Mechanism of Stimulation of Myogenesis under the Action of Succinic Acid through the Succinate Receptor SUCNR1.

Effect of succinic acid on the processes of myogenesis was investigated in the study with the cells of C2C12 line. In the concentration range 10-1000 µM, succinic acid stimulated the process of myogenic differentiation, increasing the levels of myogenesis factors MyoD (at all stages of myogenesis) and myogenin (at the stage of terminal differentiation). Presence of the succinate receptors SUCNR1 was revealed in the C2C12 cells using Western blotting, level of which decreased during myogenesis. When succinic acid was added to the cells, the level of intracellular succinate did not change significantly and decreased during myogenic differentiation. Using a specific Gai protein inhibitor, pertussis toxin, it was found that stimulation of myogenesis in the C2C12 cells under the action of succinic acid is realized through SUCNR1-Gai interaction.

Effects of radial extracorporeal shock wave with different frequencies on acute skeletal muscle injury in rabbits.

To study the efficacy and possible mechanisms of radial extracorporeal shock wave (rESW) with different frequencies for the treatment of acute skeletal muscle injury in rabbits, 48 rabbits of acute injured biceps femoris were randomly divided into 4 groups. Except for the control group, the other groups were treated by rESW with 5 Hz, 10 Hz and 15 Hz, respectively. The injury symptom index scores (ISISs) in the rESW group were significantly lower than those in the control group, with the lowest in the 10 Hz rESW group. Histomorphological features demonstrated a decrease in mononuclear cells and an increase in new myocytes across all groups, with the rESW group showing the most significant changes. The concentrations of PGE2 and IL-1ß were significantly lower in all rESW groups by ELISA compared to the control group. Additionally, the 10 Hz group had lower concentrations than the 5 Hz and 15 Hz group. Compared with the control group, MyoD of the rESW groups was significantly increased, and the expression level of the 10 Hz group was higher than that of the other groups. In conclusion, rESW with 5 Hz, 10 Hz and 15 Hz take certain curative effects on acute biceps femoris injury in rabbits, and the 10 Hz rESW takes advantage over 5 Hz and 15 Hz rESW.

SRSF2 is a key player in orchestrating the directional migration and differentiation of MyoD progenitors during skeletal muscle development.

SRSF2 plays a dual role, functioning both as a transcriptional regulator and a key player in alternative splicing. The absence of Srsf2 in MyoD + progenitors resulted in perinatal mortality in mice, accompanied by severe skeletal muscle defects. SRSF2 deficiency disrupts the directional migration of MyoD progenitors, causing them to disperse into both muscle and non-muscle regions. Single-cell RNA-sequencing analysis revealed significant alterations in Srsf2-deficient myoblasts, including a reduction in extracellular matrix components, diminished expression of genes involved in ameboid-type cell migration and cytoskeleton organization, mitosis irregularities, and premature differentiation. Notably, one of the targets regulated by Srsf2 is the serine/threonine kinase Aurka. Knockdown of Aurka led to reduced cell proliferation, disrupted cytoskeleton, and impaired differentiation, reflecting the effects seen with Srsf2 knockdown. Crucially, the introduction of exogenous Aurka in Srsf2-knockdown cells markedly alleviated the differentiation defects caused by Srsf2 knockdown. Furthermore, our research unveiled the role of Srsf2 in controlling alternative splicing within genes associated with human skeletal muscle diseases, such as BIN1, DMPK, FHL1, and LDB3. Specifically, the precise knockdown of the Bin1 exon17-containing variant, which is excluded following Srsf2 depletion, profoundly disrupted C2C12 cell differentiation. In summary, our study offers valuable insights into the role of SRSF2 in governing MyoD progenitors to specific muscle regions, thereby controlling their differentiation through the regulation of targeted genes and alternative splicing during skeletal muscle development.

A change in cis-regulatory logic underlying obligate versus facultative muscle multinucleation in chordates.

Vertebrates and tunicates are sister groups that share a common fusogenic factor, Myomaker (Mymk), that drives myoblast fusion and muscle multinucleation. Yet they are divergent in when and where they express Mymk. In vertebrates, all developing skeletal muscles express Mymk and are obligately multinucleated. In tunicates, Mymk is expressed only in post-metamorphic multinucleated muscles, but is absent from mononucleated larval muscles. In this study, we demonstrate that cis-regulatory sequence differences in the promoter region of Mymk underlie the different spatiotemporal patterns of its transcriptional activation in tunicates and vertebrates. Although in vertebrates myogenic regulatory factors (MRFs) such as MyoD1 alone are required and sufficient for Mymk transcription in all skeletal muscles, we show that transcription of Mymk in post-metamorphic muscles of the tunicate Ciona requires the combinatorial activity of MRF, MyoD and Early B-cell Factor (Ebf). This macroevolutionary difference appears to be encoded in cis, likely due to the presence of a putative Ebf-binding site adjacent to predicted MRF binding sites in the Ciona Mymk promoter. We further discuss how Mymk and myoblast fusion might have been regulated in the last common ancestor of tunicates and vertebrates, for which we propose two models.

