Induction of Skeletal Muscle Injury by Intramuscular Injection of Cardiotoxin in Mouse.

Skeletal muscle is the most abundant tissue in the human body and has a tremendous capability to regenerate in response to muscle injuries and diseases. Induction of acute muscle injury is a common method to study muscle regeneration in vivo. Cardiotoxin (CTX) belongs to the family of snake venom toxins and is one of the most common reagents to induce muscle injury. Intramuscular injection of CTX causes overwhelming muscle contraction and lysis of myofibers. The induced acute muscle injury triggers muscle regeneration, allowing in-depth studies on muscle regeneration. This protocol describes a detailed procedure of intramuscular injection of CTX to induce acute muscle injury that could be also applied in other mammalian models.

The asymmetrical ESR1 signaling in muscle progenitor cells determines the progression of adolescent idiopathic scoliosis.

Adolescent Idiopathic Scoliosis (AIS) is a common pediatric skeletal disease highly occurred in females. The pathogenesis of AIS has not been fully elucidated. Here, we reveal that ESR1 (Estrogen Receptor 1) expression declines in muscle stem/progenitor cells at the concave side of AIS patients. Furthermore, ESR1 is required for muscle stem/progenitor cell differentiation and disrupted ESR1 signaling leads to differentiation defects. The imbalance of ESR1 signaling in the para-spinal muscles induces scoliosis in mice, while reactivation of ESR1 signaling at the concave side by an FDA approved drug Raloxifene alleviates the curve progression. This work reveals that the asymmetric inactivation of ESR1 signaling is one of the causes of AIS. Reactivation of ESR1 signaling in para-spinal muscle by Raloxifene at the concave side could be a new strategy to treat AIS.

Chemical reprogramming of melanocytes to skeletal muscle cells.

BACKGROUND: Direct cell-fate conversion by chemical reprogramming is promising for regenerative cell therapies. However, this process requires the reactivation of a set of master transcription factors (TFs) of the target cell type, which has proven challenging using only small molecules. METHODS: We developed a novel small-molecule cocktail permitting robust skin cell to muscle cell conversion. By single cell sequencing analysis, we identified a Pax3 (Paired box 3)-expressing melanocyte population holding a superior myogenic potential outperforming other seven types of skin cells. We further validated the single cell sequencing analysis results using immunofluorescence staining, in situ hybridization and FACS sorting and confirmed the myogenic potential of melanocytes during chemical reprogramming. We used single cell RNA-seq that detect the potential converted cell type, uncovering a unique role of Pax3 in facilitating chemical reprogramming from melanocytes to muscle cells. RESULTS: In this study, we demonstrated that the Pax3-expressing melanocytes to be a skin cell type for skeletal muscle cell fate conversion in chemical reprogramming. By developing a small-molecule cocktail, we showed an efficient melanocyte reprogramming to skeletal muscle cells (40%, P < 0.001). The endogenous expression of specific TFs may circumvent the additional requirement for TF reactivation and form a shortcut for cell fate conversion, suggesting a basic principle that could ease cell fate conversion. CONCLUSIONS: Our study demonstrates the first report of melanocyte-to-muscle conversion by small molecules, suggesting a novel strategy for muscle regeneration. Furthermore, skin is one of the tissues closely located to skeletal muscle, and therefore, our results provide a promising foundation for therapeutic chemical reprogramming in vivo treating skeletal muscle degenerative diseases.

Myogenesis controlled by a long non-coding RNA 1700113A16RIK and post-transcriptional regulation.

Long non-coding (lnc) RNA plays important roles in many cellular processes. The function of the vast majority of lncRNAs remains unknown. Here we identified that lncRNA-1700113A16RIK existed in skeletal muscle stem cells (MuSCs) and was significantly elevated during MuSC differentiation. Knockdown of 1700113A16RIK inhibits the differentiation of muscle stem cells. In contrast, overexpression of 1700113A16RIK promotes the differentiation of muscle stem cells. Further study shows the muscle specific transcription factor Myogenin (MyoG) positively regulates the expression of 1700113A16RIK by binding to the promoter region of 1700113A16RIK. Mechanistically, 1700113A16RIK may regulate the expression of myogenic genes by directly binding to 3'UTR of an important myogenic transcription factor MEF2D, which in turn promotes the translation of MEF2D. Taken together, our results defined 1700113A16RIK as a positive regulator of MuSC differentiation and elucidated a mechanism as to how 1700113A16RIK regulated MuSC differentiation.

Testis-enriched circular RNA circ-Bbs9 plays an important role in Leydig cell proliferation by regulating a CyclinD2-dependent pathway.

Circular RNAs belong to a new category of non-coding RNAs, characterised by a circular structure, conservation, stability and high expression in eukaryotes. They often show tissue- or cell-specific expression. Here, we identified a testis-enriched circular RNA (circRNA), circular Bbs9 (circ-Bbs9) that is highly expressed in mouse testis. An RNase R treatment experiment confirmed that circ-Bbs9 is indeed a circRNA. In situ hybridisation experiments showed that circ-Bbs9 is expressed in Leydig cells along seminiferous tubules and in the cytoplasm of the TM3 Leydig cell line. Knocking down the circ-Bbs9 in TM3 cells by lentivirus vectors arrested cell proliferation, whereas overexpression of circ-Bbs9 induced cell proliferation significantly. Knocking down circ-Bbs9 inhibited the protein level of cyclin D2 (Ccnd2) and RNA immunoprecipitation results showed that circ-Bbs9 interacts with Ccnd2. Our results show that use of the Hedgehog pathway Smoothened Agonist (SAG) HCl and antagonists cyclopamine and gant6 affects the expression levels of Glioma-Associated Oncogene Homolog 1 (Gli1), Ccnd2 and other genes in this pathway. Our research reveals that a Leydig cell-specific circRNA, circ-Bbs9, plays a critical role in Leydig cell proliferation through regulating the levels of cell cycle-related Ccnd2. Thus, our results emphasise the important role of circRNA in the male reproductive system.