Search Results (15)

Meat yield, determined by muscle growth and development, is an important economic trait for the swine industry and a focus of research in animal genetics and breeding. PDZ and LIM domain 5 (PDLIM5) are cytoskeleton-related proteins that play key roles in various tissues and cells. These proteins have multiple isoforms, primarily categorized as short (PDLIM5-short) and long (PDLIM5-long) types, distinguished by the absence and presence of an LIM domain, respectively. However, the expression patterns of swine PDLIM5 isoforms and their regulation during porcine skeletal muscle development remain largely unexplored. We observed that PDLIM5-long was expressed at very low levels in pig muscles and that PDLIM5-short and total PDLIM5 were highly expressed in the muscles of slow-growing pigs, suggesting that PDLIM5-short, the dominant transcript in pigs, is associated with a slow rate of muscle growth. PDLIM5-short suppressed myoblast proliferation and myogenic differentiation in vitro. We also identified two single nucleotide polymorphisms (−258 A > T and −191 T > G) in the 5′ flanking region of PDLIM5, which influenced the activity of the promoter and were associated with muscle growth rate in pigs. In summary, we demonstrated that PDLIM5-short negatively regulates myoblast proliferation and differentiation, providing a theoretical basis for improving pig breeding programs. Full article

Cell division cycle 23 (CDC23) is a component of the tetratricopeptide repeat (TPR) subunit in the anaphase-promoting complex or cyclosome (APC/C) complex, which participates in the regulation of mitosis in eukaryotes. However, the regulatory model and mechanism by which the CDC23 gene regulates muscle production in pigs are largely unknown. In this study, we investigated the expression of CDC23 in pigs, and the results indicated that CDC23 is widely expressed in various tissues and organs. In vitro cell experiments have demonstrated that CDC23 promotes the proliferation of myoblasts, as well as significantly positively regulating the differentiation of skeletal muscle satellite cells. In addition, Gene Set Enrichment Analysis (GSEA) revealed a significant downregulation of the cell cycle pathway during the differentiation process of skeletal muscle satellite cells. The protein–protein interaction (PPI) network showed a high degree of interaction between genes related to the cell cycle pathway and CDC23. Subsequently, in differentiated myocytes induced after overexpression of CDC23, the level of CDC23 exhibited a significant negative correlation with the expression of key factors in the cell cycle pathway, suggesting that CDC23 may be involved in the inhibition of the cell cycle signaling pathway in order to promote the differentiation process. In summary, we preliminarily determined the function of CDC23 with the aim of providing new insights into molecular regulation during porcine skeletal muscle development. Full article

Muscular insufficiency is observed in many conditions after injury, chronic inflammation, and especially in elderly populations. Causative cell therapies for muscle deficiencies are not state of the art. Animal models to study the therapy efficacy are, therefore, needed. We developed an improved protocol to produce myoblasts suitable for pre-clinical muscle therapy studies in a large animal model. Myoblasts were isolated from the striated muscle, expanded by employing five different protocols, and characterized on transcript and protein expression levels to determine procedures that yielded optimized regeneration-competent myoblasts and multi-nucleated myotubes. We report that swine skeletal myoblasts proliferated well under improved conditions without signs of cellular senescence, and expressed significant levels of myogenic markers including Pax7, MyoD1, Myf5, MyoG, Des, Myf6, CD56 (p ≤ 0.05 each). Upon terminal differentiation, myoblasts ceased proliferation and generated multi-nucleated myotubes. Injection of such myoblasts into the urethral sphincter complex of pigs with sphincter muscle insufficiency yielded an enhanced functional regeneration of this muscle (81.54% of initial level) when compared to the spontaneous regeneration in the sham controls without myoblast injection (67.03% of initial level). We conclude that the optimized production of porcine myoblasts yields cells that seem suitable for preclinical studies of cell therapy in a porcine large animal model of muscle insufficiency. Full article

