Effect of β-hydroxy-β-methylbutyrate on miRNA expression in differentiating equine satellite cells exposed to hydrogen peroxide
Chodkowska et al. Genes & Nutrition
Effect of β-hydroxy-β-methylbutyrate on miRNA expression in differentiating equine satellite cells exposed to hydrogen peroxide
Karolina A. Chodkowska 0
Anna Ciecierska 0
Kinga Majchrzak 0
Piotr Ostaszewski 0
Tomasz Sadkowski 0
0 Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - SGGW , Nowoursynowska 159, 02-776 Warsaw , Poland
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: Our 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.
miRNA; HMB; Equine satellite cells; Muscle injury; Skeletal muscle
β-Hydroxy-β-methylbutyrate (HMB) is a metabolite of
the essential amino acid leucine and is naturally
synthesized in animals, plants, and humans [
supplementation of HMB is used to enhance gains in strength
and lean body mass associated with resistance training
and for increasing lean mass in cancer-related cachexia
]. Unlike anabolic hormones which only increase
muscle protein synthesis to accelerate muscular
hypertrophy, HMB increases dynamic strength [
] and lean
body mass [
] acting as an anti-catabolic agent, reducing
protein breakdown [
] and cellular damage which may
accompany intense exercise [
]. Moreover, previous
studies have demonstrated that HMB supplementation
decreased plasma post-exercise creatine kinase and lactic
acid in thoroughbreds [
Reactive oxygen species (ROS), such a hydrogen
peroxide (H2O2), exert a critical regulatory role on skeletal
muscle function [
]. In resting muscle cells, free
radicals and ROS are rapidly and efficiently neutralized by
antioxidants. Exercise creates an imbalance between
ROS and activates natural antioxidant mechanisms.
Moreover, ROS produced during exercise by
inflammatory cells may also be involved in delayed onset of
muscle damage observed during inflammation . The
inflammatory response coincides with muscle repair,
regeneration, and growth, involving activation and
proliferation of satellite cells followed by their terminal
differentiation. In response to the damage, quiescent
satellite cells are activated and undergo several cycles of
cell division prior to their withdrawal from the cell cycle
through terminal differentiation and finally fusion with
the damaged skeletal muscle fibers [
trainingrelated tissue microdamage, activation of satellite cells is
considered to play a crucial role in injured muscle fibers
by incorporating new myonuclei and thus increasing
muscle size and strength (by hypertrophy) [
MicroRNAs (miRNAs) are small non-coding interfering
RNA molecules (18–25 nucleotides) able to
posttranscriptionally regulate gene expression through
sequence-specific base pairing to messenger ribonucleic
acid (mRNA). These molecules have been shown to be
important key players in a variety of physiological and
pathological processes (proliferation, differentiation,
apoptosis, hypertrophy, timing development, inflammation,
cancer, etc.). A group of miRNAs, highly enriched in
skeletal and/or cardiac muscles (myomiRs), has recently been
identified and includes miR-1, miR-133a, miR-133b,
miR206, miR-208, miR-208b, miR-486, and miR-499 [
which regulate skeletal muscle development.
Szcześniak et al. [
] were the first who demonstrated
the effect of HMB in ESC. Our study was performed to
evaluate miRNA profile and relevant target genes in
differentiating equine satellite cells incubated with HMB and
also exposed to H2O2 an in vitro factor initiating cellular
response similar to that observed in vivo during a short
intensive physical exercise and post-exercise injury.
Muscle samples and cell culture
Samples of skeletal muscles (m. semitendinosus) were
collected from 6 months old healthy stallions in a slaughter
house. Muscle samples (0.5 × 0.5 × 0.5 cm) were taken
immediately, washed in phosphate-buffered saline (PBS) with
gradually decreasing antibiotic concentration [40.000 and
20.000 IU Penicillium crystalicum (PC; Polfa, Poland) per
100 ml PBS], cleaned from connective and fat tissue, cut
and immediately suspended in sterile fetal bovine serum
(FBS; Life Technologies, USA) with 10% addition of
dimethylsulfoxide (DMSO), gradually frozen to − 80 °C, and
finally stored in liquid nitrogen until use.
Satellite cell isolation, proliferation, and differentiation
Equine satellite cells (ESC) were isolated according to
the following protocol. Protease from Streptomyces
griseus (Pronase®, Sigma-Aldrich, USA) was reconstituted
in low-glucose Dulbecco's modified Eagle’s medium
(DMEM), GlutaMAX™, Pyruvate (Life Technologies,
USA), and stirred for 1 h, pH 7.3. The incubation buffer
(IB) consisted per sample of Pronase 0.5 mg/ml, 18 ml
of DMEM, FBS 2 ml (Life Technologies, USA), and PC
(20.000 IU). IB was filtered through a cellulose acetate
membrane syringe filter (Sigma-Aldrich, USA). The
fragmented muscle tissue was thawed, washed in PBS with
PC (20.000 IU), and suspended in IB for 1.5 h at 37 °C,
shaken every 15 min. Then, samples were sieved through
cell strainer (70 μm, nylon, Falcon, USA). The filtrate
was centrifuged for 20 min (350 g), which was repeated
three times. After each centrifugation, supernatant was
discarded, cell pellet was re-suspended in growth
medium (GM; 10%FBS/10% horse serum (HS) in
DMEM (Life Technologies, USA) and antibiotics (AB;
0.5% amphotericin B (Fungizone, Life Technologies,
USA), 1% penicillin-streptomycin (Life Technologies,
USA)). After the last centrifugation, cell suspension was
transferred to polystyrene Petri dishes (Becton
Dickinson, USA) for 1.5 h to allow adhesion of fibroblast. After
that, supernatant with satellite cells was transferred into
culture dishes (Primaria Cell Culture Flask, Becton
Dickinson, USA) and cultured in GM. The growth medium
was changed every 2 days. On the tenth day of
proliferation, cells were trypsinized, counted by Scepter Cell
Counter (Merck Millipore, Germany), transferred
(30,000 cells from each isolation) to Collagen I Cellware
six-well plate (Greiner Bio-One, USA), and cultured in
GM. After reaching 80% confluency, the proliferation
media was replaced by the differentiation media (DM;
2%HS in DMEM with AB).
