Abstract

   Myostatin (MSTN), a member of the transforming growth factor-β
   superfamily, inhibits the activation of muscle satellite cells.
   However, the role and regulatory network of MSTN in equine muscle cells
   are not well understood yet. We discovered that MSTN knockdown
   significantly reduces the proliferation rate of equine muscle satellite
   cells. In addition, after the RNA sequencing of equine satellite cells
   transfected with MSTN-interference plasmid and control plasmid, an
   analysis of the differentially expressed genes was carried out. It was
   revealed that MSTN regulatory networks mainly involve genes related to
   muscle function and cell-cycle regulation, and signaling pathways, such
   as Notch, MAPK, and WNT. Subsequent real-time PCR in equine satellite
   cells and immunohistochemistry on newborn and adult muscle also
   verified the MSTN regulatory network found in RNA sequencing analysis.
   The results of this study provide new insight into the regulatory
   mechanism of equine MSTN.

   Keywords: horse, satellite cell, MSTN gene, RNA sequencing, signaling
   pathway, cell proliferation

1. Introduction

   MSTN knockout increases mice skeletal muscle mass as a result of both
   hyperplasia (increase in number) and hypertrophy (enlargement) of the
   muscle fibers [[54]1], and the mutations in the MSTN gene could result
   in the “double muscling” phenotype in cattle [[55]2,[56]3], sheep
   [[57]4], dogs [[58]5], and humans [[59]6]. MSTN, often known as the
   “speed gene” in horses [[60]7], has been acknowledged as a significant
   genetic factor influencing race distance aptitude [[61]8,[62]9].
   Numerous sequence variations have been found in the MSTN gene’s
   upstream and downstream regions, and research has examined their
   relationships to Thoroughbred horses’ race performance
   [[63]10,[64]11,[65]12]. Researchers have used CRISPR/Cas9 editing
   technologies to make MSTN-null horse embryos with the goal of
   artificially enhancing equine sports performance [[66]13,[67]14]. The
   equine’s general health is significantly impacted by skeletal muscle
   metabolism. The majority of research on equine skeletal muscle has
   concentrated on a small number of specific metabolites produced during
   acute exercise or training [[68]15]. After suffering a muscle-fiber
   injury, skeletal muscles in particular have an extraordinary ability
   for regeneration, allowing for the complete restoration of their
   structure and function within a few weeks [[69]16]. Direct trauma and
   excessive exercise can harm a horse’s myofibers over the course of a
   lifetime [[70]17], and the activation [[71]18], proliferation [[72]19],
   and terminal differentiation of muscle quiescent satellite cells (SCs)
   [[73]20] are crucial to their recovery. At present, little is known
   about the MSTN regulation network in equine muscle SCs, despite the
   fact that the impact of MSTN knockdown and MSTN-related signaling
   pathways in other mammalian skeletal muscle SCs have been examined
   [[74]20,[75]21,[76]22,[77]23,[78]24,[79]25]. In this study, we used
   miRNA interference approaches to expose the MSTN regulation network in
   equine muscle SCs because MSTN is an important gene for comprehending
   the mechanisms underpinning equine muscle development and regeneration
   [[80]26].

2. Materials and Methods

2.1. In Vitro Culture of Horse Muscle SCs

   Semitendinosus muscle samples were collected from healthy horses at the
   local slaughter house. When the muscle sample arrived at the lab, it
   was immediately sanitized with 70% ethanol and washed three to four
   times with 4-fold volume cold DPBS. After the visible adipose and
   connective tissues on the muscle mass were removed with a knife, all
   the muscles were excised and chopped into little pieces with scissors.
   With collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA) and
   trypsin (0.25%, Sigma-Aldrich) solutions, fractionated enzymatic
   digestion was carried out for 2–30 min at 37 °C while stirring in a
   water bath. The cell suspension was then progressively filtered through
   70 µm and 40 µm cell strainers. The cell pellet was then extracted from
   the filtrates using a centrifuge. The cells pellet was then transferred
   to culture disks and resuspended in media (20% FBS/DMEM/AB), which were
   then incubated at 37 °C with 5% CO[2]. In total, 1.5 h of preplating
   were utilized to reduce any potential fibroblast contamination.
   Satellite-cell-containing supernatant was then added to culture disks.
   Every two days, the growing media were changed. The cells were passaged
   at a ratio of 1:3 after reaching 80% confluence.

