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.

The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts

“…Interestingly, a subsequent study (33) showed that MuRF2 and MuRF3 deletion in mice causes severe skeletal muscle histological alterations, including sarcomeric disarrays, reinforcing the notion that MuRF2 can be involved in cytoskeleton organization. More recently, MuRF2 was also shown to be a skeletal muscle tissue organizer in mice with MuRF1 and MuRF2 deletion (21). In spite of these data clearly establishing a connection between MuRF2 and the skeletal muscle fiber cytoskeleton, no studies had so far approached fibroblasts, which are cells extremely dependent upon their cytoskeleton to function.…”

“…Cells were isolated from the hind limb muscles of male FVB mice (∼8 weeks old, 30±3 g). Cell isolation and digestion were made as previously described (20,21). After digestion, the cells were incubated at 37°C in an atmosphere of 5% CO 2 for 40-50 min on 75 cm 2 tissue culture dishes.…”

“…Both C2C12 (ATCC CRL-1772, USA) and the supernatant cells from P0 myoblast-enriched pool were plated in Matrigel-coated 24-well plates (Matrigel Matrix Growth Factor Reduced; BD Biosciences #354230, USA) and incubated until 2 days after differentiation as previously described (20,21).…”

Recent developments in the field of cachexia, sarcopenia, and muscle wasting: highlights from the 12th Cachexia Conference

“…Other studies buttress the view that increased FoxO signalling and the activation of the transcription factors nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB), MuRF1, and muscle atrophy F‐box (MAFbx) in skeletal muscle play major roles during cachexia onset and progression . MuRF1 and MAFbx are essentially involved in muscle atrophy development.…”

MicroRNAs em plasticidade muscular: efeitos da superexpressão do miR-29c na modulação da massa muscular esquelética.

“…A proteína MuRF1 em conjunto com a sua homóloga MuRF2, além de desempenharem um papel na atrofia muscular, também são importantes na formação do sarcômero e diferenciação miogênica (McElhinny et al, 2004). Nosso grupo de pesquisa demonstrou que a ablação destes MuRFs prejudica a diferenciação das células satélites, levando a um déficit no processo de regeneração muscular em camundongos (Moriscot et al, 2019), demonstrando assim que MuRFs também desempenham um papel importante na regulação da regeneração muscular esquelética.…”