Primary satellite cell cultures from semitendinosus
muscle of all horses were isolated, and the culture with
the best scores of cell viability (MTT assay) [
fusion index was selected for further analysis (data not
shown). Different stages of equine satellite cell culture
are presented in Fig. 1.
After the second day of differentiation, 50 μM HMB
(Metabolic Technologies Inc., USA) was added to the culture
media, and then, cells were incubated for an additional 24 h.
Ca-HMB was purchased from MTI (USA). The free HMB
acid was extracted by acidification and organic extraction [
HMB dose was chosen based upon previous studies [
and MTT assay results which confirmed literature data (data
not shown). During the last hour of incubation, 3 mM
hydrogen peroxide (solution 30% (w/w) in H2O
(Sigma-Aldrich, USA) was added to induce cell damage. Due to the
lack of literature data on the doses of H2O2 used in the
equine satellite cell culture, the MTT assay was performed
using doses ranging for 0.125 to 50 mM. Compared to the
previously described doses of H2O2 used in other cell culture
models, those used for ESCs were relatively large. For this
reason, we decided to use H2O2 dose 3 mM with DL-25
(Fig. 2). The experimental design is presented in Fig. 3.
After the H2O2 treatment, the differentiating ESCs were
scraped and total RNA was isolated using a miRNeasy
Mini Kit (Qiagen, USA) according to the manufacturer’s
protocol. The quantity of RNA was measured
spectrophotometrically using NanoDrop 2000 (Thermo
Scientific, USA). The quality of the total RNA was verified by
Bioanalyzer 2100 (Agilent, USA), and only samples with
RIN ≥ 9.2 were used for further analysis.
For the microRNA profiling, the Custom Equine miRNA
8x15K Microarray slides were designed using eArray
platform (https://earray.chem.agilent.com/earray, GEO
database: GPL20990) and provided by Agilent
MiRNA was isolated from eight equine satellite cell
cultures for both HMB pre-treated (n = 8) and control
group (n = 8). As recommended by Agilent Technologies
(USA), 100 ng of total RNA of each sample was taken
and labeled using miRNA Complete Labeling and Hyb
Kit (version 2.3, December 2010). For hybridization,
Microarray Hybridization Chamber (Agilent, USA) and
Hyb-Buffer (Agilent, USA) were used according to the
manufacturer’s protocol. In the next step, slides were washed
out using Gene Expression Wash Pack (Agilent, USA) and
scanned in Microarray Scanner (model G2565CA) with
SureScan High-Resolution Technology (Agilent, USA).
Microarray data were extracted, the background was
subtracted, and normalization was performed using the
standard procedures included in the Agilent Feature
Extraction (FE) Software version 10.7.3.1.
Analysis of gene expression (GE) profile was performed
using Horse Gene Expression Microarray, 4x44K (Agilent
Technologies, USA) based on the same protocol as
described by Szcześniak et al. (2016), [
twocolor microarray, with 825 ng of cRNA from
HMBexposed cells (labeled by Cy5, n = 4) and 825 ng of cRNA
from control cells (labeled by Cy3, n = 4), and RNA
SpikeIn Kit (Agilent Technologies, USA) as an internal control
were used. The background was subtracted and Linear and
Lowess normalization was performed using the standard
procedures included in the Agilent Feature Extraction (FE)
Software version 10.7.3.1. The data were statistically
analyzed using Gene Spring 13.0 software (Agilent, USA). The
statistical significance of the differences was evaluated using
Student’s t test (p < 0.05) and Benjamini and Hochberg
multiple testing correction. False Discovery Rate (FDR)
≤ 0.05 and fold change (FC) ≥ 1.3 were considered as
statistically significant. Microarray data were deposited at the
Gene Expression Omnibus data repository under the
number GSE73779 for miRNA and GSE93025 for cDNA.
The criteria for miRNA and differentially expressed gene
(DEG) selection for real-time qPCR validation and
further analysis were of biological relevance (miRNAs
linked to muscular development, hypertrophy, muscle
injuries, oxidative stress, and tissue regeneration) and
were assessed based on Pathway Studio Mammalian
(Elsevier, USA) and available literature.
For miRNA, real-time qPCR validation miRCURY
LNA™ Universal RT microRNA PCR kit (Exiqon, USA)
was used. A two-step protocol was applied: (1)
polymerase activation at 95 °C for 10 min and (2) 40
amplification cycles at 95 °C for 10s and 60 °C for 1 min,
according to the manufacturer protocol.
Primers were chosen based on the miRNA sequences
assigned to microarray probes and were provided by
Exiqon (Denmark) (Table 1). Calculation of the relative
miRNA expression using the ΔΔCt method was applied
using GenEX 6 software provided by MultiD (Sweden).
Obtained data were statistically analyzed using two-tailed
Student’s t test. Values of p ≤ 0.05 were considered
Based on previous studies in different species and the
manufacturer recommendation (Exiqon, Denmark), a U6
snRNA reference was used. To verify GE microarray
results, the real-time qPCR method was applied. All the
steps of real-time qPCR procedure were made based on
the protocols previously described by Szcześniak et al.