Muscle regeneration therapy using dedifferentiated fat cell (DFAT) for anal sphincter dysfunction.

PURPOSE: We investigated the effects of mouse-derived DFAT on the myogenic differentiation of a mouse-derived myoblast cell line (C2C12) and examined the therapeutic effects of rat-derived DFAT on anal sphincter injury using a rat model. METHODS: C2C12 cells were cultured using DMEM and DFAT-conditioned medium (DFAT-CM), evaluating MyoD and Myogenin gene expression via RT-PCR. DFAT was locally administered to model rats with anorectal sphincter dysfunction 3 days post-CTX injection. Therapeutic effects were assessed through functional assessment, including anal pressure measurement using solid-state manometry pre/post-CTX, and on days 1, 3, 7, 10, 14, 17, and 21 post-DFAT administration. Histological evaluation involved anal canal excision on days 1, 3, 7, 14, and 21 after CTX administration, followed by hematoxylin-eosin staining. RESULTS: C2C12 cells cultured with DFAT-CM exhibited increased MyoD and Myogenin gene expression compared to control. Anal pressure measurements revealed early recovery of resting pressure in the DFAT-treated group. Histologically, DFAT-treated rats demonstrated an increase in mature muscle cells within newly formed muscle fibers on days 14 and 21 after CTX administration, indicating enhanced muscle tissue repair. CONCLUSION: DFAT demonstrated the potential to enhance histological and functional muscle tissue repair. These findings propose DFAT as a novel therapeutic approach for anorectal sphincter dysfunction treatment.

The role of ZEB1 in mediating the protective effects of metformin on skeletal muscle atrophy.

Metformin is an important antidiabetic drug that has the potential to reduce skeletal muscle atrophy and promote the differentiation of muscle cells. However, the exact molecular mechanism underlying these functions remains unclear. Previous studies revealed that the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), which participates in tumor progression, inhibits muscle atrophy. Therefore, we hypothesized that the protective effect of metformin might be related to ZEB1. We investigated the positive effect of metformin on IL-1ß-induced skeletal muscle atrophy by regulating ZEB1 in vitro and in vivo. Compared with the normal cell differentiation group, the metformin-treated group presented increased myotube diameters and reduced expression levels of atrophy-marker proteins. Moreover, muscle cell differentiation was hindered, when we artificially interfered with ZEB1 expression in mouse skeletal myoblast (C2C12) cells via ZEB1-specific small interfering RNA (si-ZEB1). In response to inflammatory stimulation, metformin treatment increased the expression levels of ZEB1 and three differentiation proteins, MHC, MyoD, and myogenin, whereas si-ZEB1 partially counteracted these effects. Moreover, marked atrophy was induced in a mouse model via the administration of lipopolysaccharide (LPS) to the skeletal muscles of the lower limbs. Over a 4-week period of intragastric administration, metformin treatment ameliorated muscle atrophy and increased the expression levels of ZEB1. Metformin treatment partially alleviated muscle atrophy and stimulated differentiation. Overall, our findings may provide a better understanding of the mechanism underlying the effects of metformin treatment on skeletal muscle atrophy and suggest the potential of metformin as a therapeutic drug.

Diarylpropionitrile-stimulated ERß nuclear accumulation promotes MyoD-induced muscle regeneration in mdx mice by interacting with FOXO3A.

