Effect of β-hydroxy-β-methylbutyrate on miRNA expression in differentiating equine satellite cells exposed to hydrogen peroxide

Abstract

BackgroundSkeletal muscle injury activates satellite cells to initiate processes of proliferation, differentiation, and hypertrophy in order to regenerate muscle fibers. The number of microRNAs and their target genes are engaged in satellite cell activation. β-Hydroxy-β-methylbutyrate (HMB) is known to prevent exercise-induced muscle damage. The purpose of this study was to evaluate the effect of HMB on miRNA and relevant target gene expression in differentiating equine satellite cells exposed to H2O2. We hypothesized that HMB may regulate satellite cell activity, proliferation, and differentiation, hence attenuate the pathological processes induced during an in vitro model of H2O2-related injury by changing the expression of miRNAs.MethodsEquine satellite cells (ESC) were isolated from the samples of skeletal muscle collected from young horses. ESC were treated with HMB (24 h) and then exposed to H2O2 (1 h). For the microRNA and gene expression assessment microarrays, technique was used. Identified miRNAs and genes were validated using real-time qPCR. Cell viability, oxidative stress, and cell damage were measured using colorimetric method and flow cytometry.ResultsAnalysis of miRNA and gene profile in differentiating ESC pre-incubated with HMB and then exposed to H2O2 revealed difference in the expression of 27 miRNAs and 4740 genes, of which 344 were potential target genes for identified miRNAs. Special attention was focused on differentially expressed miRNAs and their target genes involved in processes related to skeletal muscle injury. Western blot analysis showed protein protection in HMB-pre-treated group compared to control. The viability test confirmed that HMB enhanced cell survival after the hydrogen peroxide exposition.ConclusionsOur results suggest that ESC pre-incubated with HMB and exposed to H2O2 could affect expression on miRNA levels responsible for skeletal muscle development, cell proliferation and differentiation, and activation of tissue repair after injury. Enrichment analyses for targeted genes revealed that a large group of genes was associated with the regulation of signaling pathways crucial for muscle tissue development, protein metabolism, muscle injury, and regeneration, as well as with oxidative stress response.Electronic supplementary materialThe online version of this article (10.1186/s12263-018-0598-2) contains supplementary material, which is available to authorized users.

Simultaneous miRNA and mRNA Transcriptome Profiling of Differentiating Equine Satellite Cells Treated with Gamma-Oryzanol and Exposed to Hydrogen Peroxide

“…Equine satellite cells (ESC) were isolated based on the same protocol as described by Chodkowska et al [25]. The growth medium was changed every two days.…”

“…The growth medium was changed every two days. Based on the cell viability (MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) and fusion index, the best primary cell line was chosen [25,26].…”

“…When the cells obtained 80% confluence, the proliferation medium was replaced by differentiation medium (2% HS/ Dulbecco’s Modified Eagle Medium (DMEM)/AB). After the 2nd day of differentiation, 0.125 µM of GO (TCI Chemicals, Portland, USA), dissolved in 0.04 µL/mL DMSO as a vehicle, which was also used in the control medium, was added for 24 h. The MTT assay was used to choose the concentration of GO and H 2 O 2 [25]. During the last part of the experiment (the last one hour of GO incubation), H 2 O 2 (3 mM, Sigma Aldrich, Poznań, Poland) was added.…”

“…A number of genes and miRNAs were previously described as being involved in cellular and metabolic processes, observed during effort, inflammation, or aging in muscular tissue [24,25]. We hypothesize that GO pre-incubation may change miRNA and the gene profile of differentiating equine satellite cells treated with hydrogen peroxide (H 2 O 2 ).…”

Role of satellite cells in growth and regeneration of skeletal muscles

“…W odpowiedzi na uszkodzenie będące wynikiem urazu mechanicznego, termicznego bądź rozwoju choroby, komórki satelitowe ulegają aktywacji, podziałom komórkowym, różnicowaniu oraz fuzji z uszkodzonymi włóknami mięśniowymi (ryc. 1) (7,8,23,26). Są one unipotencjalnymi komórkami macierzystymi mięśni szkieletowych zdolnymi do samoodnawiania własnej populacji (37).…”