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The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family. [ABSTRACT FROM AUTHOR]

Senescent cells are characterized by multiple features such as increased expression of senescence-associated β-galactosidase activity (SA β-gal) and cell cycle inhibitors such as p21 or p16. They accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin (Doxo) cause buildup of senescent cells, leading to the inhibition of tissue regeneration. Senescent cells influence the neighboring cells via numerous secreted factors which form the senescence-associated secreted phenotype (SASP). Lipids are emerging as a key component of SASP that can control tissue homeostasis. Arachidonic acid-derived lipids have been shown to accumulate within senescent cells, specifically 15d-PGJ2, which is an electrophilic lipid produced by the non-enzymatic dehydration of the prostaglandin PGD2. This study shows that 15d-PGJ2 is also released by Doxo-induced senescent cells as an SASP factor. Treatment of skeletal muscle myoblasts with the conditioned medium from these senescent cells inhibits myoblast fusion during differentiation. Inhibition of L-PTGDS, the enzyme that synthesizes PGD2, diminishes the release of 15d-PGJ2 by senescent cells and restores muscle differentiation. We further show that this lipid post-translationally modifies Cys184 of HRas in C2C12 mouse skeletal myoblasts, causing a reduction in the localization of HRas to the Golgi, increased HRas binding to Ras Binding Domain (RBD) of RAF Kinase (RAF-RBD), and activation of cellular Mitogen Activated Protein (MAP) kinase–Extracellular Signal Regulated Kinase (Erk) signaling (but not the Akt signaling). Mutating C184 of HRas prevents the ability of 15d-PGJ2 to inhibit the differentiation of muscle cells and control the activity of HRas. This work shows that 15d-PGJ2 released from senescent cells could be targeted to restore muscle homeostasis after chemotherapy.

Scope: Ribes fasciculatum Siebold & Zucc. var. chinense Maxim. Leave extract (RFE) has been reported to be non-toxic and suitable for consumption as a food, with documented anti-allergic and anti-obesity effects. Skeletal muscle atrophy in various chronic inflammatory conditions has been a significant concern, yet the role of RFE in ameliorating muscle atrophy remains to be demonstrated. This study aims to investigate the regulation of muscle atrophy by RFE in chronic inflammatory conditions. Methods and results: C2C12 myoblasts were treated with conditioned medium from Raw264.7 macrophages induced with LPS to simulate an inflammatory condition, or with conditioned medium containing RFE. This study found that muscle atrophy is significantly exacerbated in the inflammatory conditions, while RFE markedly improves this condition. RFE was found to recover the diminished differentiation capacity of myoblasts that occurs in chronic inflammatory conditions. Importantly, RFE mediates inhibition of polarization into pro-inflammatory M1 macrophages in Raw264.7 cells. This indicates that afzelin, active compound of RFE, interferes with the binding of LPS to the TLR4/MD2 complex. Conclusion: These results suggest that RFE can improve muscle atrophy in chronic inflammatory states by disrupting the binding of LPS to the TLR4/MD2 complex and inhibiting the polarization of macrophages into M1 phenotype.

Abstract Background Satellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin–proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined. Results Using conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter. Conclusions These results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels.

The skeletal muscle is a highly plastic tissue that shows a remarkable adaptive capacity in response to acute and resistance exercise, and modifies its composition to adapt to use and disuse, a process referred to as muscle plasticity. Heat shock proteins (HSPs), a class of evolutionarily conserved molecular chaperones, have been implicated in the regulation of skeletal muscle plasticity. Here, we summarize key findings supporting the notion that HSPs are important components required to maintain skeletal muscle integrity and functionality. HSPs participate in the transcriptional program required for myogenesis and are activated following muscle exercise and injury. Their dysfunction, either as a consequence of improper expression or genetic mutations, contributes to muscle atrophy and leads to the development of myopathies and peripheral motor neuropathies. Denervation/reinnervation and repeated rounds of nerve degeneration/regeneration have been observed in motor neuropathies, suggesting that an imbalance in HSP expression and function may impair the repair of the neuromuscular junctions. Boosting HSP activity may help preventing muscle atrophy by promoting muscle differentiation and helping the repair of NMJs. Boosting HSP function may also help to combat the development of rhabdomyosarcoma (RMS), a highly aggressive type of pediatric soft tissue sarcoma whose cells have skeletal muscle features but are unable to fully differentiate into skeletal muscle cells.

Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish neu3.2, mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1–4-cell-stage embryos injected with neu3.2 splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior–posterior axis formation. Myog and myod1 expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of Neu2 mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases. [ABSTRACT FROM AUTHOR]

This study investigated the effects and active compounds of silkworm pupae, an edible insect, on C2C12 muscle differentiation. The protein of silkworm pupae was extracted using sonication after defatting with hexane. Subsequently, the extract was rehydrated using Alcalase to obtain a protein hydrolysate. The silkworm pupae protein hydrolysate effectively promoted C2C12 myogenic differentiation without cytotoxicity. Subsequently, the hydrolysate was fractionated into four subfractions using preparative high-performance liquid chromatography (Prep-HPLC). Subfraction 1 was the most effective in promoting C2C12 myogenic differentiation and significantly upregulated the expression of myoblast transcription factors, 1.5-fold of myoblast determination protein 1 (MyoD), 2-fold of myogenin, and 3-fold of myosin heavy chain (MyHC). Liquid chromatography–tandem mass spectrometry (LC-MS/MS) and multivariate statistical analysis were used to identify the active peptides in silkworm pupae responsible for the observed effects; then, dipeptides and essential amino acids, such as isoleucine (Ile), valine (Val), and methionine (Met), were identified. In addition, Val, Ile, and two dipeptides underwent quantification to determine the potential bioactive peptides that enhanced C2C12 myogenic differentiation. This study suggests that the peptides from silkworm pupae could be used as a nutraceutical to enhance muscle growth. [ABSTRACT FROM AUTHOR]

Natural antisense transcripts (NATs) are endogenous RNAs opposite to sense transcripts, and they can significantly contribute to regulating various biological processes through multiple epigenetic mechanisms. NATs can affect their sense transcripts to regulate the growth and development of skeletal muscle. Our analysis of third-generation full-length transcriptome sequencing data revealed that NATs represented a significant portion of the lncRNA, accounting for up to 30.19%–33.35%. The expression of NATs correlated with myoblast differentiation, and genes expressing NATs were mainly involved in RNA synthesis, protein transport, and cell cycle. We found a NAT of MYOG (MYOG-NAT) in the data. We found that the MYOG-NAT could promote the differentiation of myoblasts in vitro. Additionally, knockdown of MYOG-NAT in vivo led to muscle fiber atrophy and muscle regeneration retardation. Molecular biology experiments demonstrated that MYOG-NAT enhances the stability of MYOG mRNA by competing with miR-128-2-5p, miR-19a-5p, and miR-19b-5p for binding to MYOG mRNA 3′UTR. These findings suggest that MYOG-NAT plays a critical role in skeletal muscle development and provides insights into the post-transcriptional regulation of NATs. • NATs are widespread in muscle development and are mainly involved in RNA synthesis and protein processing. • MYOG-NAT is specifically expressed in differentiated myotubes and skeletal muscle tissues. • MYOG-NAT promotes muscle differentiation and muscle regeneration. • MyoD1 promotes transcription of MYOG-NAT. • MYOG-NAT binds to MYOG mRNA and promotes its expression. [ABSTRACT FROM AUTHOR]