The introduction of single-cell RNA sequencing (scRNA-seq) technology has spurred additional advancements in analyzing the cellular composition of tissues. The longissimus dorsi (LD) in pigs serves as the primary skeletal muscle for studying meat quality in the pig industry. However, the single-cell profile of porcine LD is still in its infancy stage. In this study, we profiled the transcriptomes of 16,018 cells in the LD of a newborn Suhuai pig at single-cell resolution. Subsequently, we constructed a cellular atlas of the LD, identifying 11 distinct cell populations, including endothelial cells (24.39%), myotubes (18.82%), fibro-adipogenic progenitors (FAPs, 18.11%), satellite cells (16.74%), myoblasts (3.99%), myocytes (5.74%), Schwann cells (3.81%), smooth muscle cells (3.22%), dendritic cells (2.99%), pericytes (1.86%), and neutrophils (0.33%). CellChat was employed to deduce the cell–cell interactions by evaluating the gene expression of receptor–ligand pairs across different cell types. The results show that FAPs and pericytes are the primary signal contributors in LD. In addition, we delineated the developmental trajectory of myogenic cells and examined alterations in the expression of various marker genes and molecular events throughout various stages of differentiation. Moreover, we found that FAPs can be divided into three subclusters (NR2F2-FAPs, LPL-FAPs, and TNMD-FAPs) according to their biological functions, suggesting that the FAPs could be associated with the differentiation of tendon cell. Taken together, we constructed the cellular atlas and cell communication network in LD of a newborn Suhuai pig, and analyzed the developmental trajectory of myogenic cells and the heterogeneity of FAPs subpopulation cells. This enhances our comprehension of the molecular features involved in skeletal muscle development and the meat quality control in pigs. Full article

Mutations in the DMD gene can cause Duchenne or Becker muscular dystrophy (DMD/BMD) by affecting the giant isoform of dystrophin, a protein encoded by the DMD gene. The role of small dystrophin isoforms is not well investigated yet, and they may play a role in muscle development and molecular pathology. Here, we investigated the nuclear localization of short carboxy-terminal dystrophin isoforms during the in vitro differentiation of human, porcine, and murine myoblast cultures. We could not only confirm the presence of Dp71 in the nucleoplasm and at the nuclear envelope, but we could also identify the Dp40 isoform in muscle nuclei. The localization of both isoforms over the first six days of differentiation was similar between human and porcine myoblasts, but murine myoblasts behaved differently. This highlights the importance of the porcine model in investigating DMD. We could also detect a wave-like pattern of nuclear presence of both Dp71 and Dp40, indicating a direct or indirect involvement in gene expression control during muscle differentiation. Full article

Skeletal muscle formation is an extremely important step in animal growth and development. Recent studies have found that TMEM8c (also known as Myomaker, MYMK), a muscle-specific transmembrane protein, can promote myoblast fusion and plays a key role in the normal development of skeletal muscle. However, the effect of Myomaker on porcine (Sus scrofa) myoblast fusion and the underlying regulatory mechanisms remain largely unknown. Therefore, in this study, we focused on the role and corresponding regulatory mechanism of the Myomaker gene during skeletal muscle development, cell differentiation, and muscle injury repair in pigs. We obtained the entire 3′ UTR sequence of porcine Myomaker using the 3′ RACE approach and found that miR-205 inhibited porcine myoblast fusion by targeting the 3′ UTR of Myomaker. In addition, based on a constructed porcine acute muscle injury model, we discovered that both the mRNA and protein expression of Myomaker were activated in the injured muscle, while miR-205 expression was significantly inhibited during skeletal muscle regeneration. The negative regulatory relationship between miR-205 and Myomaker was further confirmed in vivo. Taken together, the present study reveals that Myomaker plays a role during porcine myoblast fusion and skeletal muscle regeneration and demonstrates that miR-205 inhibits myoblast fusion through targeted regulation of the expression of Myomaker. Full article

Myogenic differentiation is a complex biological process that is regulated by multiple factors, among which long noncoding RNAs (lncRNAs) play an essential role. However, in-depth studies on the regulatory mechanisms of long noncoding RNAs (lncRNAs) in myogenic differentiation are limited. In this study, we characterized the role of the novel lncRNA TCONS_00323213, which is upregulated during porcine skeletal muscle satellite cell (PSC) differentiation in myogenesis. We found that TCONS_00323213 affected the proliferation and differentiation of PSC in vitro. We performed quantitative polymerase chain reaction (qPCR), 5-ethynyl-20-deoxyuridine (EdU), western blotting, immunofluorescence staining, pull-down assays, and cleavage under targets and tagmentation (CUT and Tag) assays to clarify the effects and action mechanisms of TCONS_00323213. LncRNA TCONS_00323213 inhibited myoblast proliferation based on analyses of cell survival rates during PSC proliferation. Functional analyses revealed that TCONS_00323213 promotes cell differentiation and enhances myogenin (MyoG), myosin heavy chain (MyHC), and myocyte enhancer factor 2 (MEF2C) during myoblast differentiation. As determined by pull-down and RNA immunoprecipitation (RIP) assays, the lncRNA TCONS_00323213 interacted with PBX/Knotted Homeobox 2 (PKNOX2). CUT and Tag assays showed that PKNOX2 was significantly enriched on the MyoG promoter after lncRNA TCONS_00323213 knockdown. Our findings demonstrate that the interaction between lncRNA TCONS_00323213 and PKNOX2 relieves the inhibitory effect of PKNOX2 on the MyoG promoter, increases its expression, and promotes PSC differentiation. This novel role of lncRNA TCONS_00323213 sheds light on the molecular mechanisms by which lncRNAs regulate porcine myogenesis. Full article

