The E3 Ligases MuRF1 and MuRF2 Play a Key Role in Skeletal Muscle Fibroblast Functional Capacity

Abstract

IntroductionFibroblasts are crucial for proper matrix deposition in wound healing and it has been well established that fibroblast migration depends upon the cytoskeleton structure. Due to the large quantity of fibroblasts in skeletal muscle tissue, their close proximity to the satellite cell niche, the role they play in regeneration of skeletal muscle, and the importance of E3 ligases in cellular homeostasis, our objective was to investigate the expression of MuRF1 and MuRF2 in skeletal muscle fibroblasts and address their importance with the migration process, a key functional hallmark.Material and methodsThe protocols used in this study are in agreement with ethical principles in animal research followed by the Brazilian College of Animal Experimentation and were approved by the Institute of Biomedical Sciences/University of São Paulo ‐ Ethical Committee for Animal Research (# 151/12). For fibroblast muscle primary culture, cells were isolated from hind limb muscles of male FVB mice (~8 weeks old, 30±3g). The cells were incubated at 37°C in an atmosphere of 5% CO2 on 75 cm2 tissue culture dishes with growth medium( DMEM with 10% Fetal Bovine Serum) and the rapidly adhering fraction containing muscle fibroblasts isolated were used (adherent cells were kept after replacing of medium. For small interfering RNA (siRNA) interference, we transfected primary fibroblasts when reaching 80% confluence with MuRF1 and MuRF2 specific siRNAs. Wound‐healing assay was evaluated with a scratched wound created 12 hours after siRNA silencing. Cell migration into denuded area was monitored over a time course using a phase contrast microscopy and cell movement was calculated as decrease of the wound area over time (in percentage, initial area of the wound: 100%).ResultsOur results revealed the presence of MuRF1 in the cytoplasm and nuclei of primary fibroblasts isolated from skeletal muscle, cardiac muscle and also the permanent cell line MEF. On the other hand, the MuRF2 labeling was detected only at the cytoplasm of all cell lines analyzed. Knock down of MuRF1 by siRNA did not show a significant effect on migration capacity. However, MuRF2 siRNA knock down caused a significant decrease in fibroblast migration capacity after two (Vehicle 10% ± 0,04% vs MuRF2 3% ± 0,01%), four (Vehicle 13% ± 0,05% vs MuRF2 6% ± 0,01%) and twenty four hours (Vehicle 60% ± 0,13% vs MuRF2 38% ± 0,11%) of onset of the assay. Twenty‐four hours after the beginning of the wound‐healing assay, the distribution of polymerized actin was analyzed. Interestingly, MuRF1 and MuRF2 siRNA knocked down skeletal muscle fibroblasts exhibited a reduction in polymerized actin fibers. Accordingly, when we used fibroblasts simultaneously knocked down for MuRF1 and MuRF2 a reduction of polymerized actin fibers was observed.ConclusionWe conclude that MuRF1 and MuRF2 are expressed in fibroblasts from skeletal muscle and those proteins play an important role in the fibroblast migration activity.Support or Funding InformationGrant: 2012/13315‐7 São Paulo Research Fundation (Fapesp).