogenic deficit and deregulation of the chromatin-remodeling complex

Abstract

Background

Skeletal muscle regeneration is a powerful and highly synchronized process and it is well described that intracellular pathways related to anabolic responses are readily activated. On the other hand, the role of catabolic pathways in the skeletal muscle regenerative response is much less understood. In the present study, we hypothesized that MuRF1, a key gene involved in skeletal muscle mass loss, and its close counterpart MuRF2 play a role in skeletal muscle regeneration.

Methods and results

Wild type, single MuRF1 knock out, single MuRF2 knock out and double MuRF1&2 dKO mice had their tibialis anterior muscle injured by a single round of 4 sequential injections of cardiotoxin (CTX-10ūM, 5ūl each injection) spread along the length of the muscle. The animals were killed after 1, 3, 10 and 28 days after injury and the muscles were used to address general histology, satellite cells, myogenic markers and apoptosis. In addition, we silenced MuRF1 and MuRF2 in a primary myogenic cell culture to evaluate myogenesis and BAF57, a SWI/SNF chromatin-remodeling complex component. Finally, we employed Chromatin Immuno Precipitation assays to investigate whether MuRF1 associates with the myogenic promoters MyoD and myogenin. After cardiotoxin (CTX) injection, MuRF1 and MuRF2 expression was strongly increased and spread out inside the remaining fibers and also along satellite cells. In line with a critical role during regeneration, MuRF1&2 dKO deficient injured muscles were unable to properly regenerate. In addition, dKO injured muscle failed to express activators of the myogenic program, Myf-5, FHL2 and MARP2, while myogenin levels were normally increased. Accordingly, siRNA reduced levels of MuRF1 and MuRF2 caused a severe myogenic deficit in primary myogenic cells, without any proliferation deficiency. Finally, in MuRF1&2 siRNA knockdown studies and Chromatin Immuno Precipitation analysis of primary myogenic cultures we have shown an impaired nuclear clearance of BAF57 and a shortage in chromatin remodeling as a likely mechanism underlying the deficient myogenic differentiation.

Conclusion

In summary, our results indicate a key cooperative role of the E3 ligases MuRF1 and MuRF2 in skeletal muscle regenerative response and myogenesis by inducing chromatin opening at myogenic promoters after BAF57 removal during muscle regeneration.