The skeletal muscle is a highly plastic tissue that shows a remarkable adaptive capacity in response to acute and resistance exercise, and modifies its composition to adapt to use and disuse, a process referred to as muscle plasticity. Heat shock proteins (HSPs), a class of evolutionarily conserved molecular chaperones, have been implicated in the regulation of skeletal muscle plasticity. Here, we summarize key findings supporting the notion that HSPs are important components required to maintain skeletal muscle integrity and functionality. HSPs participate in the transcriptional program required for myogenesis and are activated following muscle exercise and injury. Their dysfunction, either as a consequence of improper expression or genetic mutations, contributes to muscle atrophy and leads to the development of myopathies and peripheral motor neuropathies. Denervation/reinnervation and repeated rounds of nerve degeneration/regeneration have been observed in motor neuropathies, suggesting that an imbalance in HSP expression and function may impair the repair of the neuromuscular junctions. Boosting HSP activity may help preventing muscle atrophy by promoting muscle differentiation and helping the repair of NMJs. Boosting HSP function may also help to combat the development of rhabdomyosarcoma (RMS), a highly aggressive type of pediatric soft tissue sarcoma whose cells have skeletal muscle features but are unable to fully differentiate into skeletal muscle cells. [ABSTRACT FROM AUTHOR]

Facioscapulohumeral muscular dystrophy (FSHD) represents the third most common form of muscular dystrophy and is characterized by muscle weakness and atrophy. FSHD is caused by the altered expression of the transcription factor double homeobox 4 (DUX4), which is involved in several significantly altered pathways required for myogenesis and muscle regeneration. While DUX4 is normally silenced in the majority of somatic tissues in healthy individuals, its epigenetic de-repression has been linked to FSHD, resulting in DUX4 aberrant expression and cytotoxicity in skeletal muscle cells. Understanding how DUX4 is regulated and functions could provide useful information not only to further understand FSHD pathogenesis, but also to develop therapeutic approaches for this disorder. Therefore, this review discusses the role of DUX4 in FSHD by examining the possible molecular mechanisms underlying the disease as well as novel pharmacological strategies targeting DUX4 aberrant expression. [ABSTRACT FROM AUTHOR]

Background: Satellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin–proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined. Results: Using conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter. Conclusions: These results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels. [ABSTRACT FROM AUTHOR]

Myoblast fusion is essential for skeletal muscle development, growth, and regeneration. However, the molecular mechanisms underlying myoblast fusion and differentiation are not fully understood. Previously, we reported that interleukin-4 (IL-4) promotes myoblast fusion; therefore, we hypothesized that IL-4 signaling might regulate the expression of the molecules involved in myoblast fusion. In this study, we showed that in addition to fusion, IL-4 promoted the differentiation of C2C12 myoblast cells by inducing myoblast determination protein 1 (MyoD) and myogenin, both of which regulate the expression of myomerger and myomaker, the membrane proteins essential for myoblast fusion. Unexpectedly, IL-4 treatment increased the expression of myomerger, but not myomaker, in C2C12 cells. Knockdown of IL-4 receptor alpha (IL-4Rα) in C2C12 cells by small interfering RNA impaired myoblast fusion and differentiation. We also demonstrated a reduction in the expression of MyoD, myogenin, and myomerger by knockdown of IL-4Rα in C2C12 cells, while the expression level of myomaker remained unchanged. Finally, cell mixing assays and the restoration of myomerger expression partially rescued the impaired fusion in the IL-4Rα-knockdown C2C12 cells. Collectively, these results suggest that the IL-4/IL-4Rα axis promotes myoblast fusion and differentiation via the induction of myogenic regulatory factors, MyoD and myogenin, and myomerger. [ABSTRACT FROM AUTHOR]

Musculoskeletal disorders represent one of the main causes of disability worldwide, and their prevalence is predicted to increase in the coming decades. Stem cell therapy may be a promising option for the treatment of some of the musculoskeletal diseases. Although significant progress has been made in musculoskeletal stem cell research, osteoarthritis, the most-common musculoskeletal disorder, still lacks curative treatment. To fine-tune stem-cell-based therapy, it is necessary to focus on the underlying biological mechanisms. Ion channels and the bioelectric signals they generate control the proliferation, differentiation, and migration of musculoskeletal progenitor cells. Calcium- and voltage-activated potassium (KCa) channels are key players in cell physiology in cells of the musculoskeletal system. This review article focused on the big conductance (BK) KCa channels. The regulatory function of BK channels requires interactions with diverse sets of proteins that have different functions in tissue-resident stem cells. In this narrative review article, we discuss the main ion channels of musculoskeletal stem cells, with a focus on calcium-dependent potassium channels, especially on the large conductance BK channel. We review their expression and function in progenitor cell proliferation, differentiation, and migration and highlight gaps in current knowledge on their involvement in musculoskeletal diseases. [ABSTRACT FROM AUTHOR]

Background: Sarcopenia and muscular dystrophy are two muscle diseases. In cancer patients, cancer cachexia induces continuous weight loss and muscle loss due to the disease itself or the use of anticancer drugs. Cachexia occurs in up to 80% of cancer patients. It is recognized as a direct cause of reduced quality of life, contributing to at least 20% of cancer-associated deaths and limiting therapeutic options for cancer patients. Cancer cachexia is associated with multiple chronic or end-stage conditions and develops similarly. There are various options for the treatment of cancer cachexia, but there are still many issues to be solved. Hence, to determine its potential to overcome the muscle wasting during cancer cachexia, we studied the effect of BST204, a refined dry ginseng extract, on muscle fiber regeneration. Experimental procedure: We checked the muscle regeneration efficacy of BST204. First, BaCl2 and freeze injury models were selected to investigate muscle regeneration after BST204 administration. In addition, after inducing muscle differentiation of C2C12 cells, the efficacy of BST204 was analyzed. In this model, we analyzed the expression of the signal pathway (PI3K-AKT signal) by Western blot and imaging methods. Results and conclusion: These results showed that BST204 induced muscle fiber regeneration in BaCl2 and freeze injury models. Also, we confirmed that BST204 could regulate the PI3K/AKT signaling pathway and regulate the differentiation of C2C12 cells. These results indicate that BST204 has the potential to facilitate the skeletal muscle regeneration during muscle wasting induced by various factors including cancer cachexia.