2.2. Immunofluorescence Assay

   After being fixed with 4% paraformaldehyde for 30 min, the cells were
   rinsed with PBS containing BSA and Triton X-100, blocked for 1 h at 37
   °C with PBS containing Triton X-100, and then incubated with the
   primary Pax7 (AB-528428, DSHBY) and desmin (LS-B3122, LSBio) antibodies
   overnight at 4°C. Following three cycles of washing, samples were
   incubated with the secondary antibody for 1 h at room temperature. The
   cells were then washed three times, exposed to Hoechst (1 mg/mL, 10 min
   at RT), incubated, rinsed once more, and mounted.

2.3. Plasmid Construction and Transfection Analysis

   The miRNAs targeting MSTN (mirRNA267 and mirRNA364) were designed with
   BLOCKit RNA DESIGN
   ([81]https://www.thermofisher.cn/cn/zh/home/life-science/rnai.html
   (accessed on 7 October 2022)) based on the sequence of Mongolian horse
   MSTN CDS.

   In order to create the MSTN knockdown plasmid, these two miRNAs with
   BglII and EcoRI restriction sites were separately ligated with Td
   Tomato-C1 vector and then transformed into the competent cells DH5α.
   The plasmid was verified by restriction digestion and sequencing, and
   finally the correct interference plasmid was purified and named Td
   Tomato-mirRNA267(364)-eMSTN.

   SCs were transfected with the Td Tomato-miRNA plasmid using
   Lipofectamine 2000 (Life Science) at a plasmid density of 60,000
   cells/cm^2. At 36 h post transfection, the fluorescence signal was
   observed with a fluorescence microscope to verify the transfection
   efficiency.

2.4. RNA Sequencing (RNA-seq)

   At 36 h, the cell culture fluid was removed and the cells were washed
   with PBS three times. Finally, each well was collected with 1 mL Trizol
   reagent (Invitrogen, Waltham, MA, USA) to collect the cell sample;
   then, the total RNA could be extracted immediately, or it could be
   stored in an ultra-low temperature refrigerator at −80 °C for later
   use. The RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) was
   used to create a library, and the Illumina HiseqTM2500 was used to
   perform the sequencing.

2.5. Screening of Differentially Expressed Genes (DEGs)

   DEGs in MSTN knockdown group compared with controls of equine SCs were
   identified using the DESeq R package from
   ww.bioinfo.au.tsinghua.edu.cn/software/degseq, accessed on 7 October
   2022. Differential gene screening mainly refers to the difference fold
   (Fold change value) and q value (padj value, corrected p value) as
   related indicators; the genes with expression |log2 fold change| ≥ 1
   and q < 0.05 were selected as DEGs.

2.6. Gene Ontology (GO) and KEGG Pathway Enrichment Analysis

   DEGs were annotated using the GO enrichment analysis, in which gene
   length bias was corrected. GO terms with the corrected value p < 0.05
   were considered significantly enriched. Signaling pathways were
   investigated using KEGG (Kyoto Encyclopedia of Genes and Genomes), and
   KOBAS version 2.0 software. q < 0.05 was considered a significant
   value.

2.7. Quantitative PCR (qPCR) Assay

   Trizol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA)
   was used to separate the RNA from the MSTN knockdown and control
   groups, and a reverse transcription kit was used to convert the RNA
   samples into cDNA (Fermentas; Thermo Fisher Scientific, Inc.,
   Pittsburgh, PA, USA). Using β-actin as a control, 14 genes were chosen
   for validation of the RNA sequencing gene profiling results. In total,
   10 µL of SYBR Green PCR Master Mix, 2 µL of cDNA, 1.2 µL of each primer
   (10 µM), and 6.8 µL of RNase-free water made up the SYBR Green PCR
   experiment. The cycling schedules were as follows: 95 °C for 1 min, 95
   °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Using the 2^−∆∆CT
   approach, the levels of gene expression were calculated. The parallel
   experiment has at least three replicates, and the data’s mean value of
   Mean ± S.D. is shown. In this study, a significance level of p < 0.05
   is accepted. [82]Table S1 contains a list of the primers utilized for
   this study.