]. The sequences of primers are listed in
Table 2. Gapdh was used as a reference gene.
Target gene prediction and ontological analyses
MicroRNA target gene prediction was performed using the
TargetScan database. The analysis was performed for all
identified HMB-affected miRNAs. For each predicted target
of individual miRNA, the sum of the context + scores was
automatically calculated. Predicted targets of each miRNA
family were automatically sorted by total context + score.
Analysis was performed for the context score percentile
(50) and conserved/non-conserved miRNA families and
target sites [
]. For further analysis, common genes for
those identified genes using GE microarray and predicted
miRNA target genes were selected and considered as
targets for HMB treatment-influenced miRNAs.
Ontological analyses revealing molecular functions,
biological processes, and pathways of miRNA targets were
performed in DAVID 6.7 using Fisher’s exact test with
p ≤ 0.05. Detailed analysis of the role of HMB-modulated
miRNAs, genes identified using GE, and target genes in
various metabolic and signal pathways was performed
using Pathway Studio Web (Elsevier, USA). Relationships
between all differentially expressed miRNAs were
visualized with Pathway Studio’s Build Pathway functionality
which is based on the wave-propagation algorithm
developed for the navigation through complex networks. Find
Direct Links/All objects Directions Algorithm was used in
Western blot analysis
The procedure of Western blot analysis was performed
based on the previously described methodology by
Zielniok et al. [
]. Antibodies used in Western blot
were against the following: SOD1 (ab62800), SOD2
(ab13534), TGFβ2 (sc-90), α-tubulin (ab176560), BDNF
Cell viability, cell damage, and oxidative stress
Hydrogen peroxide, used in the experiment as a
damage factor, is known to affect various cellular processes.
Several tests related to the cell viability, cell damage,
and oxidative stress were performed to assess the
impact of HMB on the cellular processes following
incubation with H2O2. Experimental conditions
(incubation time, doses of HMB and H2O2) were the same as
previously in the part related to the microarray and
real-time qPCR analysis.
CellROX® Green Reagent Kit (Life Technologies) was
used to measure oxidative stress and cell death in ESC’s
based on the manufacturer protocol. Cells were seeded on
24-well plates at 0.05 × 106 cells/cm2. Cells were incubated
for 60 min with CellROX reagent in a final concentration
250 μM. During the last 15 min of staining, SYTOX Red
Dead Cell was added (at the final concentration 5 nM). The
samples were analyzed immediately after staining using
FACS Aria II (BD Biosciences) flow cytometer. A total of
50,000 events per sample (n = 3) were collected. This
staining was performed on live cells during the proliferative
phase (90% confluency). Data were analyzed using FlowJo
(TreeStar, USA) and GraphPad Prism software.
The second test related to oxidative stress called Total
Antioxidant Capacity (TAC) Assay Kit (Abcam, UK) was
used according to the manufacturer protocol. This test
can measure either the combination of both small
molecule antioxidants and proteins or small molecules alone
Annealing time and temperature
15 s/60 °C
15 s/60 °C
15 s/60 °C
15 s/60 °C
15 s/60 °C
15 s/60 °C
15 s/60 °C
in the presence of our proprietary Protein Mask. Cells
were seeded on 96-well plates at 2 × 106 cells (n = 6).
After a 90-min incubation, the plate was read on Tekan
System reader at 570 nm wavelength. Data were
analyzed using GraphPad Prism software.
Lipid peroxidation is the degradation of lipids that
may accompany the activity of several cell damage
factors including hydrogen peroxide. It is also one of the
popular markers for oxidative stress. Lipid peroxidation
Assay Kit (Sigma-Aldrich) was used to measure lipid
peroxidation. All the procedure was performed based on
the provided manufacturer protocol. The concentration
of MDA was measured for n = 6. The staining was
performed on live cells during differentiation phase.
In order to increase the reliability of the obtained
results related to cell survival, the MTT test was also
performed (n = 6) based on the previously published protocol
]. Data for both tests were analyzed using GraphPad
Qualitative flow cytometry assay for mitochondrial
depolarization was also performed according to the
manufacturer protocol. The
5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocyanine iodide (JC-1,
SigmaAldrich) was used. It is a cationic, lipophilic dye that
accumulates in mitochondria and exhibits green
fluorescence (525 nm) in its monomeric state. The mostly
implemented application of JC-1 is detection of
mitochondrial depolarization occurring in the early stages of
apoptosis. JC-1 was dissolved in DMSO and medium II for a
final concentration of 0.6 μM. The cells were incubated at
37 °C, washed, trypsinized, and resuspended in 2%FBS/PBS
medium. Fifty thousand events were collected for each
sample using FACS Aria II (BD Biosciences) flow
cytometer. Fluorescence compensation was done for 525 nm.
This staining was performed on live cells during the
proliferative phase (90% of confluency; n = 3). Data were
analyzed using FlowJo (TreeStar, USA) and GraphPad
In the “Results” and “Discussion” sections, gene symbols
are marked in italics and lowercase. The arrows indicate
the direction of expression change: ↓ and ↑ for
downand upregulation, respectively.
Analysis of the miRNA expression in differentiating
equine satellite cells incubated with HMB (24 h) and
exposed to H2O2 (1 h) revealed differences in 27 miRNAs.
Among them, eight demonstrated higher expression and
19 lower expression when compared to control (Table 3).