Abstract Background Muscle regeneration includes proliferation and differentiation of muscle satellite cells, which involves the mammalian target of rapamycin (mTOR). We identified the C‐terminal unique attached sequence motif (UNE) domain of leucyl‐tRNA synthetase (LRS‐UNE‐L) as an mTORC1 (mTOR complex1)‐activating domain that acts through Vps34 and phospholipase D1 (PLD1) when introduced in the form of a muscle‐enhancing peptide. Methods In vitro Vps34 lipid kinase assay, phosphatidylinositol 3‐phosphate (PI(3)P) measurement, in vivo PLD1 assay, and western blot assay were performed in HEK293 cells to test the effect of the LRS‐UNE‐L on the Vps34‐PLD1‐mTOR pathway. Adeno‐associated virus (AAV)‐LRS‐UNE‐L was transduced in C2C12 cells in vitro, in BaCl2‐injured tibialis anterior (TA) muscles, and in 18‐month‐old TA muscles to analyse its effect on myogenesis, muscle regeneration, and aged muscle, respectively. The muscle‐specific cell‐permeable peptide M12 was fused with LRS‐UNE‐L and tested for cell integration in C2C12 and HEK293 cells using FACS analysis and immunocytochemistry. Finally, M12‐LRS‐UNE‐L was introduced into BaCl2‐injured TA muscles of 15‐week‐old Pld1+/+ or Pld1−/− mice, and its effect was analysed by measurement of cross‐sectional area of regenerating muscle fibres. Results The LRS‐UNE‐L expression restored amino acid‐induced S6K1 phosphorylation in LRS knockdown cells in a RagD GTPases‐independent manner (421%, P = 0.007 vs. LRS knockdown control cells). The LRS‐UNE‐L domain was directly bound to Vps34; this interaction was accompanied by increases in Vps34 activity (166%, P = 0.0352), PI(3)P levels (146%, P = 0.0039), and PLD1 activity (228%, P = 0.0294) compared with amino acid‐treated control cells, but it did not affect autophagic flux. AAV‐delivered LRS‐UNE‐L domain augmented S6K1 phosphorylation (174%, P = 0.0013), mRNA levels of myosin heavy chain (MHC) (122%, P = 0.0282) and insulin‐like growth factor 2 (IGF2) (146%, P = 0.008), and myogenic fusion (133%, P = 0.0479) in C2C12 myotubes. AAV‐LRS‐UNE‐L increased the size of regenerating muscle fibres in BaCl2‐injured TA muscles (124%, P = 0.0279) (n = 9–10), but it did not change the muscle fibre size of TA muscles in old mice. M12‐LRS‐UNE‐L was preferentially delivered into C2C12 cells compared with HEK293 cells and augmented regeneration of BaCl2‐injured TA muscles in a PLD1‐dependent manner (116%, P = 0.0022) (n = 6). Conclusions Our results provide compelling evidence that M12‐LRS‐UNE‐L could be a muscle‐enhancing protein targeting mTOR.

The coordinate regulation of metabolism and epigenetics to establish cell state-specific gene expression patterns during lineage progression is a central aspect of cell differentiation, but the factors that regulate this elaborate interplay are not well-defined. The imprinted Dlk1-Dio3 noncoding RNA (ncRNA) cluster has been associated with metabolism in various progenitor cells, suggesting it functions as a regulator of metabolism and cell state. Here, we directly demonstrate that the Dlk1-Dio3 ncRNA cluster coordinates mitochondrial respiration and chromatin structure to maintain proper cell state. Stable muscle cell lines were generated harboring two distinct deletions in the proximal promoter region resulting in either greatly upregulated or downregulated expression of the entire Dlk1-Dio3 ncRNA cluster. Both mutant lines displayed impaired muscle differentiation along with dysregulated structural gene expression and abnormalities in mitochondrial respiration. Genome-wide chromatin accessibility and histone methylation patterns were also severely affected in these mutants. Our results strongly suggest that muscle cells are sensitive to Dlk1-Dio3 ncRNA dosage and that the cluster coordinately regulates metabolic activity and the epigenome to maintain proper cell state in the myogenic lineage., (© 2024. Published by The Company of Biologists Ltd.)

Abstract Background Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions. Results In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation. Conclusions Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.

This study investigated the effects and active compounds of silkworm pupae, an edible insect, on C2C12 muscle differentiation. The protein of silkworm pupae was extracted using sonication after defatting with hexane. Subsequently, the extract was rehydrated using Alcalase to obtain a protein hydrolysate. The silkworm pupae protein hydrolysate effectively promoted C2C12 myogenic differentiation without cytotoxicity. Subsequently, the hydrolysate was fractionated into four subfractions using preparative high-performance liquid chromatography (Prep-HPLC). Subfraction 1 was the most effective in promoting C2C12 myogenic differentiation and significantly upregulated the expression of myoblast transcription factors, 1.5-fold of myoblast determination protein 1 (MyoD), 2-fold of myogenin, and 3-fold of myosin heavy chain (MyHC). Liquid chromatography–tandem mass spectrometry (LC-MS/MS) and multivariate statistical analysis were used to identify the active peptides in silkworm pupae responsible for the observed effects; then, dipeptides and essential amino acids, such as isoleucine (Ile), valine (Val), and methionine (Met), were identified. In addition, Val, Ile, and two dipeptides underwent quantification to determine the potential bioactive peptides that enhanced C2C12 myogenic differentiation. This study suggests that the peptides from silkworm pupae could be used as a nutraceutical to enhance muscle growth.

The development of skeletal muscle (myogenesis) is a well-orchestrated process where myoblasts withdraw from the cell cycle and differentiate into myotubes. Signaling by fluxes in intracellular calcium (Ca2+) is known to contribute to myogenesis, and increased mitochondrial biogenesis is required to meet the metabolic demand of mature myotubes. However, gaps remain in the understanding of how intracellular Ca2+ signals can govern myogenesis. Polycystin-2 (PC2 or TRPP1) is a nonselective cation channel permeable to Ca2+. It can interact with intracellular calcium channels to control Ca2+ release and concurrently modulates mitochondrial function and remodeling. Due to these features, we hypothesized that PC2 is a central protein in mediating both the intracellular Ca2+ responses and mitochondrial changes seen in myogenesis. To test this hypothesis, we created CRISPR/Cas9 knockout (KO) C2C12 murine myoblast cell lines. PC2 KO cells were unable to differentiate into myotubes, had impaired spontaneous Ca2+ oscillations, and did not develop depolarization-evoked Ca2+ transients. The autophagic-associated pathway beclin-1 was downregulated in PC2 KO cells, and direct activation of the autophagic pathway resulted in decreased mitochondrial remodeling. Re-expression of full-length PC2, but not a calcium channel dead pathologic mutant, restored the differentiation phenotype and increased the expression of mitochondrial proteins. Our results establish that PC2 is a novel regulator of in vitro myogenesis by integrating PC2-dependent Ca2+ signals and metabolic pathways. [ABSTRACT FROM AUTHOR]

Myoblast fusion is essential for the formation, growth, and regeneration of skeletal muscle, but the molecular mechanisms that govern fusion and myofiber formation remain poorly understood. Past studies have shown an important role of the actin cytoskeleton and actin regulators in myoblast fusion. The Cyclase-Associated Proteins (CAP) 1 and 2 recently emerged as critical regulators of actin treadmilling in higher eukaryotes including mammals. Whilst the role of CAP2 in skeletal muscle development and function is well characterized, involvement of CAP1 in this process remains elusive. Here we report that CAP1, plays a critical role in cytoskeletal remodeling during myoblast fusion and formation of myotubes. Cap1 mRNA and protein are expressed in both murine C2C12 and human LHCN-M2 myoblasts, but their abundance decreases during myogenic differentiation. Perturbing the temporally controlled expression of CAP1 by overexpression or CRISPR-Cas9 mediated knockout impaired actin rearrangement, myoblast alignment, expression of profusion molecules, differentiation into multinucleated myotubes, and myosin heavy chain expression. Endogenous Cap1 expression is post-transcriptionally downregulated during differentiation by canonical myomiRs miR-1, miR-133, and miR-206, which have conserved binding sites at the 3′ UTR of the Cap1 mRNA. Deletion of the endogenous 3′ UTR by CRISPR-Cas9 in C2C12 cells phenocopies overexpression of CAP1 by inhibiting myotube formation. Our findings implicates Cap1 and its myomiR-mediated downregulation in the myoblast fusion process and the generation of skeletal muscle.