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive degenerative disease of skeletal muscle, characterized by intramuscular inflammation, muscle regeneration disorder and replacement of muscle with fibroadipose tissue. DMD is caused by the absence of normal dystrophy. Impaired self-renew ability and limited differentiation capacity of satellite cells are proved as main reasons for muscle regeneration failure. The deficiency of estrogen impedes the process of muscle regeneration. However, the role of estrogen receptor ß (ERß) in muscle regeneration is still unclear. This study aims to investigate the role and the pharmacological effect of ERß activation on muscle regeneration in mdx mice. This study showed that mRNA levels of ERß and myogenic-related genes both witnessed increasing trends in dystrophic context. Our results revealed that treatment with selective ERß agonist (DPN, diarylpropionitrile) significantly increased myogenic differentiation 1 (MyoD-1) level and promoted muscle regeneration in mdx mice. Similarly, in mdx mice with muscle-specific estrogen receptor α (ERα) ablation, DPN treatment still promoted muscle regeneration. Moreover, we demonstrated that myoblasts differentiation was accompanied by raised nuclear accumulation of ERß. DPN treatment augmented the nuclear accumulation of ERß and, thus, contributed to myotubes formation. One important finding was that forkhead box O3A (FOXO3A), as a pivotal transcription factor in Myod-1 transcription, participated in the ERß-promoted muscle regeneration. Overall, we offered an interesting explanation about the crucial role of ERß during myogenesis.

Expression mechanisms of mir-486-5p and its host gene sANK1 in porcine muscle.

BACKGROUND: MiR-486-5p has been identified as a crucial regulator of the PI3K/AKT signalling pathway, which plays a significant role in skeletal muscle development. Its host gene, sANK1, is also essential for skeletal muscle development. However, the understanding of porcine miR-486-5p and sANK1 has been limited. METHODS AND RESULTS: In this study, PCR analyses revealed a positive correlation between the expression of miR-486-5p and sANK1 in the longissimus dorsi muscle of the Bama mini-pig and Landrace-pig, as well as during myoblast differentiation. Furthermore, the expression of miR-486-5p/sANK1 was higher in the Bama mini-pig compared to the Landrace-pig. There was a total of 18 single nucleotide polymorphisms (SNP) present in the sANK1 promoter region. Among these SNPs, 14 of them resulted in alterations in transcription factor binding sites (TFBs). Additionally, the promoter fluorescence assay demonstrated that the activity of the sANK1 promoter derived from the Bama mini-pig was significantly higher compared to Landrace-pig. It is worth noting that ten regulatory SNPs have the potential to influence the activity of the sANK1 promoter. A nuclear mutation A-G located at position - 401 (relative to the transcription start site) in the Bama mini-pig was identified, which creates a putative TFB motif for MyoD. CONCLUSIONS: The findings presented in this study offer fundamental molecular knowledge and expression patterns of miR-486-5p/sANK1, which can be valuable for gaining a deeper understanding of the gene's involvement in porcine skeletal muscle development, and meat quality.

CAR T-cells targeting FGFR4 and CD276 simultaneously show potent antitumor effect against childhood rhabdomyosarcoma.

Chimeric antigen receptor (CAR) T-cells targeting Fibroblast Growth Factor Receptor 4 (FGFR4), a highly expressed surface tyrosine receptor in rhabdomyosarcoma (RMS), are already in the clinical phase of development, but tumour heterogeneity and suboptimal activation might hamper their potency. Here we report an optimization strategy of the co-stimulatory and targeting properties of a FGFR4 CAR. We replace the CD8 hinge and transmembrane domain and the 4-1BB co-stimulatory domain with those of CD28. The resulting CARs display enhanced anti-tumor activity in several RMS xenograft models except for an aggressive tumour cell line, RMS559. By searching for a direct target of the RMS core-regulatory transcription factor MYOD1, we identify another surface protein, CD276, as a potential target. Bicistronic CARs (BiCisCAR) targeting both FGFR4 and CD276, containing two distinct co-stimulatory domains, have superior prolonged persistent and invigorated anti-tumor activities compared to the optimized FGFR4-specific CAR and the other BiCisCAR with the same 4-1BB co-stimulatory domain. Our study thus lays down the proof-of-principle for a CAR T-cell therapy targeting both FGFR4 and CD276 in RMS.

Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

Muscle regeneration depends on muscle stem cell (MuSC) activity. Myogenic regulatory factors, including myoblast determination protein 1 (MyoD), regulate the fate transition of MuSCs. However, the direct target of MYOD in the process is not completely clear. Using previously established MyoD knock-in (MyoD-KI) mice, we revealed that MyoD targets dual-specificity phosphatase (Dusp) 13 and Dusp27. In Dusp13:Dusp27 double knock-out mice, the ability for muscle regeneration after injury was reduced. Moreover, single-cell RNA sequencing of MyoD-high expressing MuSCs from MyoD-KI mice revealed that Dusp13 and Dusp27 are expressed only in specific populations within MyoD-high MuSCs, which also express Myogenin. Overexpressing Dusp13 in MuSCs causes premature muscle differentiation. Thus, we propose a model where DUSP13 and DUSP27 contribute to the fate transition of MuSCs from proliferation to differentiation during myogenesis.