Analysis of gene expression profile for the same
experimental conditions as those mentioned above revealed
difference in the expression of 4740 transcripts. After
removing all duplicate values and unknown sequences,
1923 unique genes were found (Additional file 1: Table S1).
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 (miR-1, miR-133a/b, miR-206, miR-128, miR-146a/b,
miR-204, miR-155, miR-193a, miR-221/222, miR-324,
miR331, miR-374b, miR-486, miR-675), (2) muscle regeneration
and hypertrophy (miR-1, miR-133a/b, miR-142, miR-128,
miR-146b, miR-208b, miR-675), (3) oxidative stress and
inflammation (miR-146a/b), and (4) others (miR-149,
miR30c, miR-532-3p, miR-532-5p, miR-542) (Fig. 4).
Functional analysis showed that GE microarray
identified genes were significantly associated with the
following biological processes: cellular processes, muscle organ
development, proteolysis involved in cellular protein
catabolic process, muscle cell differentiation, positive
regulation of biological processes, cell death, apoptosis,
regulation of cell proliferation, and positive regulation of
inflammatory process (Additional file 2: Table S2).
Among identified genes (DEG), special attention has
been focused on a few important groups which are
known to be HMB affected: muscle organ development
(e.g., six1, myf5, acta1, cav1, myh3, myh7, myl2, myl3,
sgcd, tgfb2), response to wounding/injury (e.g., jak2, igf2,
several members of cxcl and interleukin genes, sod1,
sod2), inflammatory response/innate immune response/
oxidative stress (tlr3, tlr4, tlr10, cd40, cd44, igf2, itgb6,
il-5, il-6, il-15, il-23, sod1, sod2, and a large group of
chemokine ligand: ccl-1, ccl-2, ccl-5, ccl-8).
From the microarray results, six miRNAs and six genes
were selected as a single representative for the
aforementioned processes for further RT-qPCR validation.
The analysis confirmed statistically significant
differences in the expression of six miRNAs (miR-204,
miR208b, miR-222, miR-675, miR-146a, and miR-146b) and
six genes (sod1, sod2, tgfb2, myf5, bdnf, otud4) in
HMBtreated ESC when compared to control condition
(CTRL) (Fig. 5). All RT-qPCR validated miRNAs and
genes presented the same trend as microarray results.
Prediction and ontological analysis of miRNA target genes (DET)
TargetScan analysis was performed to predict potential
target genes for all identified miRNAs. The analysis
revealed unique 3310 targets for downregulated and 2117
unique targets for upregulated miRNAs. We compared
all identified HMB-regulated DEG and aforementioned
predicted miRNA target genes to find those which could
be regulated by HMB-induced miRNAs in ESC cultures
exposed to H2O2. Finally, 344 differentially expressed
target genes (DET) were identified.
Functional analysis showed that DET were associated
significantly with several processes which plays an
important role in the physiological (protein metabolism,
muscle tissue development, cellular homeostasis,
apoptosis) and pathological (inflammation, cancer) conditions
in muscular tissue (Table 4).
Signaling pathway analysis showed that 27 identified
miRNAs could affected target genes involved in several
important signaling pathways related to the processes previously
described as modified by HMB and also some other which
HMB was suspected to affect. The most meaningful
pathways are the following: MAPK, RIG-I, Toll-like
receptor, hypertrophic cardiomyopathy, ubiquitin-mediated
proteolysis, Ras, and response to oxidative stress.
Western blot analysis
Western blot analysis of the level of reference proteins
and five proteins related to the muscle tissue, muscle
damage, and oxidative stress was performed. However, the
results are difficult to interpret. Protein degradation at
different levels was observed in all samples treated only
with hydrogen peroxide (Fig. 6). In groups pre-incubated
with HMB and H2O2, protein degradation was smaller or
not observed. It is related to the protein degradation
which is strongly linked to hydrogen peroxide effect.
Cell viability, cell damage, and oxidative stress
To measure cell viability, two tests were used—MTT
and SYTOX Red Dead Cell (as a component of CellROX
Green Reagent kit). In both tests, increased cell viability
and decreased amount of dead cells were observed in a
group pre-treated with HMB and incubated with H2O2
than in a control group (incubated only with H2O2). All
the results from these two tests were statistically
significant (p < 0.05). The results of SYTOX Red Dead Cell (A)
and MTT test (B) are presented in Fig. 7.
Oxidative stress was measured using CellROX® Green
Reagent. There was no significant difference between
groups (Fig. 8a). Similar results were obtained with the
test for lipid peroxidation. There were no statistically
significant differences between HMB pre-treated group
and control. However, surprisingly, higher lipid
peroxidation trend was observed in a HMB pre-treated group
compared to control (Fig. 8b).
Qualitative flow cytometry assay for mitochondrial
depolarization (JC-1) showed significant differences
between the Q2 population (monomers + aggregates in %)
and Q4 population (JC-1) in control and HMB
pretreated group. There was no significant difference
between Q1 population (% of aggregates) and Q3
population (% of monomers) (Fig. 9a).
Results obtained in a total antioxidant capacity (TAC)
assay showed significant differences between HMB
pretreated and control group. Higher antioxidant capacity
was observed in HMB pre-treated group (Fig. 9b).