Skeletal muscle development is a complex and highly orchestrated biological process mediated by a series of myogenesis regulatory factors. Numerous studies have demonstrated that circular RNAs (circRNAs) are involved in muscle differentiation, but the exact molecular mechanisms involved remain unclear. Here, we analyzed the expression of circRNAs at the adult and embryo development stages of cattle musculus longissimus. A stringent set of 1,318 circRNAs candidates were identified, and we found that 495 circRNAs were differentially expressed between embryonic and adult tissue libraries. We subsequently focused on one of the most downregulated circRNAs (using the adult stage expression as control), and this was named muscle differentiation-associated circular RNA (circMYBPC1). With RNA binding protein immunoprecipitation (RIP) and RNA pull-down assays, circMYBPC1 was identified to promote myoblast differentiation by directly binding miR-23a to relieve its inhibition on myosin heavy chain (MyHC). In addition, RIP assays demonstrated that circMYBPC1 could directly bind MyHC protein. In vivo observations also suggested that circMYBPC1 may stimulate skeletal muscle regeneration after muscle damage. These results revealed that the novel non-coding circRNA circMYBPC1 promotes differentiation of myoblasts and may promote skeletal muscle regeneration. Our results provided a basis for in-depth analysis of the role of circRNA in myogenesis and muscle diseases.

Abstract Purpose and background Recently, we found that maximal medial rectus recession and lateral rectus resection in patients with complete lateral rectus paralysis resulted in a partial restoration of abduction. In an attempt to understand some of the mechanisms involved with this effect we examined gene expression profiles of lateral recti from these patients, with our focus being directed to genes related to myogenesis. Materials and methods Lateral recti resected from patients with complete lateral rectus paralysis and those from concomitant esotropia (controls) were collected. Differences in gene expression profiles between these two groups were examined using microarray analysis and quantitative Reverse-transcription PCR (qRT-PCR). Results A total of 3056 differentially expressed genes (DEGs) were identified between these two groups. Within the paralytic esotropia group, 2081 genes were up-regulated and 975 down-regulated. The results of RT-PCR revealed that PAX7, MYOG, PITX1, SIX1 and SIX4 showed higher levels of expression, while that of MYOD a lower level of expression within the paralytic esotropia group as compared with that in the control group (p

Translation acts as an additional layer of regulation that has an important role in gene expression and function. Due to a phenomenon called codon usage bias, highly expressed genes are thought to be codon-biased to support efficient translation. As more than one codon can code for the same amino acid (synonymous codons), organisms may exhibit preferences for specific codons that facilitate increased expression of important genes due to variations in the availability of corresponding tRNAs. Advances in sequencing technologies have recently permitted the study of the translatome, which refers to the entire population of mRNA associated with ribosomes for protein synthesis and can be investigated through ribosome profiling. This cutting-edge technique allows the identification of actively translated regions, but can also reveal translational pausing events that stem from the presence of SNPs. Our previous research has demonstrated variation in tRNA expression across tissues and different states of health, leading us to consider the connection between tRNA abundance and translational stalling due to SNPs. By coupling ribosome profiling with tRNA sequencing and RNA sequencing, we have investigated all elements of translational machinery to predict translational efficiency, estimate proteome composition, and evaluate tRNA abundance as a source of genetic variation. In this work, we utilized ribosome profiling, tRNAseq, and RNAseq and performed an integrative analysis in bovine tissues (kidney, liver and muscle) as well as murine myoblast cell lines (C2C12). By applying these methods to different bovine tissues, we were able to explore the interplay between tRNA availability and translational stalling events. Moreover, we have identified translationally regulated genes underlying tissue-specific biological processes and found that many upregulated and downregulated genes coincided with high and low translational efficiency respectively. We have also successfully defined stalling sites that depict the regulatory information encoded within the coding sequence of transcripts, which could control translation rate and facilitate proper protein folding. Through the implementation of these next generation sequencing (NGS) methods to cell culture, we were able to evaluate the translatome at various time points (0-min, 30-min, 60-min, and 4-h) post-induction of differentiation. Where most studies have focused on molecular changes between differentiating myoblasts and multinucleated myotubes that are present 7 d after differentiation, we focus on the earliest stages of muscle differentiation that initiate muscle development and therefore kickstart the process of meat production. This work offers an atlas of distinctive stalling sites across bovine tissues and various stages of muscle differentiation, which provides an opportunity to predict codon optimality and understand tissue-specific or time-specific mechanisms of regulating protein synthesis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Ribes fasciculatum var. chinense extract (RFE) alleviates muscle atrophy under chronic inflammatory conditions. • Muscle regeneration capacity is recovered by RFE under chronic inflammatory conditions. • RFE inhibits the polarization of macrophages from M0 to M1 macrophages. • Afzelin, the active component in RFE, interferes the binding of LPS to the TLR4/MD2 complex. Ribes fasciculatum Siebold & Zucc. var. chinense Maxim. Leave extract (RFE) has been reported to be non-toxic and suitable for consumption as a food, with documented anti-allergic and anti-obesity effects. Skeletal muscle atrophy in various chronic inflammatory conditions has been a significant concern, yet the role of RFE in ameliorating muscle atrophy remains to be demonstrated. This study aims to investigate the regulation of muscle atrophy by RFE in chronic inflammatory conditions. C2C12 myoblasts were treated with conditioned medium from Raw264.7 macrophages induced with LPS to simulate an inflammatory condition, or with conditioned medium containing RFE. This study found that muscle atrophy is significantly exacerbated in the inflammatory conditions, while RFE markedly improves this condition. RFE was found to recover the diminished differentiation capacity of myoblasts that occurs in chronic inflammatory conditions. Importantly, RFE mediates inhibition of polarization into pro-inflammatory M1 macrophages in Raw264.7 cells. This indicates that afzelin, active compound of RFE, interferes with the binding of LPS to the TLR4/MD2 complex. These results suggest that RFE can improve muscle atrophy in chronic inflammatory states by disrupting the binding of LPS to the TLR4/MD2 complex and inhibiting the polarization of macrophages into M1 phenotype. [ABSTRACT FROM AUTHOR]

Myoblast fusion is essential for skeletal muscle development, growth, and regeneration. However, the molecular mechanisms underlying myoblast fusion and differentiation are not fully understood. Previously, we reported that interleukin-4 (IL-4) promotes myoblast fusion; therefore, we hypothesized that IL-4 signaling might regulate the expression of the molecules involved in myoblast fusion. In this study, we showed that in addition to fusion, IL-4 promoted the differentiation of C2C12 myoblast cells by inducing myoblast determination protein 1 (MyoD) and myogenin, both of which regulate the expression of myomerger and myomaker, the membrane proteins essential for myoblast fusion. Unexpectedly, IL-4 treatment increased the expression of myomerger, but not myomaker, in C2C12 cells. Knockdown of IL-4 receptor alpha (IL-4Rα) in C2C12 cells by small interfering RNA impaired myoblast fusion and differentiation. We also demonstrated a reduction in the expression of MyoD, myogenin, and myomerger by knockdown of IL-4Rα in C2C12 cells, while the expression level of myomaker remained unchanged. Finally, cell mixing assays and the restoration of myomerger expression partially rescued the impaired fusion in the IL-4Rα-knockdown C2C12 cells. Collectively, these results suggest that the IL-4/IL-4Rα axis promotes myoblast fusion and differentiation via the induction of myogenic regulatory factors, MyoD and myogenin, and myomerger.