Muscle development is closely related to meat quality and production. CircRNAs, with a closed-ring structure, have been identified as a key regulator of muscle development. However, the roles and mechanisms of circRNAs in myogenesis are largely unknown. Hence, in order to unravel the functions of circRNAs in myogenesis, the present study explored circRNA profiling in skeletal muscle between Mashen and Large White pigs. The results showed that a total of 362 circRNAs, which included circIGF1R, were differentially expressed between the two pig breeds. Functional assays showed that circIGF1R promoted myoblast differentiation of porcine skeletal muscle satellite cells (SMSCs), while it had no effect on cell proliferation. In consideration of circRNA acting as a miRNA sponge, dual-luciferase reporter and RIP assays were performed and the results showed that circIGF1R could bind miR-16. Furthermore, the rescue experiments showed that circIGF1R could counteract the inhibitory effect of miR-16 on cell myoblast differentiation. Thus, circIGF1R may regulate myogenesis by acting as a miR-16 sponge. In conclusion, this study successfully screened candidate circRNAs involved in the regulation of porcine myogenesis and demonstrated that circIGF1R promotes myoblast differentiation via miR-16, which lays a theoretical foundation for understanding the role and mechanism of circRNAs in regulating porcine myoblast differentiation. Full article

Biochemical and biophysical properties instruct cardiac tissue morphogenesis. Here, we are reporting on a blend of cardiac decellularized extracellular matrix (dECM) from porcine ventricular tissue and fibrinogen that is suitable for investigations employing an in vitro 3D cardiac cell culture model. Rapid and specific coagulation with thrombin facilitates the gentle inclusion of cells while avoiding sedimentation during formation of the dECM-fibrin composite. Our investigations revealed enhanced cardiogenic differentiation in the H9c2 myoblast cells when using the system in a co-culture with Nor-10 fibroblasts. Further enhancement of differentiation efficiency was achieved by 3D embedding of rat neonatal cardiomyocytes in the 3D system. Calcium imaging and analysis of beating motion both indicate that the dECM-fibrin composite significantly enhances recovery, frequency, synchrony, and the maintenance of spontaneous beating, as compared to various controls including Matrigel, pure fibrin and collagen I as well as a fibrin-collagen I blend. Full article

The growth and development of skeletal muscle is regulated by many factors, and recent studies have shown that circular RNAs (circRNAs) can participate in this process. The model of porcine skeletal muscle injury was constructed to search for circRNAs that can regulate the growth and development of skeletal muscle in pigs. Using whole-transcriptome sequencing and bioinformatics analysis, a novel circRNA (circCSDE1) was screened out, which is highly expressed in skeletal muscle. Functional studies in C2C12 cells demonstrated that circCSDE1 could promote proliferation and inhibit myoblast differentiation, while opposing changes were observed by circCSDE1 knockdown. A dual-luciferase reporter assay revealed that circCSDE1 directly targeted miR-21-3p to regulate the expression of the downstream target gene (Cyclin-dependent kinase 16, CDK16). Moreover, miR-21-3p could inhibit proliferation and promote myoblast differentiation in C2C12 cells, opposite with the effects of circCSDE1. Additionally, the rescue experiments offered further evidence that circCSDE1 and its target, miR-21-3p, work together to regulate myoblast proliferation and differentiation. This study provides a theoretical basis for further understanding the regulatory mechanisms of circRNAs. Full article

DLK1 is paternally expressed and is involved in metabolism switching, stem cell maintenance, cell proliferation, and differentiation. Porcine DLK1 was identified in our previous study as a candidate gene that regulates muscle development. In the present study, we characterized DLK1 expression in pigs, and the results showed that DLK1 was highly expressed in the muscles of pigs. In-vitro cellular tests showed that DLK1 promoted myoblast proliferation, migration, and muscular hypertrophy, and at the same time inhibited muscle degradation. The expression of myogenic and fusion markers and the formation of multinucleated myotubes were both upregulated in myoblasts with DLK1 overexpression. DLK1 levels in cultured myocytes were negatively correlated with the expression of key factors in the Notch pathway, suggesting that the suppression of Notch signaling pathways may mediate these processes. Collectively, our results suggest a biological function of DLK1 as an enhancer of muscle development by the inhibition of Notch pathways. Full article