MicroRNAs are essential regulators for numerous
biological processes by modulating gene expression at the
post-transcriptional level. Several muscle-specific miRNAs
(myomiRs) have been shown to play an important role in
normal myoblast proliferation, differentiation, and muscle
remodeling in response to different type of factors. Recent
studies have begun to link miRNAs and certain
musclerelated diseases [
]. Modulation of miRNAs by dietary
factors and miRNA-based gene therapies seems to be a
promising option for the treatment of cardiac and skeletal
muscle diseases [
]. Among dietary additives, HMB
seems to be an interesting potential myoprotectant for
]. Previous studies suggest that HMB may be
involved in the regeneration processes of skeletal muscles
]. Moreover, HMB stimulates skeletal muscle satellite
cell activation and may potentially increase skeletal muscle
regenerative capacity after damage induction [
Our objective was to determine the influence of HMB on
miRNA and gene expression in differentiating equine
satellite cells subjected to damaging activity of hydrogen
peroxide, as an in vitro model of short extreme effort-related
muscle damage observed in racing and sport horses.
Microarray analysis of total RNA in differentiating ESC
incubated with HMB (24 h) and treated with H2O2 (1 h)
revealed the difference in the expression of 27 miRNAs
(Table 3) and 4740 DEG (Additional file 1: Table S1) from
which 344 DET were chosen (Table 4). Identified miRNAs
and a large group of identified genes were previously
described as these involved in the pathological and
physiological processes in skeletal muscles as well as in
other tissues. Selected miRNAs (miR-204 (↑), miR-208b (↓),
miR-222 (↑), miR-675 (↓), miR-146a (↑), miR-146b (↑)) and
genes (bdnf (↓), sod1 (↓), sod2 (↑), tgfb2 (↓), myf5 (↓), otud4
(↑)) were validated by RT-qPCR showing the same trend as
in microarray analysis.
HMB effects on miRNAs related to satellite/muscle cell proliferation and differentiation
Of the 27 identified miRNAs, 9 are related to cell
proliferation and 13 to differentiation in muscle tissue (Fig. 4).
Some of miRNAs seem to be particularly interesting in the
context of previous publications confirming proven and
potential HMB effect on muscle. Among them, family of
miR-146a/b able to balance the induction of muscle
proliferation or differentiation with miR-146 up- and
downregulation, respectively [
]. The miR-146a was one of the
highest differentially expressed molecules showing 120.92
fold change in HMB-treated cells. It could suggest their
possible involvement in promotion of HMB-induced
myoblast proliferation. It is well-known that activation and
proliferation of satellite cells is a prerequisite of skeletal
muscle injury repair [
], and it is possible that HMB is
capable to influence miRNA expression, increasing
myoblast proliferation rate and thus facilitating the myofiber
regeneration. Similar observations were done for miR-133, in
which upregulation was described as proliferation-inducing
while its downregulation was responsible for differentiation
]. Interestingly, miRNA-222/221 which
over-expression was noticed in myoblasts undergoing
differentiation with its downregulation after differentiation
] was downregulated in ESC cultures exposed to H2O2
and pretreated with HMB, when compared to control. The
same expression trend (↓) was observed in miR-374b
which over-expression is known to impair C2C12 cell
differentiation, while inhibition promoted this process [
Moreover, three miRNAs (miR-675, miR-324, and
miR331) known to be over-expressed in muscle cell
] were downregulated in our experiment. Two
other miRNAs, miR-206 and miR-1, known to be
downregulated in muscle cell proliferation and upregulated
during differentiation , have manifested downregulation in
ESC cultures treated with HMB. Moreover, some of the
identified miRNAs showed the opposite trend of
expression change to this mentioned above (miR-1↓, miR-133↓,
miR-206↓), promoting cell differentiation and proliferation
in case of miRNA upregulation and downregulation,
respectively. They were represented by miR-204 which was
upregulated in differentiated human cardiomyocyte
progenitor cells [
] and miR-155 (↑) and miR-193a (↑),
known to regulate cell differentiation in muscle cells [
and brown fat cells [
], respectively. All of them possessed
the same expression trend which was observed in our
experiment in the case of HMB-treated group.
The search of DET for the aforementioned miRNAs was
done using Pathway Studio Web and has revealed a large
group of genes involved in proliferation and
differentiation, the processes previously described to be HMB
modulated. The following cell proliferation-related genes were
identified: jak2 (target of identified miR-101, miR-155),
rarg (miR-142-3p, miR-30c), pten (miR-146a, miR-374b,
miR-193a), ets1 (miR-221/222), and rarb (miR-146a,
miR146b); cell differentiation-related target genes: jak2
(miR155), pten (miR-1), klf4 (miR-1, miR-146a, miR-206) and
ets1 (miR-221/222). Moreover, we identified several target
genes which are involved in muscle organ development:
sgcd (miR-142-3p), scd (miR-1, miR-128), cav3 (miR-101),
tcf12 (miR-101, miR-142-3p, miR-155, miR-204, miR-208,
miR-221/222), and col19a1 (miR-1, miR-206), as
modulated in ESC treated with HMB. Special attention deserves
miR-206 together with described above miR-1 and
miR133 which regulate expression of one of its potential target
genes cx43 involved not only in muscle development but
also in muscle regeneration where its upregulation was
]. The same expression trend of cx43 was
observed in our experiment in HMB-treated group. MiR-206
decreased expression in our experiment may be related to
the fact that inhibition of miR-206 robustly increases
myotube development [
Taken together, changes in expression of
proproliferative (miR-133a/b, miR-146a/b, miR-222/221) and
differentiation-related miRNAs (miR-1, miR-133a/b,
miR155, miR-193a, miR-204, miR-206, miR-221/222, miR-331,
miR-324, miR-374, miR-675) were observed following
HMB incubation and exposition of ESC cultures to H2O2,
with concomitant changes in expression of their
corresponding DET. These results, presenting the
proproliferation and pro-differentiation effects of the
aforementioned miRNAs, could be considered as contradictory,
but in fact, both processes are important for proper
myogenesis—satellite cell proliferation necessary for proper
myofiber regeneration manifested by myoblast fusion with
damaged fibers or new myofiber formation, here shown at
the very early stage of this process.