2.8. Immunohistochemistry

   Equine muscle cryosections of 8 µm were prepared from the Tissue-Tek
   embedded samples and were collected onto coated glass slides (Thermo
   Scientific SuperFrost Plus Adhesion slides, Fisher scientific,
   Brussels, Belgium) and stored at −20 °C. The muscle cryosections were
   air-dried and then blocked for 120 min in 5% defat milk in PBS solution
   at room temperature. The slides were washed with permeabilization
   solution (BSA and 0.2% triton in PBS solution) and incubated overnight
   at −4 °C with primary antibodies for MSTN (DF13273, Affinity, Hong
   Kong, China), MyoD1 (MA5-12902, Thermo Fisher), and MyoG (A17427,
   ABclonal, Wuhan, China) at a dilution of 1:500. After the slides were
   rinsed 5 min in PBS, they were washed briefly with permeabilization
   solution and incubated with the secondary antibodies dissolved in 0.5%
   BSA in PBS for 1 h at room temperature. Subsequently, after rinsing the
   slides for 5 min in PBS, the DAB Horseradish Peroxidase Color
   Development Kit (HZ-0010, Luzhen Biology, China) was applied. The
   staining intensity of 10 random units at a 200× amplification (with
   each muscle fascicle as the measure unit) was quantified by the
   well-established Image-J-software-based method indicated in the
   previous studies [[83]27,[84]28,[85]29].

2.9. Statistics

   There are at least three replicates of the parallel experiment, and the
   mean value of Mean ± S.D is indicated for the data. All data were
   analyzed by SPSS16.0 using two-way ANOVA with repeated measures,
   followed by Tukey’s test. A difference was deemed statistically
   significant if the p-value was less than 0.05.

3. Results

3.1. The Characteristics of Horse Muscle SCs

   We were able to successfully separate and purify the Mongolian domestic
   horse muscle satellite cells (HMSC) in vitro using the methods of
   enzyme digestion and differential adhesion. The horse fetal fibroblast
   (HFF) is a polygonal cell type, as can be seen in ([86]Figure 1A),
   whereas the HMSC have finer edges and are primarily spindle-shaped.
   Additionally, HMSC demonstrated a different rate of in vitro
   proliferation, demonstrating that its proliferation is slower than that
   of HFF (p < 0.05). ([87]Figure 1B). Additionally, the results of qPCR
   ([88]Figure 1C) and cell immunofluorescence test ([89]Figure 1D–F)
   jointly demonstrated that HMSC, but not HFF, exhibits significant
   expression of genes specific to mammalian SCs, such as Desmin, Pax7,
   and MyoD1.

Figure 1.

   [90]Figure 1
   [91]Open in a new tab

   HMSC characterization. (A) Morphology of HFF and HMSC in vitro. (B)
   Proliferation curves (p < 0.05) of HFF (blue line) and HMSC (red line).
   (C) qPCR determination of Pax7 and MyoD1 mRNA expression in HFF and
   HMSC * p < 0.05. (D–F) Immunofluorescence results of Pax7, Desmin, and
   MyoD1 in HFF and HMSC.

3.2. MSTN Knockdown Accelerates HMSC Cell Growth

   The interference plasmids miR267 and miR364 were created in this
   investigation by designing two miRNAs (miR267, miR364) that target
   horse MSTN mRNA and cloning them into the Td Tomato-C1 plasmid
   ([92]Figure 2A). The CtrlmiR plasmid containing a control miRNA was
   employed in this investigation as a control. Based on the red
   fluorescence of Td Tomato, the transfection efficiencies of three
   different plasmid types reached 80% after 24 h ([93]Figure 2B), and the
   expression of MSTN mRNA was dramatically repressed (p < 0.05)
   ([94]Figure 2C). Furthermore, we discovered that MSTN knockdown might
   greatly improve the cell proliferation rate of HMSC during subsequent
   culture, particularly during the first 48 to 72 h ([95]Figure 2D).

Figure 2.