A number of studies have utilized blood waste as a bioresource by enzymatic hydrolysis to obtain amino acids, such as branched-chain amino acids, which can increase muscle mass or prevent muscle loss during weight loss. Although a significantly high content of branched-chain amino acids has been reported in porcine whole-blood protein hydrolysate (PWBPH), the effects of PWBPH on skeletal muscle differentiation and exercise function remain unclear. In this study, we investigated the effects of PWBPH on exercise endurance in ICR mice and muscle differentiation in C2C12 mouse myoblasts and gastrocnemius (Gas) muscle of mice. Supplementation with PWBPH (250 and 500 mg/kg for 5 weeks) increased the time to exhaustion on a treadmill. PWBPH also increased the Gas muscle weight to body weight ratio. In addition, PWBPH treatment increased skeletal muscle differentiation proteins and promoted the Akt/mTOR-dependent signaling pathway in vitro and in vivo. These results suggest that PWBPH can be utilized as a bioresource to enhance exercise function and skeletal muscle differentiation. Full article

Although thousands of long noncoding RNAs (lncRNAs) have been identified in porcine growth and development, the regulation mechanisms of functional lncRNAs have not been well explored. In this study, using 5′- and 3′-rapid amplification of cDNA ends (RACE) assays, we obtained two different variants of lncRNA maternally expressed gene 3 (MEG3), namely, MEG3 v1 and MEG3 v2, that were both highly expressed in porcine skeletal muscle and in the early stage of the differentiation of porcine satellite cells. Moreover, we identified the core transcript MEG3 v2. Functional analyses showed that MEG3 overexpression could effectively arrest myoblasts in the G1 phase, inhibit DNA replication, and promote myoblast differentiation, whereas MEG3 knockdown resulted in the opposite effects. Interestingly, the expression of serum response factor (SRF), a crucial transcription factor for myogenesis process, remarkably increased and decreased in mRNA and protein levels with the respective overexpression and knockdown of MEG3. Dual luciferase reporter assay showed that MEG3 could attenuate the decrease of luciferase activity of SRF induced by miR-423-5p in a dose-dependent manner. MEG3 overexpression could relieve the inhibitory effect on SRF and myoblast differentiation induced by miR-423-5p. In addition, results of RNA immunoprecipitation analysis suggested that MEG3 could act as a ceRNA for miR-423-5p. Our findings initially established a novel connection among MEG3, miR-423-5p, and SRF in porcine satellite cell differentiation. This novel role of MEG3 may shed new light on understanding of molecular regulation of lncRNA in porcine myogenesis. Full article

The differences of pork quality characteristics among different pig breeds mainly came from the differences in myofiber type compositions. Growing evidence indicated the key role of miRNAs in myofiber specification. In the present study, we found that miR-152 is more abundant in the slow-twitch myofiber-enriched muscles. However, its role in myofiber type transformation and myogenesis is largely unknown. Overexpression of miR-152 in porcine myotubes promoted the formation of slow-twitch myofibers and myogenesis. While, inhibition of miR-152 expression showed the opposite effect to miR-152 mimics transfection. The luciferase reporter analysis confirmed that miR-152 straightly targets the 3′-untranslated region (3’-UTR) of uncoupling protein 3 (UCP3) to cause its post-transcriptional inhibition in the protein level. The knockdown of UCP3 by siRNA showed the similar effect of miR-152 on myofiber type transition. Furthermore, the rescue experiment in the porcine myotube transfected with miR-152 mimics or/and UCP3 overexpression plasmid with or without the 3’UTR revealed that UCP3 mediates the action of miR-152 in slow-twitch myofiber formation. Taken together, our findings proposed a novel molecular mechanism through which miR-152 epigenetically regulates meat quality via promoting slow-twitch myofiber formation and skeletal myogenesis. Full article

Akirin2 plays an important role in skeletal myogenesis. In this study, we found that porcine Akirin2 (pAkirin2) mRNA level was significantly higher in fast extensor digitorum longus (EDL) and longissimus lumborum (LL) muscles than in slow soleus (SOL) muscle of pigs. Overexpression of pAkirin2 increased the number of myosin heavy chain (MHC)-positive cells, indicating that pAkirin2 promoted myoblast differentiation. We also found that overexpression of pAkirin2 increased the mRNA expressions of MHCI and MHCIIa and decreased the mRNA expression of MHCIIb. Myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) are the major downstream effectors of calcineurin. Here we also observed that the mRNA expressions of MEF2C and NFATc1 were notably elevated by pAkirin2 overexpression. Together, our data indicate that the role of pAkirin2 in modulating MHCI and MHCIIa expressions may be achieved through calcineurin/NFATc1 signaling pathway. Full article