HMB involvement in oxidative stress and inflammation
In our study, we also observed HMB-related changes in
the expression of miRNAs playing an important role in
modulation of inflammation and oxidative stress. The
acute inflammatory response is protective and stimulates
repairing of injured tissue [
]. The inflammatory
infiltrate is a component of the satellite cell niche and also
a source of locally released cytokines which regulate
One of the most interesting miRNAs involved in
oxidative stress and inflammation appears to be miR-146 family
which members are known as negative regulators of
inflammatory cytokine expression during immune response
]. Curtale et al.  showed that miR-146b may
mediate anti-inflammatory activities and modulates the
TLR4 signaling pathway by direct targeting several genes
which are most likely targets for our identified miRNAs
(cxcl10, tlr4). Their study also provides evidence for a link
between miR-146b and IL-10, indicating that miR-146b
induction depends on the activity of IL-10, which is
suspected to be realized by muscle cells, both in vivo and
in vitro [
]. We did not observe changes in il-10 mRNA
expression in our experiment; however, other interleukin
and cytokine gene expression was changed (e.g., il-5, il-6,
il-13, il-15, il-18, cxcl10, and ccl11).
In muscles, inflammatory response coincides with
repair, regeneration, and growth, which involve activation
and proliferation of satellite cells, followed by their
terminal differentiation. Until now, limited number of data
is available to distinguish features of muscle
inflammation that promote injury from those that
promote growth or repair of muscle. Moreover, dietary
supplementation is known to be one of the ways to reduce
skeletal and cardiac muscle damage by decreasing the
inflammatory and oxidative stress response to exercise in
sport horses [
]. In muscles, anti-inflammatory
substances (e.g., NSAIDs) are used to control excess local
tissue damage by limiting proteolysis from infiltrating
inflammatory cells [
]. HMB has been suggested to
inhibit inflammation . However, its anti-inflammatory
mechanism is still not fully understood. Recent study
conducted by Yakabe et al. [
] suggested that HMB has
anti-inflammatory potential by downregulation of IL-6
expression. Surprisingly, il-6 was upregulated in our
experiment (FC = 20.01). Interestingly, the local production
of IL-6 by skeletal muscle cells and stromal cells
promotes activation of satellite cells, thereby increasing
myotube regeneration [
]. It is known that IL-6 mediates
many aspects of the exercise-induced acute-phase
response, including the upregulation of antioxidant
defenses as response to oxidative stress [
]. Similar to
the aforementioned authors who demonstrated that
miR146b may inhibit pro-inflammatory cytokine secretion,
we observed over-expression of both miR-146a and
miR-146b. Moreover, miR-155, known to be an
immunomodulatory miRNA, acts as a broad limiter of
pro-inflammatory gene expression in muscle [
possessing the same trend which was observed in our
experiment in the case of HMB-treated group. This, in turn,
suggests that HMB may play an important role in the
inflammation processes as an anti-inflammatory factor
which could be related to the inhibition of
proinflammatory cytokine secretion by HMB-induced
miR146 over-expression and activate innate immunity
response by miR-155 over-expression.
Interestingly, among DEG (several of them were
classified as DET), a large group with the highest FC was
involved in different kind of processes related to
immunity-acute phase of inflammatory, activation of
immunity cells, innate immunity, and pro-inflammatory
activity (Table 5). Several of them (e.g., ccl11, ccl2, cxcl10,
and saa1) were strongly upregulated, and this tendency
was previously described as associated with
proinflammatory activity status [
] which is not fully
consistent with tendency of identified in our experiment
miRNA expression. Moreover, that a large group of DEG
is involved in the inflammatory response and innate
immune response in different kind of tissues (tlr3, tlr4,
tlr10, cd40, cd44, igf2, itgb6, il-5, il-6, il-15, il-23, and a
group of chemokine ligand: ccl-1, ccl-2, ccl-5, ccl-8). Both
processes are necessary during regeneration, upon injury
when immune cells rapidly infiltrate the muscle tissue to
remove injured necrotic cells and secrete factors that are
essential to activate satellite cells.
The search of target genes for the identified
aforementioned miRNAs has revealed a number of genes
involved in innate immunity and processes
accompanying inflammation, which HMB affects,
represented by jak2 (miR-101, miR-155), tlr4 (miR-146a/b,
miR-155), cxcl10, cxcl11 (miR-146a/b), and
cd47-target for oxidative stress and inflammation-related
miRNAs (miR-221/222 and miR-155). HMB impact on
the inflammatory processes and oxidative stress is not
fully understood. However, our results show that this
substance can modulate in the opposite way
expression of pro- and anti-inflammatory miRNAs and
genes. We assume this could be linked not only to
the potential anti-inflammatory effect of HMB but
also to the activation of early (innate) immune
response (associated with H2O2-related damage), which
is the initial phase of the regeneration process.