   [96]Figure 2
   [97]Open in a new tab

   The effect of MSTN knockdown on HMSC proliferation. (A) Plasmid
   construction images in SnapGeneTM1.1.3 Software for equine MSTN
   knockdown plasmid. (B) After 24h transfection in Mongolian horse SCs in
   monolayer cultures. (C) qPCR validation of the interference efficiency.
   (D) Cell proliferation curves for different groups. * represents p <
   0.05.

3.3. MSTN Knockdown Alters the Transcriptome

   Two samples of each cell line were sequenced, and RNA-seq libraries
   were created for the control (transfected with CtrlmiR) and
   experimental (transfected with miR267 and 364) groups to profile gene
   expression after MSTN knockdown. After eliminating adapters and
   removing low-quality reads, the sequencing provided a high-quality
   dataset ([98]Figure 3A–C) and about 78,134,605 clean reads.

Figure 3.

   [99]Figure 3
   [100]Open in a new tab

   Analysis of differentially expressed genes (DEGs) after MSTN knockdown
   in HMSC. (A) Clean base number of all sample readings. (B) Filtering
   distribution map indicates the high quality of readings. (C) The
   location of readings indicates the normal distribution ratio of read
   sequences. (D) Clustering of DEGs among samples. (E) Volcano plot for
   the DEGs between control group and MSTN knockdown group. (F) Histogram
   of GO enrichment of DEGs. (G) KEGG pathway analysis on DEGs. (H) qPCR
   validation of DEGs. Red line represents the gene expression level in
   control group, and all bars represent the expression levels of tested
   genes in experimental groups treated with different miRNA targeting
   MSTN. All tested genes in experimental groups showed significant
   changes at p < 0.05 compared to the control group.

3.4. DEG Analysis Reveals the MSTN Regulatory Networks in HSMCs

   To examine variations in gene expression, read count data from the
   transcriptome were used. There were 598 DEGs in all that showed a
   difference expression in the experimental and control groups. When the
   threshold values were q < 0.05 and |log2 Fold change| ≥ 1, 427 of them
   were up-regulated and 171 were down-regulated in the experimental
   groups compared to the control group ([101]Figure 3D,E). Three
   categories were created from the results of the GO enrichment study of
   DEGs: biological process (BP), cellular component (CC), and molecular
   function (MF). The cellular activities, developmental processes, and
   biological regulation in terms of BP; the cell part, organelles, and
   membranes in terms of CC; and the catalytic activity, signal
   transduction activity, and binding in terms of MF were shown to be
   enriched in the down- and up-regulated genes ([102]Figure 3F).
   According to KEGG analysis, DEGs are involved in 37 pathways, including
   the mitogen-activated protein kinase (MAPK) signaling pathway, the
   TGF-β signaling pathway, the signaling pathway that controls stem cell
   pluripotency, the signaling pathway that controls the actin skeleton,
   the PI3A-Akt signaling pathways, the cancer pathways, and the signaling
   pathway for cell adhesion molecules ([103]Figure 3G). In total, 14 DEGs
   associated with myogenesis and the satellite cell cycle were chosen for
   qPCR experiments to confirm the RNA-seq results. Overall, the qPCR and
   RNA-seq results were well correlated, proving the accuracy and
   reliability of the RNA-seq data ([104]Figure 3H).

3.5. The MSTN Regulatory Network in the Horse Muscle Was Verified by
Immunohistochemistry Data

   We used immunohistochemistry to examine the expression of MSTN, MyoG,
   and MyoD1 in adult and newborn equine muscle tissues because the in
   vivo expression of MSTN in muscle tissue declines with age. This
   allowed us to determine whether the expression of the positively
   regulated DEG MyoG, and the negatively regulated DEG MyoD1 also changed
   in a manner that was consistent with age. The immunohistochemistry
   results confirmed the findings of RNA-seq and qPCR, demonstrating that
   MSTN negatively regulates MyoD1 expression while positively regulating
   MyoG at the protein level ([105]Figure 4).

Figure 4.

   [106]Figure 4
   [107]Open in a new tab

   Immunohistochemistry staining of MSTN, MyoG, and MyoD1 expression in
   newborn and adult equine muscles. The positive signals are quantified
   in left bar charts. * Indicates the significance at the level of p <
   0.05.