Long noncoding RNAs (lncRNAs) are involved in a variety of biological processes and illnesses. While a considerable number of lncRNAs have been discovered in skeletal muscle to far, their role and underlying processes during myogenesis remain mostly unclear. In this study, we described a new functional lncRNA named lncR-133a. Gene overexpression and interference studies in goat skeletal muscle satellite cells (MuSCs) were used to establish its function. The molecular mechanism by which lncR-133a governs muscle differentiation was elucidated primarily using quantitative real-time PCR (qRT-PCR), Western blotting, dual-luciferase activity assays, RNA immunoprecipitation, biotin-labeled probe, and RNA fluorescence in situ hybridization analyses. LncR-133a was found to be substantially expressed in longissimus thoracis et lumborum muscle, and its expression levels changed during MuSC differentiation in goats. We validated that lncR-133a suppresses MuSC differentiation in vitro. Dual-luciferase reporter screening, Argonaute 2 (AGO2) RNA immunoprecipitation assays, biotin-labeled lncR-133a capture, and fluorescence in situ hybridization showed that lncR-133a interacted with miR-133a-3p. Additionally, miR-133a-3p facilitated MuSC differentiation, but lncR-133a reversed this effect. The luciferase reporter assay and functional analyses established that miR-133a-3p directly targets fibroblast growth factor receptor 1 (FGFR1). Moreover, lncR-133a directly reduced miR-133a-3p's capacity to suppress FGFR1 expression, and positively regulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In summary, our results suggested that lncR-133a suppresses goat muscle differentiation by targeting miR-133a-3p and activating FGFR1/ERK1/2 signaling pathway. [ABSTRACT FROM AUTHOR]

Cell Res. 2002; 12: 9-18. 10.1038/sj.cr.7290105 41 Xie S.J., Li J.H., Chen H.F., Tan Y.Y., Liu S.R., Zhang Y., Xu H., Yang J.H., Liu S., Zheng L.L. Inhibition of the JNK/MAPK signaling pathway by myogenesis-associated miRNAs is required for skeletal muscle development. Many gene expression profiling studies in agriculturally significant animals have been carried out since the introduction of high-throughput sequencing tools, and many genes or factors associated to skeletal muscle development have been found [[8], [10]]. Although progress has been made in understanding how genes work during myogenesis in pigs [[8], [10]], cattle [[27]], goats [[11], [28]], and sheep [[9]], our understanding of the genes involved in goat skeletal muscle development is still limited and incomplete. Among them, we indeed identified many terms related to muscle development, such as muscle structure development, muscle tissue development, muscle contraction, muscle cell development, and muscle cell differentiation. [Extracted from the article]

Muscle atrophy in postmenopausal women is caused by estrogen deficiency and a variety of inflammatory factors, including tumor necrosis factor alpha (TNFα). Paeoniflorin (PNF), a natural compound with anti-inflammatory properties, improves estradiol synthesis. Here, we demonstrate that PNF inhibits the progression of TNFα-induced skeletal muscle atrophy after menopause by restoring mitochondrial biosynthesis. Differentiated myoblasts damaged by TNFα were restored by PNF, as evident by the increase in the expression of myogenin (MyoG) and myosin heavy chain 3 (Myh3)—the markers of muscle differentiation. Moreover, diameter of atrophied myotubes was restored by PNF treatment. TNFα-repressed nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) (a major regulator of mitochondrial biosynthesis) were restored by PNF, via regulation by estrogen receptor alpha (ERα), an upregulator of NRF1. This mechanism was confirmed in ovariectomized (OVX) mice with a ~40% reduction in the cross-sectional area of the anterior tibialis muscle. OVX mice administered PNF (100, 300 mg/kg/day) for 12 weeks recovered more than ~20%. Behavioral, rotarod, and inverted screen tests showed that PNF enhances reduced muscle function in OVX mice. ERα restored expression of mitofusin 1 (MFN1) and mitofusin 2 (MFN2) (mitochondrial fusion markers) and dynamin-related protein (DRP1) and fission 1 (FIS1) (mitochondrial fission markers). Therefore, PNF can prevent muscle atrophy in postmenopausal women by inhibiting dysfunctional mitochondrial biogenesis. [ABSTRACT FROM AUTHOR]

Skeletal muscle demonstrates a high degree of regenerative capacity repeating the embryonic myogenic program under strict control. Rhabdomyosarcoma is the most common sarcoma in childhood and is characterized by impaired muscle differentiation. In this study, we observed that silencing the expression of syndecan-4, the ubiquitously expressed transmembrane heparan sulfate proteoglycan, significantly enhanced myoblast differentiation, and fusion. During muscle differentiation, the gradually decreasing expression of syndecan-4 allows the activation of Rac1, thereby mediating myoblast fusion. Single-molecule localized superresolution direct stochastic optical reconstruction microscopy (dSTORM) imaging revealed nanoscale changes in actin cytoskeletal architecture, and atomic force microscopy showed reduced elasticity of syndecan-4-knockdown cells during fusion. Syndecan-4 copy-number amplification was observed in 28% of human fusion-negative rhabdomyosarcoma tumors and was accompanied by increased syndecan-4 expression based on RNA sequencing data. Our study suggests that syndecan-4 can serve as a tumor driver gene in promoting rabdomyosarcoma tumor development. Our results contribute to the understanding of the role of syndecan-4 in skeletal muscle development, regeneration, and tumorigenesis. [ABSTRACT FROM AUTHOR]

Wings are probably the most advanced evolutionary novelty in insects. In the fruit fly Drosophila melanogaster, proper development of wings requires the activity of so-called wing hearts located in the scutellum of the thorax. Immediately after the imaginai ecdysis, these accessory circulatory organs remove hemolymph and apoptotic epidermal cells from the premature wings through their pumping action. This clearing process is essential for the formation of functional wing blades. Mutant flies that lack intact wing hearts are flightless and display malformed wings. The embryonic wing heart progenitors originate from two adjacent parasegments corresponding to the later second and third thoracic segments. However, adult dipterian flies harbor only one pair of wings and only one pair of associated wing hearts in the second thoracic segment. Here we show that the specification of WHPs depends on the regulatory activity of the Hox gene Ultrabithorax. Furthermore, we analyzed the development of wing hearts in the famous four-winged Ultrabithorax (Ubx) mutant, which was first discovered by Ed Lewis in the 1970s. In these flies, the third thoracic segment is homeotically transformed into a second thoracic segment resulting in a second pair of wings instead of the club-shaped halteres. We show that a second pair of functional wing hearts is formed in the transformed third thoracic segment and that all wing hearts originate from the wild-type population of wing heart progenitor cells. [ABSTRACT FROM AUTHOR]