11 parp14 Up
12 gsdmd Up
17 psmb9 Up
MicroRNAs related to cell reaction to injury—potential
role of HMB as myoprotectant
Among all identified miRNAs, several were known to be
involved in cell reaction to injury and different phases of
MiR-675 seems to be one of the most interesting
miRNAs which is the second of the most downregulated
in HMB-treated group and is closely related to
regeneration processes. Previous studies showed that
miR675 is expressed in skeletal muscles during myoblast
differentiation and muscle regeneration [
miRNA, miR-146, is related to satellite cell activation [
myoblast differentiation, and muscle regeneration in vivo
]. Moreover, also, miR-208 is known to be involved in
injury-induced satellite cell activation [
]. We suspect
that HMB may stimulate and/or accelerate activation of
equine satellite cells at very early stage of the regeneration
process. These observations may suggest that HMB acting
by the aforementioned miRNA induction may be involved
in the satellite cell activation which accompanies
regeneration. Hypertrophy is also an important phenomenon of
regeneration process in muscle; however, it is related to
the final stage of regeneration [
We identified several miRNAs that were previously
described in relation to muscle hypertrophy. They were
represented by downregulation in skeletal muscle hypertrophy
miR-1 and miR-133a/b [
] which possessed the same
trend as was noticed in our experiment in the case of
HMB-treated differentiating ESC cultures. Similar
observation was done for miR-142 (↓), which as mentioned above
presented one of the highest fold changes (FC = 105.23)
among the identified miRNAs, and its downregulation was
described during cardiac hypertrophy and is able to inhibit
cytokine signaling and function in the myocardium . It
is possible that HMB incubation changes the expression of
the aforementioned miRNAs facilitating for more efficient
late regeneration (not observed during the first day after
injury). More research is needed to evaluate the role of
miR133a/miR-1/miR-142-dependent hypertrophy mechanisms
of HMB action in the activation of skeletal hypertrophy and
muscle regeneration in different physiological and
Several potential target genes for identified miRNAs
were classified as follow: hypertrophy: tpm3 (miR-1,
miR-206), tpm1 (miR-142-5p), sgcd (miR-142-5p),
cacnb1 (miR-208b), itgav (miR-142-5p), itga2 (miR-30c,
miR-128); muscle regeneration-related processes and
pathways: cx43 (miR-206), klf4 (miR-30c), and vegfc
Other HMB-related miRNAs and genes
The effect of several identified in our experiment
miRNAs in muscle tissue is still not clear. However, our
results suggest that HMB modulates miRNA expression
which was previously described in connection with
various physiological and pathological conditions and could
also be affected by HMB in injured muscle tissue:
miR374b—related to lipid and protein metabolism [
miR-14—regulator of mitochondria function in C2C12
], miR-532-3p—regulates mitochondrial fission in
cardiac muscle , miR-30c—cellular adipogenesis and
cardiac hypertrophy and ischemia [
], miR-450a/c and
miR-142—negative regulators of cardiac hypertrophy
], and miR-542 family—regeneration of various
]. Interestingly, several identified genes (among
them potential target genes for the aforementioned
miRNAs) are also related to amino acid metabolism and
protein ubiquitination and proteasome pathways (rnf6,
cand2, ube2b, rnf138, rnf19b, rnf38, ube2l6, birc3,
rnf114, mib1, trim36, and rnf4) which are closely related
to the protein degradation during muscle atrophy in
cachexia, and HMB is known to inhibit this process in
humans and animals [
2, 5, 6
Target gene-related signaling pathways
Furthermore, ontological analysis of miRNA DET
revealed that a large group of genes was associated with
biological processes and signaling pathways that are
closely related to the muscle cell, muscle tissue
development, and protein metabolism presented in Tables 6
and 7, respectively.
Among the most strongly regulated pathways were
these previously described as engaged in immunity and
inflammation: Toll-like receptor, RIG-I, Ras, MAPK, and
ubiquitin-mediated proteolysis. Toll-like receptor pathway
is related to the inflammatory activity [
] and different
kind of myopathies and could be modulated by miR-155
]. RIG-I-like receptor pathway is known to play a
crucial role in innate response [
] which is necessary to
activate early muscle regeneration and may be modulated by
three identified miRNAs: miR-146a, miR-146b, and
miR155. Ras and MAPK pathways promote protein
degradation in muscle cells [
] and oxidative stress (closely
related to miR-146 activity) [
]. Moreover, MAPK pathway
plays a pivotal role in the energy metabolism through
modulating lipid metabolism, skeletal muscle growth, and
different kind of muscular myopathies and atrophy [
Finally, ubiquitin-mediated proteolysis identified as a
potential target gene-related pathway is closely related to
muscle atrophy by protein degradation.
Western blot analysis—HMB protective effect on proteins
Western blot analysis was performed to check the level of
proteins corresponding with several genes and miRNA.
However, the results that were obtained are impossible to
use in this way. It is related to the protein degradation
which is strongly linked to hydrogen peroxide effect.
Previous studies related to hydrogen peroxide effect on
different kind of proteins showed that this substance cause
protein degradation. For example, exposure of skeletal
muscle myotubes to ROS (i.e., hydrogen peroxide) can
activate proteases leading to protein degradation as it was
described by Li et al. [
] and Clung et al. [
et al. [
] observed the interactions of a liquid and gaseous
H2O2 with amino acids and proteins (bovine serum
albumin and aldolase). In this study, authors observed that
two dosages of hydrogen peroxide cause total degradation
of BSA (the absence of bands). In our study, we observed
similar situation. We checked three different
housekeeping genes: β-actin, gapdh, and α-tubulin. For all of them,
we noticed the same trend of protein degradation in
samples where the cells were exposed to hydrogen peroxide
(Fig. 6). Moreover, we checked several different proteins
where we also noticed protein degradation on a similar
level as in samples mentioned above. We suspect that
samples where the cells were previously incubated with
HMB were protected from protein degradation related to
hydrogen peroxide. This degradation has already occurred
at the cellular level, not during the sample preparation for
western blotting. During the sample preparation
procedure, we used RIPA buffer with protease inhibitor cocktail.
