Inhibition of Sesn2 has negative regulatory effects on the myogenic differentiation of C2C12 myoblasts.

Sestrin2 (Sesn2) has been previously confirmed to be a stress-response molecule. However, the influence of Sesn2 on myogenic differentiation remains elusive. This study was conducted to analyze the role of Sesn2 in the myogenic differentiation of C2C12 myoblasts and related aspects in mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Our results showed that knockdown of Sesn2 reduced the myogenic differentiation capacity of C2C12 myoblasts. Predictive analysis from two databases suggested that miR-182-5p is a potential regulator of Sesn2. Further experimental validation revealed that overexpression of miR-182-5p decreased both the protein and mRNA levels of Sesn2 and inhibited myogenesis of C2C12 myoblasts. These findings suggest that miR-182-5p negatively regulates myogenesis by repressing Sesn2 expression. Extending to an in vivo model of DMD, knockdown of Sesn2 led to decreased Myogenin (Myog) expression and increased Pax7 expression, while its overexpression upregulated Myog levels and enhanced the proportion of slow-switch myofibers. These findings indicate the crucial role of Sesn2 in promoting myogenic differentiation and skeletal muscle regeneration, providing potential therapeutic targets for muscular dystrophy.

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.

A molecular pathway for cancer cachexia-induced muscle atrophy revealed at single-nucleus resolution.

Cancer cachexia is a prevalent and often fatal wasting condition that cannot be fully reversed with nutritional interventions. Muscle atrophy is a central component of the syndrome, but the mechanisms whereby cancer leads to skeletal muscle atrophy are not well understood. We performed single-nucleus multi-omics on skeletal muscles from a mouse model of cancer cachexia and profiled the molecular changes in cachexic muscle. Our results revealed the activation of a denervation-dependent gene program that upregulates the transcription factor myogenin. Further studies showed that a myogenin-myostatin pathway promotes muscle atrophy in response to cancer cachexia. Short hairpin RNA inhibition of myogenin or inhibition of myostatin through overexpression of its endogenous inhibitor follistatin prevented cancer cachexia-induced muscle atrophy in mice. Our findings uncover a molecular basis of muscle atrophy associated with cancer cachexia and highlight potential therapeutic targets for this disorder.

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.

Photobiomodulation mitigates Bothrops jararacussu venom-induced damage in myoblast cells by enhancing myogenic factors and reducing cytokine production.

BACKGROUND: Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are not yet fully understood. Herein, the effectiveness of photobiomodulation effects on regenerative response of C2C12 myoblast cells following exposure to Bothrops jararacussu venom (BjsuV), as well as the mechanisms involved was investigated. METHODOLOGY/PRINCIPAL FINDINGS: C2C12 myoblast cells were exposed to BjsuV (12.5 µg/mL) and irradiated once for 10 seconds with laser light of 660 nm (14.08 mW; 0.04 cm2; 352 mW/cm2) or 780 nm (17.6 mW; 0.04 cm2; 440 mW/ cm2) to provide energy densities of 3.52 and 4.4 J/cm2, and total energies of 0.1408 and 0.176 J, respectively. Cell migration was assessed through a wound-healing assay. The expression of MAPK p38-α, NF-Кß, Myf5, Pax-7, MyoD, and myogenin proteins were assessed by western blotting analysis. In addition, interleukin IL1-ß, IL-6, TNF-alfa and IL-10 levels were measured in the supernatant by ELISA. The PBM applied to C2C12 cells exposed to BjsuV promoted cell migration, increase the expression of myogenic factors (Pax7, MyF5, MyoD and myogenin), reduced the levels of proinflammatory cytokines, IL1-ß, IL-6, TNF-alfa, and increased the levels of anti-inflammatory cytokine IL-10. In addition, PBM downregulates the expression of NF-kB, and had no effect on p38 MAKP. CONCLUSION/SIGNIFICANCE: These data demonstrated that protection of the muscle cell by PBM seems to be related to the increase of myogenic factors as well as the modulation of inflammatory mediators. PBM therapy may offer a new therapeutic strategy to address the local effects of snakebite envenoming by promoting muscle regeneration and reducing the inflammatory process.

Comparing the efficacy of concomitant treatment of resistance exercise and creatine monohydrate versus multiple individual therapies in age related sarcopenia.

