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.
Highlights
Skeletal muscle injury activates satellite cells to initiate processes of proliferation, differentiation, and hypertrophy in order to regenerate muscle fibers
Functional analysis of identified miRNAs and differentially expressed genes (DEG) Based upon the Pathway Studio Web Software (Elsevier, USA) and available literature, the results were divided into groups containing miRNAs related to the specific cellular processes, as follows: (1) cell proliferation and differentiation, (2) muscle regeneration and hypertrophy
We focused on HMB-induced miRNA expression changes previously described as those associated with the muscle tissue injury, regeneration, and the accompanying processes such as cell activation, proliferation, migration, and differentiation
Summary
Skeletal muscle injury activates satellite cells to initiate processes of proliferation, differentiation, and hypertrophy in order to regenerate muscle fibers. 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, attenuate the pathological processes induced during an in vitro model of H2O2-related injury by changing the expression of miRNAs. Hydroxy-β-methylbutyrate (HMB) is a metabolite of the essential amino acid leucine and is naturally synthesized in animals, plants, and humans [1]. The inflammatory response coincides with muscle repair, regeneration, and growth, involving activation and proliferation of satellite cells followed by their terminal differentiation. Activation of satellite cells is considered to play a crucial role in injured muscle fibers by incorporating new myonuclei and increasing muscle size and strength (by hypertrophy) [13]
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