Based on this observation and previous studies [
related to HMB, we think that this substance may
decrease protein degradation in equine satellite cells
exposed to hydrogen peroxide; however, the comparison of
protein levels between experimental and control
conditions is impossible.
Cell viability, cell damage, and oxidative stress
Five different tests were performed to check the effect
of potential antioxidant properties of HMB and its
cell protection activity. Based on our results from the
test where we observed direct cell oxidative stress
together with typical damage (lipid peroxidation
Number of List of genes
together with mitochondria activity changes) related
to oxidative stress, it cannot be unambiguously
determined whether HMB in experimental dosage has
properties that limit oxidative stress (Figs. 8a, b and
9a). However, we observed that HMB increase total
antioxidant capacity (Fig. 9b). According to our
knowledge, there are limited data considering direct
antioxidant activity of HMB; however, as it was
mentioned, our study revealed several miRNAs, genes,
and pathways related to oxidative stress and
antioxidant activity which were modulated by HMB.
Increased total antioxidant capacity may represent an
adaptive response to the enhanced generation of ROS
related to hydrogen peroxide activity or likely
responsible for the attenuation of oxidative damage.
Two independent tests showed that HMB
increased cell viability: MTT and SYTOX Red Dead
Cell (Fig. 7). These observations are consistent with
the study presented by Vallejo et al. [
also observed that HMB enhanced myoblast viability
(in C2C12 cell line). Moreover, earlier study by
Pacini et al. [
] showed that HMB significantly
prevented dexamethasone-induced cell mortality. Based
on our results, we think that HMB may prevents
cells from the hydrogen peroxide-induced mortality
and enhance ESC’s viability.
Our study presents several new findings of the
mechanisms of action of HMB and its potential role in muscle
physiology and pathology. We focused on HMB-induced
miRNA expression changes previously described as
those associated with the muscle tissue injury
(inflammation), regeneration, and the accompanying processes
such as cell activation, proliferation, migration, and
differentiation. The scheme of putative mode of miRNAs
and DEG-depended HMB action is proposed in Fig. 10.
We demonstrated for the first time that HMB-treated
equine satellite cells exposed to H2O2 have modulated
expression of 27 miRNAs which could affect the
abovementioned processes. Many of them were not known for
being differentially expressed during myogenic
proliferation, differentiation, or processes related to muscle
injury and activation in early stage of regeneration. That is
why, it would also be interesting to investigate whether
some of the abovementioned miRNAs participate in
skeletal muscle degeneration/regeneration process as
well as degeneration-related equine muscular diseases,
such as muscle inflammation related to the extreme
effort or recurrent rhabdomyopathy.
Moreover, we found DET for identified
HMBmodulated miRNAs which are related to key processes
in muscle physiology and pathology. Also, identified
Pathway Number of
P value genes
Fig. 10 Potential role of HMB-induced miRNAs and selected target genes in muscle regeneration process
pathways MAPK, Ras, and RIG-I together with those
involved in oxidative stress response seem to support our
knowledge about the potential mechanisms of HMB
action. Based on the obtained results, we also believe that
HMB increases the survival of cells treated with
hydrogen peroxide. Further analyses evaluating the effect of
HMB on injured, recovering muscle tissue are needed to
verify the collected data.
Additional file 1: Table S1. Genes differentially expressed in
HMBincubated equine satellite cells exposed to H2O2, compared to control.
FDR ≤ 0.05, FC ≥ 1.3, n = 4. (XLSX 550 kb)
Additional file 2: Table S2. Biological function of differentially expressed
genes (DEG). (XLSX 28 kb)
CTRL: Control condition; DEG: Differentially expressed genes;
DET: Differentially expressed target genes; DM: Differentiation medium;
DMSO: Dimethylsulfoxide; ESCs: Equine satellite cells; FBS: Fetal bovine
serum; GE: Gene expression; GM: Growth medium; HMB:
β-Hydroxy-βmethylbutyrate; HS: Horse serum; IB: Incubation buffer; PBS:
Phosphatebuffered saline; ROS: Reactive oxygen species
The authors would like to thank Dr. Małgorzata Gajewska and Emmanuel
Gonzalez Escobar for their help in preparing the manuscript.
This research was funded by National Science Centre (Poland), Grant No.
2011/03/B/NZ5/05697. Publication of this manuscript was supported by
KNOW (Leading National Research Centre) Scientific Consortium “Healthy
Animal - Safe Food,” decision of the Ministry of Science and Higher
Education No. 05-1/KNOW2/2015.
Availability of data and materials
All data generated or analyzed during this study are included in this
published article (and its additional files).
KCH carried out the muscle sampling, cell viability, oxidative stress and lipid
peroxidation assays, RT-qPCR validation of microarray results, western blot,
ontological analysis, and interpretation of the obtained data and wrote the
manuscript. AC carried out the equine satellite cell isolation and culture, RNA
isolation and microarray analysis, and western blot. KM carried out the flow
cytometry analysis and data interpretation. PO participated in the study
design and helped in the manuscript revision. TS participated in the study
design; supervised the project; performed the muscle sampling, statistical
analyses of microarray and RT-qPCR data, cell viability, oxidative stress, and
lipid peroxidation; assisted in the manuscript preparation and revision; and
prepared the figures. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study complies with the national and institutional guidelines of the use
of animals in research according to the Polish legal act from January 21,
2005. Since sample collection was performed during the routine slaughter
and no additional procedures that would be harmful and painful for animals
were applied, this study did not require formal ethics approval.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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