Myostatin (Mstn) and GDF11 are critical factors that are involved in muscle atrophy in the young and sarcopenia in the elderly, respectively. These TGF-β superfamily proteins activate not only Smad signalling but also non-Smad signalling including the Ras-mediated ERK pathway (Raf–MEK–ERK phosphorylation cascade). Although Mstn and GDF11 have been shown to induce muscle atrophy or sarcopenia by Smad2/3-mediated Akt inhibition, participation of the non-Smad Ras–ERK pathway in atrophy and sarcopenia has not been well determined. We show here that both Mstn and GDF11 prevented skeletal myocyte differentiation but that the MEK inhibitor U0126 or trametinib restored differentiation in Mstn- or GDF11-treated myocytes. These MEK inhibitors induced the expression of DA-Raf1 (DA-Raf), which is a dominant-negative antagonist of the Ras–ERK pathway. Exogenous expression of DA-Raf in Mstn- or GDF11-treated myocytes restored differentiation. Furthermore, administration of trametinib to aged mice resulted in an increase in myofiber size or recovery from muscle atrophy. The trametinib administration downregulated ERK activity in these muscles. These results imply that the Mstn/GDF11-induced Ras–ERK pathway plays critical roles in the inhibition of myocyte differentiation and muscle regeneration, which leads to muscle atrophy. Trametinib and similar approved drugs might be applicable to the treatment of muscle atrophy in sarcopenia or cachexia. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the rate of Myostatin (MSTN) and Follistatin (FLST) gene expression in fast and slow twitch muscles in response to 8 weeks of high intensity interval training (HIIT). To this end, 12 male 8-week old Wistar rats were randomly classified into two groups of exercise (n=6) and control (n=6). Exercise group performed a progressive HIIT protocol, five times weekly for 8 weeks using alternating sprint running for 30 min/session that divided into three 4min bouts (35-50m/min speed, >90% VO2 max) separated by a 2min active recovery period (30-50% VO2 max). Gene expression levels for MSTN and FLST in Soleus (SOL) and Extensor digitorum longus (EDL) muscles were measured by real-time RT-PCR. MSTN mRNA levels has slightly (7%); yet significantly (p

Abstract Background Functional genomics employs several experimental approaches to investigate gene functions. High-throughput techniques, such as loss-of-function screening and transcriptome profiling, allow to identify lists of genes potentially involved in biological processes of interest (so called hit list). Several computational methods exist to analyze and interpret such lists, the most widespread of which aim either at investigating of significantly enriched biological processes, or at extracting significantly represented subnetworks. Results Here we propose a novel network analysis method and corresponding computational software that employs the shortest path approach and centrality measure to discover members of molecular pathways leading to the studied phenotype, based on functional genomics screening data. The method works on integrated interactomes that consist of both directed and undirected networks – HIPPIE, SIGNOR, SignaLink, TFactS, KEGG, TransmiR, miRTarBase. The method finds nodes and short simple paths with significant high centrality in subnetworks induced by the hit genes and by so-called final implementers – the genes that are involved in molecular events responsible for final phenotypic realization of the biological processes of interest. We present the application of the method to the data from miRNA loss-of-function screen and transcriptome profiling of terminal human muscle differentiation process and to the gene loss-of-function screen exploring the genes that regulates human oxidative DNA damage recognition. The analysis highlighted the possible role of several known myogenesis regulatory miRNAs (miR-1, miR-125b, miR-216a) and their targets (AR, NR3C1, ARRB1, ITSN1, VAV3, TDGF1), as well as linked two major regulatory molecules of skeletal myogenesis, MYOD and SMAD3, to their previously known muscle-related targets (TGFB1, CDC42, CTCF) and also to a number of proteins such as C-KIT that have not been previously studied in the context of muscle differentiation. The analysis also showed the role of the interaction between H3 and SETDB1 proteins for oxidative DNA damage recognition. Conclusion The current work provides a systematic methodology to discover members of molecular pathways in integrated networks using functional genomics screening data. It also offers a valuable instrument to explain the appearance of a set of genes, previously not associated with the process of interest, in the hit list of each particular functional genomics screening.

Abstract Background mTOR signaling is an essential nutrient and energetic sensing pathway. Here we describe AIMTOR, a sensitive genetically encoded BRET (Bioluminescent Resonance Energy Transfer) biosensor to study mTOR activity in living cells. Results As a proof of principle, we show in both cell lines and primary cell cultures that AIMTOR BRET intensities are modified by mTOR activity changes induced by specific inhibitors and activators of mTORC1 including amino acids and insulin. We further engineered several versions of AIMTOR enabling subcellular-specific assessment of mTOR activities. We then used AIMTOR to decipher mTOR signaling in physio-pathological conditions. First, we show that mTORC1 activity increases during muscle cell differentiation and in response to leucine stimulation in different subcellular compartments such as the cytosol and at the surface of the lysosome, the nucleus, and near the mitochondria. Second, in hippocampal neurons, we found that the enhancement of neuronal activity increases mTOR signaling. AIMTOR further reveals mTOR-signaling dysfunctions in neurons from mouse models of autism spectrum disorder. Conclusions Altogether, our results demonstrate that AIMTOR is a sensitive and specific tool to investigate mTOR-signaling dynamics in living cells and phenotype mTORopathies.

Defects in achieving a fully differentiated state and aberrant expression of genes and microRNAs (miRs) involved in differentiation are common to virtually all tumor types. Here, we demonstrate that the zinc finger transcription factor ZNF281/Zfp281 is down‐regulated during epithelial, muscle, and granulocytic differentiation in vitro. The expression of this gene is absent in terminally differentiated human tissues, in contrast to the elevated expression in proliferating/differentiating ones. Analysis of the 3’UTR of ZNF281/Zfp281 revealed the presence of numerous previously undescribed miR binding sites that were proved to be functional for miR‐mediated post‐transcriptional regulation. Many of these miRs are involved in differentiation pathways of distinct cell lineages. Of interest, ZNF281/Zfp281 is able to inhibit muscle differentiation promoted by miR‐1, of which ZNF281/Zfp281 is a direct target. These data suggest that down‐regulation of ZNF281/Zfp281 during differentiation in various cell types may occur through specific miRs whose expression is tissue‐restricted. In addition, we found that in rhabdomyosarcoma and leiomyosarcoma tumors, the expression of ZNF281/Zfp281 is significantly higher compared with normal counterparts. We extended our analysis to other human soft tissue sarcomas, in which the expression of ZNF281 is associated with a worse prognosis. In summary, we highlight here a new role of ZNF281/Zfp281 in counteracting muscle differentiation; its down‐regulation is at least in part mediated by miR‐1. The elevated expression of ZNF281/Zfp281 in soft tissue sarcomas warrants further analysis for its possible exploitation as a prognostic marker in this class of tumors.