4. Discussion

   An ancient horse breed known as the Mongolian horse, which has long
   been a staple of the nomadic pastoral herders’ culture in North Asia
   [[108]30,[109]31], is renowned for its exceptional endurance and robust
   genetic variety [[110]30,[111]32]. As a result, the Inner Mongolia
   Autonomous Region of China and Mongolia consider the Mongolian horse to
   be one of the most significant breeds of traditional long-distance
   races [[112]32].

   The procedure of harvesting SCs from equine muscles was first described
   in 1992 [[113]33] and has been used to examine the impact of exercise
   on muscle tissue [[114]34]. However, there haven’t been many
   investigations into the processes that underlie MSTN’s function in the
   horse model. When tissue is damaged or injured, SCs change to a
   proliferative state, which enables the production of a large cell pool
   suited for myogenic differentiation [[115]35]. Our research shows that,
   similar to SCs from other species, SCs from horses exclusively express
   Pax7, MyoD1, and Desmin [[116]36,[117]37,[118]38]. In the population of
   adult SCs, Pax7 is expressed in both quiescent and active conditions
   [[119]39]. Equine SCs grown in culture have “wedge” morphologies and
   have condensed interphase chromatin, which is in line with the idea
   that the majority of SCs in resting muscles are quiescent and
   transcriptionally inactive [[120]40,[121]41].

   The MSTN CDS region’s full-length sequence, which was cloned from a
   muscle sample from a Mongolian horse in the current work, is 1134 bp,
   and it is entirely consistent with the Thoroughbred MSTN CDS sequence
   supplied by NCBI (Accession number: [122]NM_001081817.1). We discovered
   that MSTN knockdown could boost the SCs’ proliferation rate through the
   very effective RNAi studies. The MSTN knockdown investigations on the
   SCs of other animals likewise reported the same behavior. A previous
   mouse study discovered that MSTN functions through a complex regulatory
   network that includes PAX, Myosin family proteins, WNT, MAPKmTOR family
   members, and numerous genes related to CDKN1C (p57, Kip2) that control
   the cell cycle [[123]42]. Similar to this, our RNA-seq analysis
   following MSTN knockdown revealed that MSTN is involved in the
   regulation of multiple pathways crucial for the activities of muscle
   cells in horse muscle SCs.

   According to our findings, we discovered that a number of DEGs are
   connected to the PI3K/AKT/mTOR signaling pathways, which are crucial
   for mediating a variety of cellular functions, including nutrition
   uptake, anabolic responses, cell development, and survival.
   Phosphatidylinositol 3-kinase (PI3Ks), AKT, and the mammalian target of
   rapamycin (mTOR, also known as mechanistic TOR), which make up the
   heart of this pathway, are frequently over-activated in most
   malignancies and have thus come to be the subject of research in this
   area [[124]43]. Additionally, it has been noted that the MSTN-Smad
   pathway affects protein kinase AKT’s activity, preventing the mTOR
   pathway and protein synthesis [[125]44]. DEGs belonging to WNT
   signaling also emphasize the critical functions of WNT members in
   muscles. The previous study found that WNT signaling might be activated
   after muscle damage and that myogenic cells’ TCF reporter activity
   increased two days after muscle damage [[126]45]. Unlike Wnt1, Wnt3a,
   and Wnt5a, which promote SCs proliferation, Wnt4 and Wnt6 inhibit it
   [[127]19]. Although Wnt4 has a beneficial role in controlling the
   proliferation of SCs, MSTN can operate as Wnt4’s upstream antagonist to
   prevent Wnt4-mediated SCs growth. Additionally, it has been
   demonstrated that MSTN increases the expression of the WNT signaling
   pathway inhibitors sFRP1 and sFRP2 [[128]46]. We discovered that Wnt5a
   greatly increases following MSTN interference, suggesting that WNT is
   also involved in the SCs regulation controlled by MSTN. We also focused
   on aspects of cell cycle regulation in this work. SCs are in a
   quiescent state in the adult resting muscle [[129]47], which is
   characterized by a low rate of metabolism, a lack of cell cycling (G[0]
   phase), and low RNA concentration [[130]48]. In cell-culture studies,
   mechanistically, MSTN interacts with the cell cycle machinery to induce
   the cell cycle exit during the gap phases (G[1] and G[2]) [[131]49]. In
   bovine skeletal muscle SCs, MSTN knockdown resulted in an increase in
   CDK2 expression, a decrease in P21 expression, and a stimulation of
   proliferation [[132]50]. The increased production of the
   cyclin-dependent kinase inhibitor, P21, which further suppresses the
   gene essential for the G[1]/S transition, Cdk2, is thought to be the
   cause of the MSTN-triggered halt of the cell cycle and the inhibition
   of proliferation [[133]51,[134]52,[135]53]. Compared with
   activated/cycling SCs, many negative regulators of the cell cycle and
   myogenic inhibitors, including cyclin-dependent kinase inhibitors 1B
   (Cdkn1b; also known as p27 or p27Kip1 [[136]54]) and 1C (Cdkn1c, also
   known as p57 or p57Kip2); Rb [[137]55] (also known as Rb1); and Rgs2,
   Rgs5, Pmp22, and FGF suppressor gene Spry1 [[138]56], were highly
   up-regulated in quiescent SCs [[139]57,[140]58]. Our RNA-seq results
   mainly agree with those from studies on different species and models,
   suggesting that one of the main roles of MSTN is cell-cycle regulation.