Aging-related sarcopenia is a degenerative loss of strength and skeletal muscle mass that impairs quality of life. Evaluating NUDT3 gene and myogenin expression as new diagnostic tools in sarcopenia. Also, comparing the concomitant treatment of resistance exercise (EX) and creatine monohydrate (CrM) versus single therapy by EX, coenzyme Q10 (CoQ10), and CrM using aged rats. Sixty male rats were equally divided into groups. The control group, aging group, EX-treated group, the CoQ10 group were administered (500 mg/kg) of CoQ10, the CrM group supplied (0.3 mg/kg of CrM), and a group of CrM concomitant with resistance exercise. Serum lipid profiles, certain antioxidant markers, electromyography (EMG), nudix hydrolase 3 (NUDT3) expression, creatine kinase (CK), and sarcopenic index markers were measured after 12 weeks. The gastrocnemius muscle was stained with hematoxylin-eosin (H&E) and myogenin. The EX-CrM combination showed significant improvement in serum lipid profile, antioxidant markers, EMG, NUDT3 gene, myogenin expression, CK, and sarcopenic index markers from other groups. The NUDT3 gene and myogenin expression have proven efficient as diagnostic tools for sarcopenia. Concomitant treatment of CrM and EX is preferable to individual therapy because it reduces inflammation, improves the lipid serum profile, promotes muscle regeneration, and thus has the potential to improve sarcopenia.

FoxP1 Represses MEF2A in Striated Muscle.

Myocyte enhancer factor 2 (MEF2) proteins are involved in multiple developmental, physiological, and pathological processes in vertebrates. Protein-protein interactions underlie the plethora of biological processes impacted by MEF2A, necessitating a detailed characterization of the MEF2A interactome. A nanobody based affinity-purification/mass spectrometry strategy was employed to achieve this goal. Specifically, the MEF2A protein complexes were captured from myogenic lysates using a GFP-tagged MEF2A protein immobilized with a GBP-nanobody followed by LC-MS/MS proteomic analysis to identify MEF2A interactors. After bioinformatic analysis, we further characterized the interaction of MEF2A with a transcriptional repressor, FOXP1. FOXP1 coprecipitated with MEF2A in proliferating myogenic cells which diminished upon differentiation (myotube formation). Ectopic expression of FOXP1 inhibited MEF2A driven myogenic reporter genes (derived from the creatine kinase muscle and myogenin genes) and delayed induction of endogenous myogenin during differentiation. Conversely, FOXP1 depletion enhanced MEF2A transactivation properties and myogenin expression. The FoxP1:MEF2A interaction is also preserved in cardiomyocytes and FoxP1 depletion enhanced cardiomyocyte hypertrophy. FOXP1 prevented MEF2A phosphorylation and activation by the p38MAPK pathway. Overall, these data implicate FOXP1 in restricting MEF2A function in order to avoid premature differentiation in myogenic progenitors and also to possibly prevent re-activation of embryonic gene expression in cardiomyocyte hypertrophy.

Identification of Peucedanum japonicum Thunb. extract components and their protective effects against dexamethasone-induced muscle atrophy.

BACKGROUND: Peucedanum japonicum Thunb. (PJ) is a vegetable widely consumed in East Asia and is known to have anticancer and anti-inflammatory effects. However, the effect of PJ on muscle atrophy remains elusive. PURPOSE: This study aimed to investigate the effect of PJ and its active compound on dexamethasone (DEX)-induced muscle atrophy. METHODS: We performed qualitative and quantitative analysis of PJ using ultra-performance liquid chromatography-mass spectrometry tandem mass spectrometry (UPLC-MS/MS) and high-performance liquid chromatography (HPLC), respectively. The efficacy of PJ and its main compound 4-caffeoylquinic acid (CQA) on muscle atrophy was evaluated in DEX-induced myotube atrophy and DEX-induced muscle atrophy in mouse myoblasts (C2C12) and C57BL/6 mice, in vitro and in vivo, respectively. RESULTS: The UPLC-MS/MS and HPLC data showed that the concentration of 4-CQA in PJ was 18.845 mg/g. PJ and 4-CQA treatments significantly inhibited DEX-induced myotube atrophy by decreasing protein synthesis and glucocorticoid translocation to the nucleus in C2C12 myotubes. In addition, PJ enhanced myogenesis by upregulating myogenin and myogenic differentiation 1 in C2C12 cells. PJ supplementation effectively increased muscle function and mass, downregulated atrogenes, and decreased proteasome activity in C57BL/6 mice. Additionally, PJ effectively decreased the nuclear translocation of forkhead transcription factor 3 alpha by inhibiting glucocorticoid receptor. CONCLUSION: Overall, PJ and its active compound 4-CQA alleviated skeletal muscle atrophy by inhibiting protein degradation. Hence, our findings present PJ as a potential novel pharmaceutical candidate for the treatment of muscle atrophy.

Leu promotes C2C12 cell differentiation by regulating the GSK3ß/ß-catenin signaling pathway through facilitating the interaction between SESN2 and RPN2.