In this study, we evaluated the effects of yeast (Saccharomyces cerevisiae) hydrolysate (YH) on muscle differentiation and muscle atrophy inhibitory activity in C2C12 cells and atrophy inhibitory activity in a dexamethasone (DEX)-induced muscle atrophy mouse model. Jenner-Giemsa staining revealed that the length and area of ​​myotubes increased in the YH groups. Moreover, the gene expression of myogenic differentiation 1 and myogenin was significantly increased in the YH groups. In the DEX-induced muscle atrophy C2C12 model, the expression of muscle ring-finger protein-1 (MuRF-1), forkhead box class O3a, and Atrogin-1 were significantly reduced in the YH groups. Furthermore, in the muscle-atrophy mice model, grip strength and lean mass were increased by 1.28-fold and 3.5%, respectively compared to the control in the YH group (300 mg/kg). These results were confirmed by the downregulation of MuRF-1 in muscle. In conclusion, YH promoted muscle cell differentiation and exhibited an inhibitory effect on DEX-induced muscle atrophy.

Muscle differentiation is an essential link in animal growth and development, and microRNA and mRNA are indispensable in skeletal muscle differentiation. To improve the meat quality and production of the Leizhou goat, it is vital to understand the molecular mechanism by which its skeletal muscle differentiates. By RNA sequencing (RNA-SEQ), we established miRNA-mRNA profiles of Leizhou goats at three stages: fetal day 70, 90, and 120. There were 991 differently expressed mRNAs and 39 differentially expressed miRNAs found, with the differentially expressed mRNAs mainly enriched in calcium ion binding, ECM-receptor interaction, and Focal adhesion. CKM and MYH3, two muscle differentiation markers, were significantly differentially expressed during this period. In addition, we found that chi-miR-129-5p, chi-miR-433, and chi-miR-24-3p co-regulate muscle differentiation with their target genes. Finally, we can confirm that muscle differentiation occurred in Leizhou goat between 90 and 120 days of the fetus. This study is helpful to better explore the molecular mechanism of goat muscle differentiation.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder affecting 1:3500 male births and is associated with myofiber degeneration, regeneration, and inflammation. Glucocorticoid treatments have been the standard of care due to immunomodulatory/immunosuppressive properties but novel genetic approaches, including exon skipping and gene replacement therapy, are currently being developed. The identification of additional biomarkers to assess DMD-related inflammatory responses and the potential efficacy of these therapeutic approaches are thus of critical importance. The current study uses RNA sequencing of skeletal muscle from two mdx mouse models to identify high mobility group box 1 (HMGB1) as a candidate biomarker potentially contributing to DMD-related inflammation. HMGB1 protein content was increased in a human iPSC-derived skeletal myocyte model of DMD and microdystrophin treatment decreased HMGB1 back to control levels. In vivo, HMGB1 protein levels were increased in vehicle treated B10-mdx skeletal muscle compared to B10-WT and significantly decreased in B10-mdx animals treated with adeno-associated virus (AAV)-microdystrophin. However, HMGB1 protein levels were not increased in D2-mdx skeletal muscle compared to D2-WT, demonstrating a strain-specific difference in DMD-related immunopathology., Competing Interests: Competing interests M.W.L. is the founder, CEO, and owner of Diverge Translational Science Laboratory. M.W.L. is or has recently been a member of advisory boards for Solid Biosciences, Taysha Gene Therapies, Astellas Gene Therapies (formerly Audentes Therapeutics), and Ichorion Therapeutics. M.W.L. is also a consultant for Astellas Gene Therapies (formerly Audentes Therapeutics), Encoded Therapeutics, Modis Therapeutics, Lacerta Therapeutics, Dynacure, AGADA Biosciences, Affinia Therapeutics, Biomarin, Locanabio, Vertex Pharmaceuticals, Voyager Therapeutics, and Entrada Therapeutics. M.W.L. receives or recently received research support from Astellas Gene Therapies, Solid Biosciences, Kate Therapeutics, Prothelia, Ecogenome, Cure Rare Disease, Rocket Pharma, Ultragenyx, Carbon Biosciences, Locanabio, Regenxbio, Vita Therapeutics, and Lexeo Therapeutics. J.S., M.H., M.J.P., and M.B. are full-time employees of Diverge Translational Science Laboratory. J.P.G. is a full-time employee and shareholder of Solid Biosciences. K.N. is co-founder of Agada Biosciences and ReveragenBiopharma., (© 2024. Published by The Company of Biologists Ltd.)

Senescent cells are characterized by multiple features such as increased expression of senescence-associated β-galactosidase activity (SA β-gal) and cell cycle inhibitors such as p21 or p16. They accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin (Doxo) cause buildup of senescent cells, leading to the inhibition of tissue regeneration. Senescent cells influence the neighboring cells via numerous secreted factors which form the senescence-associated secreted phenotype (SASP). Lipids are emerging as a key component of SASP that can control tissue homeostasis. Arachidonic acid-derived lipids have been shown to accumulate within senescent cells, specifically 15d-PGJ 2 , which is an electrophilic lipid produced by the non-enzymatic dehydration of the prostaglandin PGD 2 . This study shows that 15d-PGJ 2 is also released by Doxo-induced senescent cells as an SASP factor. Treatment of skeletal muscle myoblasts with the conditioned medium from these senescent cells inhibits myoblast fusion during differentiation. Inhibition of L-PTGDS, the enzyme that synthesizes PGD 2 , diminishes the release of 15d-PGJ 2 by senescent cells and restores muscle differentiation. We further show that this lipid post-translationally modifies Cys184 of HRas in C2C12 mouse skeletal myoblasts, causing a reduction in the localization of HRas to the Golgi, increased HRas binding to Ras Binding Domain (RBD) of RAF Kinase (RAF-RBD), and activation of cellular Mitogen Activated Protein (MAP) kinase-Extracellular Signal Regulated Kinase (Erk) signaling (but not the Akt signaling). Mutating C184 of HRas prevents the ability of 15d-PGJ 2 to inhibit the differentiation of muscle cells and control the activity of HRas. This work shows that 15d-PGJ 2 released from senescent cells could be targeted to restore muscle homeostasis after chemotherapy., Competing Interests: SP, AB, AV, SS, MJ, RM, HL, MH, AR No competing interests declared, (© 2024, Pundlik et al.)

Objective: The primary objective of this study was to investigate the role and regulatory mechanisms of Platelet-derived Growth Factor Subunit B (PDGFB) in muscle differentiation., Methods: In this study, a vector for PDGFB was designed and transfected into quail muscle cells to investigate its role and regulatory mechanism during muscle formation. To investigate the inhibitory mechanisms of PDGFB on myogenic differentiation, the mRNA expression levels of various genes and the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2), both known to regulate muscle development and differentiation were compared., Results: PDGFB-overexpressed (OE) cells formed morphologically shorter and thinner myotubes and demonstrated a smaller total myotube area than did the control cells. This result was also confirmed at the molecular level by a reduced amount of myosin heavy chain protein in the PDGFB-OE cells. Therefore, PDGFB inhibits the differentiation of muscle cells. Additionally, the expression of myogenin (MYOG) significantly decreased in the PDGFB-OE cells on days 2 and 4 compared with that in the control cells. The phosphorylation of ERK 1/2, an upstream protein that inhibits MYOG expression, increased in the PDGFB-OE cells on day 4 compared with that in the control cells. The decreased expression of MYOG in the PDGFB-OE cells increased by inhibition ERK 1/2 phosphorylation., Conclusion: PDGFB may suppress myogenesis by reducing MYOG expression through ERK 1/2 phosphorylation. These findings can help understand muscle differentiation and potentially improve poultry meat production.

