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Effect of Β-Hydroxy-Β-Methylbutyrate on Mirna Expression in Differentiating Equine Satellite Cells Exposed to Hydrogen Peroxide Karolina A

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Effect of Β-Hydroxy-Β-Methylbutyrate on Mirna Expression in Differentiating Equine Satellite Cells Exposed to Hydrogen Peroxide Karolina A Chodkowska et al. Genes & Nutrition (2018) 13:10 https://doi.org/10.1186/s12263-018-0598-2 RESEARCH Open Access Effect of β-hydroxy-β-methylbutyrate on miRNA expression in differentiating equine satellite cells exposed to hydrogen peroxide Karolina A. Chodkowska, Anna Ciecierska, Kinga Majchrzak, Piotr Ostaszewski and Tomasz Sadkowski* Abstract Background: Skeletal 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. Methods: Equine 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. Results: Analysis 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. Conclusions: OurresultssuggestthatESCpre-incubatedwithHMBandexposedtoH2O2 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. Keywords: miRNA, HMB, Equine satellite cells, Muscle injury, Skeletal muscle * Correspondence: [email protected] Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chodkowska et al. Genes & Nutrition (2018) 13:10 Page 2 of 20 Background also exposed to H2O2 an in vitro factor initiating cellular β-Hydroxy-β-methylbutyrate (HMB) is a metabolite of response similar to that observed in vivo during a short the essential amino acid leucine and is naturally synthe- intensive physical exercise and post-exercise injury. sized in animals, plants, and humans [1]. Dietary supple- mentation of HMB is used to enhance gains in strength Methods and lean body mass associated with resistance training Muscle samples and cell culture and for increasing lean mass in cancer-related cachexia Samples of skeletal muscles (m. semitendinosus)werecol- [2, 3]. Unlike anabolic hormones which only increase lected from 6 months old healthy stallions in a slaughter muscle protein synthesis to accelerate muscular hyper- house. Muscle samples (0.5 × 0.5 × 0.5 cm) were taken im- trophy, HMB increases dynamic strength [4, 5] and lean mediately, washed in phosphate-buffered saline (PBS) with body mass [6] acting as an anti-catabolic agent, reducing gradually decreasing antibiotic concentration [40.000 and protein breakdown [5] and cellular damage which may 20.000 IU Penicillium crystalicum (PC; Polfa, Poland) per accompany intense exercise [7]. Moreover, previous 100 ml PBS], cleaned from connective and fat tissue, cut studies have demonstrated that HMB supplementation and immediately suspended in sterile fetal bovine serum decreased plasma post-exercise creatine kinase and lactic (FBS; Life Technologies, USA) with 10% addition of acid in thoroughbreds [8]. dimethylsulfoxide (DMSO), gradually frozen to − 80 °C, and Reactive oxygen species (ROS), such a hydrogen per- finally stored in liquid nitrogen until use. oxide (H2O2), exert a critical regulatory role on skeletal muscle function [9, 10]. In resting muscle cells, free radi- Satellite cell isolation, proliferation, and differentiation cals and ROS are rapidly and efficiently neutralized by Equine satellite cells (ESC) were isolated according to antioxidants. Exercise creates an imbalance between the following protocol. Protease from Streptomyces gri- ROS and activates natural antioxidant mechanisms. seus (Pronase®, Sigma-Aldrich, USA) was reconstituted Moreover, ROS produced during exercise by inflamma- in low-glucose Dulbecco's modified Eagle’s medium tory cells may also be involved in delayed onset of (DMEM), GlutaMAX™, Pyruvate (Life Technologies, muscle damage observed during inflammation [11]. The USA), and stirred for 1 h, pH 7.3. The incubation buffer inflammatory response coincides with muscle repair, re- (IB) consisted per sample of Pronase 0.5 mg/ml, 18 ml generation, and growth, involving activation and prolif- of DMEM, FBS 2 ml (Life Technologies, USA), and PC eration of satellite cells followed by their terminal (20.000 IU). IB was filtered through a cellulose acetate differentiation. In response to the damage, quiescent sat- membrane syringe filter (Sigma-Aldrich, USA). The frag- ellite cells are activated and undergo several cycles of mented muscle tissue was thawed, washed in PBS with cell division prior to their withdrawal from the cell cycle PC (20.000 IU), and suspended in IB for 1.5 h at 37 °C, through terminal differentiation and finally fusion with shaken every 15 min. Then, samples were sieved through the damaged skeletal muscle fibers [12]. During training- cell strainer (70 μm, nylon, Falcon, USA). The filtrate related tissue microdamage, activation of satellite cells is was centrifuged for 20 min (350 g), which was repeated considered to play a crucial role in injured muscle fibers three times. After each centrifugation, supernatant was by incorporating new myonuclei and thus increasing discarded, cell pellet was re-suspended in growth muscle size and strength (by hypertrophy) [13]. medium (GM; 10%FBS/10% horse serum (HS) in MicroRNAs (miRNAs) are small non-coding interfering DMEM (Life Technologies, USA) and antibiotics (AB; RNA molecules (18–25 nucleotides) able to post- 0.5% amphotericin B (Fungizone, Life Technologies, transcriptionally regulate gene expression through USA), 1% penicillin-streptomycin (Life Technologies, sequence-specific base pairing to messenger ribonucleic USA)). After the last centrifugation, cell suspension was acid (mRNA). These molecules have been shown to be transferred to polystyrene Petri dishes (Becton Dickin- important key players in a variety of physiological and son, USA) for 1.5 h to allow adhesion of fibroblast. After pathological processes (proliferation, differentiation, apop- that, supernatant with satellite cells was transferred into tosis, hypertrophy, timing development, inflammation, culture dishes (Primaria Cell Culture Flask, Becton Dick- cancer, etc.). A group of miRNAs, highly enriched in skel- inson, USA) and cultured in GM. The growth medium etal and/or cardiac muscles (myomiRs), has recently been was changed every 2 days. On the tenth day of prolifera- identified and includes miR-1, miR-133a, miR-133b, miR- tion, cells were trypsinized, counted by Scepter Cell 206, miR-208, miR-208b, miR-486, and miR-499 [14] Counter (Merck Millipore, Germany), transferred which regulate skeletal muscle development. (30,000 cells from each isolation) to Collagen I Cellware Szcześniak et al. [15] were the first who demonstrated six-well plate (Greiner Bio-One, USA), and cultured in the effect of HMB in ESC. Our study was performed to GM. After reaching 80% confluency, the proliferation evaluate miRNA profile and relevant target genes in differ- media was replaced by the differentiation media (DM; entiating equine satellite cells incubated with HMB and 2%HS in DMEM with AB). Chodkowska et al. Genes & Nutrition (2018) 13:10 Page 3 of 20 Primary satellite cell cultures from semitendinosus MiRNA was isolated from eight equine satellite cell muscle of all horses were isolated, and the culture with cultures for both HMB pre-treated (n = 8) and control the best scores of cell viability (MTT assay) [16] and fu- group (n = 8). As recommended by Agilent Technologies sion
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