   Pax7 is robustly and consistently expressed in the SCs of adult muscle,
   whereas Pax3 is often expressed at very low levels, with the exception
   of muscles such as the diaphragm [[141]59]. In our study, Pax7 and Pax3
   expression did not alter significantly; however, Pax5 expression
   significantly decreased. This distinction leads us to believe that
   different species have different regulatory roles for PAX family
   proteins in the SCs. However, more research is required to make this
   clear. Myogenic cells can selectively express the genes Myf5 and MyoD1,
   whereas resting SCs cannot. When SCs enter the cell cycle again and
   differentiate into highly proliferating myoblasts, Myf5 and MyoD1 are
   elevated. Some cells exit the cell cycle during the proliferation
   phase, start to express MyoG, and then differentiate into new muscle
   fibers, while other cells lose their myogenic capabilities by lowering
   the level of MyoD1 expression and return to a resting state to refill
   the SCs pool [[142]60]. In our research, after MSTN knockdown in horse
   muscle SCs, MyoD1 increased, whereas MyoG decreased. Together with the
   age-related MSTN downregulation-induced MyoD1 increase and MyoG
   decrease in horse muscle, the critical role of MSTN in the entire life
   cycle of muscle cells is highlighted here.

   In conclusion, the MSTN regulatory network described here advances our
   understanding of the function and activity of equine muscle SCs and
   encourages the use of this information in tissue engineering,
   regenerative medicine, and related fields of study.

Supplementary Materials

   The following supporting information can be downloaded at:
   [143]https://www.mdpi.com/article/10.3390/genes13101836/s1, Table S1:
   Q-PCR primers for DEG validations.
   [144]Click here for additional data file.^ (75.6KB, zip)

Author Contributions

   Conceptualization: U.B., T.U., Y.S. and G.L.; assistance with
   completing the experiment: T.D., M.Y. and D.B. (Demuul Bold);
   writing–review and editing; M.D. (Ming Du), B.L., T.J., G.D., D.D.,
   D.B. (Dongyi Bai), X.Z. and G.B.; and management and coordination
   responsibility for the research and making important revisions to the
   manuscript: M.D. (Manglai Dugarjav) and G.B. All authors have read and
   agreed to the published version of the manuscript.

Institutional Review Board Statement

   All procedures involving animals were approved and authorized by the
   Key Laboratory of Equine Genetics Inner Mongolia Agricultural
   University. All experiments and methods were carried out according to
   guidelines and regulations of Inner Mongolia Agricultural University.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The datasets used and/or analyzed during the current study are
   available from the corresponding author on reasonable request.

Conflicts of Interest

   The authors declare no competing interests.

Funding Statement

   This study was supported by the National Natural Science Foundation of
   China and the Mongolian Foundation of Science and Technology
   (NSFC-MFST) Joint Project (31961143025), NSFC-regional fund (31960657).

Footnotes

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   claims in published maps and institutional affiliations.

References