Background: Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions. Results: In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation. Conclusions: Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation. [ABSTRACT FROM AUTHOR]

Black chokeberry or aronia (the fruit of Aronia melanocarpa) has been reported to having pharmacological activities against metabolic syndrome, such as hypertension, obesity, diabetes, and pro-inflammatory conditions. However, the effects of aronia on myogenic differentiation and muscle homoeostasis are uncharacterized. In this study, we investigated the effects of aronia (black chokeberry) on myogenic differentiation and muscle metabolic functions in young mice. Aronia extract (AR) promotes myogenic differentiation and elevates the formation of multinucleated myotubes through Akt activation. AR protects dexamethasone (DEX)-induced myotube atrophy through inhibition of muscle-specific ubiquitin ligases mediated by Akt activation. The treatment with AR increases muscle mass and strength in mice without cardiac hypertrophy. AR treatment enhances both oxidative and glycolytic myofibers and muscle metabolism with elevated mitochondrial genes and glucose metabolism-related genes. Furthermore, AR-fed muscle fibers display increased levels of total OxPHOS and myoglobin proteins. Taken together, AR enhances myogenic differentiation and improves muscle mass and function, suggesting that AR has a promising potential as a nutraceutical remedy to intervene in muscle weakness and atrophy.

Non-centrosomal microtubule-organizing centers (MTOCs) are pivotal for the function of multiple cell types, but the processes initiating their formation are unknown. Here, we find that the transcription factor myogenin is required in murine myoblasts for the localization of MTOC proteins to the nuclear envelope. Moreover, myogenin is sufficient in fibroblasts for nuclear envelope MTOC (NE-MTOC) formation and centrosome attenuation. Bioinformatics combined with loss- and gain-of-function experiments identified induction of AKAP6 expression as one central mechanism for myogenin-mediated NE-MTOC formation. Promoter studies indicate that myogenin preferentially induces the transcription of muscle- and NE-MTOC-specific isoforms of Akap6 and Syne1, which encodes nesprin-1α, the NE-MTOC anchor protein in muscle cells. Overexpression of AKAP6β and nesprin-1α was sufficient to recruit endogenous MTOC proteins to the nuclear envelope of myoblasts in the absence of myogenin. Taken together, our results illuminate how mammals transcriptionally control the switch from a centrosomal MTOC to an NE-MTOC and identify AKAP6 as a novel NE-MTOC component in muscle cells.

Muscles of patients with facioscapulohumeral dystrophy (FSHD) are characterized by sporadic DUX4 expression and oxidative stress which is at least partially induced by DUX4 protein. Nevertheless, targeting oxidative stress with antioxidants has a limited impact on FSHD patients, and the exact role of oxidative stress in the pathology of FSHD, as well as its interplay with the DUX4 expression, remain unclear. Here we set up a screen for genes that are upregulated by DUX4 via oxidative stress with the aim to target these genes rather than the oxidative stress itself. Immortalized human myoblasts expressing DUX4 (MB135-DUX4) have an increased level of reactive oxygen species (ROS) and exhibit differentiation defects which can be reduced by treating the cells with classic (Tempol) or mitochondria-targeted antioxidants (SkQ1). The transcriptome analysis of antioxidant-treated MB135 and MB135-DUX4 myoblasts allowed us to identify 200 genes with expression deregulated by DUX4 but normalized upon antioxidant treatment. Several of these genes, including PITX1, have been already associated with FSHD and/or muscle differentiation. We confirmed that PITX1 was indeed deregulated in MB135-DUX4 cells and primary FSHD myoblasts and revealed a redox component in PITX1 regulation. PITX1 silencing partially reversed the differentiation defects of MB135-DUX4 myoblasts. Our approach can be used to identify and target redox-dependent genes involved in human diseases.

The first and second branchiomeric (branchial arch) muscles are craniofacial muscles that derive from branchial arch mesoderm. In mammals, this set of muscles is indispensable for jaw movement and facial expression. Defects during embryonic development that result in congenital partial absence of these muscles can have significant impact on patients’ quality of life. However, the detailed molecular and cellular mechanisms that regulate branchiomeric muscle development remains poorly understood. Herein we investigated the role of retinoic acid (RA) signaling in developing branchiomeric muscles using mice as a model. We administered all-trans RA (25 mg/kg body weight) to Institute of Cancer Research (ICR) pregnant mice by gastric intubation from E8.5 to E10.5. In their embryos at E13.5, we found that muscles derived from the first branchial arch (temporalis, masseter) and second branchial arch (frontalis, orbicularis oculi) were severely affected or undetectable, while other craniofacial muscles were hypoplastic. We detected elevated cell death in the branchial arch mesoderm cells in RA-treated embryos, suggesting that excessive RA signaling reduces the survival of precursor cells of branchiomeric muscles, resulting in the development of hypoplastic craniofacial muscles. In order to uncover the signaling pathway(s) underlying this etiology, we focused on Pitx2, Tbx1, and MyoD1, which are critical for cranial muscle development. Noticeably reduced expression of all these genes was detected in the first and second branchial arch of RA-treated embryos. Moreover, elevated RA signaling resulted in a reduction in Dlx5 and Dlx6 expression in cranial neural crest cells (CNCCs), which disturbed their interactions with branchiomeric mesoderm cells. Altogether, we discovered that embryonic craniofacial muscle defects caused by excessive RA signaling were associated with the downregulation of Pitx2, Tbx1, MyoD1, and Dlx5/6, and reduced survival of cranial myogenic precursor cells.

Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, cross linking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline cross links, was increased compared with the cross-linked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, whereas MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying extracellular matrix (ECM) structures and enhancing differentiation on substrates with less collagen cross linking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting cross linking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration. [ABSTRACT FROM AUTHOR]

Adaptive responses of skeletal muscle regulate the nuclear shuttling of the sarcomeric protein Ankrd2 that can transduce different stimuli into specific adaptations by interacting with both structural and regulatory proteins. In a genome-wide expression study on Ankrd2-knockout or -overexpressing primary proliferating or differentiating myoblasts, we found an inverse correlation between Ankrd2 levels and the expression of proinflammatory genes and identified Ankrd2 as a potent repressor of inflammatory responses through direct interaction with the NF-κB repressor subunit p50. In particular, we identified Gsk3β as a novel direct target of the p50/Ankrd2 repressosome dimer and found that the recruitment of p50 by Ankrd2 is dependent on Akt2-mediated phosphorylation of Ankrd2 upon oxidative stress during myogenic differentiation. Surprisingly, the absence of Ankrd2 in slow muscle negatively affected the expression of cytokines and key calcineurin-dependent genes associated with the slow-twitch muscle program. Thus, our findings support a model in which alterations in Ankrd2 protein and phosphorylation levels modulate the balance between physiological and pathological inflammatory responses in muscle.