BACKGROUND: Leucine (Leu) is an essential amino acid that facilitates skeletal muscle satellite cell differentiation, yet its mechanism remains underexplored. Sestrin2 (SESN2) serves as a Leu sensor, binding directly to Leu, while ribophorin II (RPN2) acts as a signaling factor in multiple pathways. This study aimed to elucidate Leu's impact on mouse C2C12 cell differentiation and skeletal muscle injury repair by modulating RPN2 expression through SESN2, offering a theoretical foundation for clinical skeletal muscle injury prevention and treatment. RESULTS: Leu addition promoted C2C12 cell differentiation compared to the control, enhancing early differentiation via myogenic determinant (MYOD) up-regulation. Sequencing revealed SESN2 binding to and interacting with RPN2. RPN2 overexpression up-regulated MYOD, myogenin and myosin heavy chain 2, concurrently decreased p-GSK3ß and increased nuclear ß-catenin. Conversely, RPN2 knockdown yielded opposite results. Combining RPN2 knockdown with Leu rescued increased p-GSK3ß and decreased nuclear ß-catenin compared to Leu absence. Hematoxylin and eosin staining results showed that Leu addition accelerated mouse muscle damage repair, up-regulating Pax7, MYOD and RPN2 in the cytoplasm, and nuclear ß-catenin, confirming that the role of Leu in muscle injury repair was consistent with the results for C2C12 cells. CONCLUSION: Leu, bound with SESN2, up-regulated RPN2 expression, activated the GSK3ß/ß-catenin pathway, enhanced C2C12 differentiation and expedited skeletal muscle damage repair. © 2024 Society of Chemical Industry.

Myogenin Regulates DUSP13 to Inhibit Apoptosis Induced by Reactive Oxygen Species.

BACKGROUND: Myogenin is well known as a crucial transcription factor in skeletal muscle development, yet its other biological functions remain unexplored. Previous research showed that myogenin suppresses apoptosis induced by angiotensin II in human induced pluripotent stem cell-derived cardiomyocytes, and offered a new perspective on myogenin's role in cardioprotection. However, the detailed mechanism of this cardioprotection, especially under oxidative stress, is still unclear. METHODS: In this study, hydrogen peroxide (H2O2) was used to generate reactive oxygen species in myogenin-overexpressing cardiomyocytes. The apoptosis was examined by flow cytometry. Transcriptome sequencing (RNA-seq) was performed to identify genes regulated by myogenin. Western blotting was used to detect the protein level of DUSP13 and the phosphorylation level of p38 mitogen-activated protein kinase (MAPK). The dual-luciferase reporter assay and ChIP assay were used to confirm the binding of myogenin to the promoter region of DUSP13. DUSP13 overexpression and knockdown assays were performed to study its anti-apoptotic role. RESULTS: Flow cytometry analysis of apoptosis showed that overexpressing myogenin for 24 and 48 hours decreased the apoptotic ratio by 47.9% and 63.5%, respectively, compared with untreated controls. Transcriptome sequencing performed on cardiomyocytes that expressed myogenin for different amounts of time (6, 12, 24, and 48 hours) identified DUSP13 as being up-regulated by myogenin. Western blotting showed that overexpression of myogenin increased the expression of DUSP13 and decreased the phosphorylation level of p38 MAPK. A dual-luciferase reporter assay proved that myogenin bound directly to the promoter region of DUSP13 and led to strong relative luciferase activity. Direct expression of DUSP13A and DUSP13B significantly reduced the rates of apoptosis and necrosis in cells treated with H2O2. Knockdown of DUSP13B significantly increased the rate of apoptosis in cells treated with H2O2. CONCLUSIONS: The present findings suggest that myogenin might attenuate apoptosis induced by reactive oxygen species by up-regulating DUSP13 and inactivating the p38 MAPK pathway.

The RNA-binding protein Msi2 regulates autophagy during myogenic differentiation.

Skeletal muscle development is a highly ordered process orchestrated transcriptionally by the myogenic regulatory factors. However, the downstream molecular mechanisms of myogenic regulatory factor functions in myogenesis are not fully understood. Here, we identified the RNA-binding protein Musashi2 (Msi2) as a myogenin target gene and a post-transcriptional regulator of myoblast differentiation. Msi2 knockdown in murine myoblasts blocked differentiation without affecting the expression of MyoD or myogenin. Msi2 overexpression was also sufficient to promote myoblast differentiation and myocyte fusion. Msi2 loss attenuated autophagosome formation via down-regulation of the autophagic protein MAPL1LC3/ATG8 (LC3) at the early phase of myoblast differentiation. Moreover, forced activation of autophagy effectively suppressed the differentiation defects incurred by Msi2 loss. Consistent with its functions in myoblasts in vitro, mice deficient for Msi2 exhibited smaller limb skeletal muscles, poorer exercise performance, and muscle fiber-type switching in vivo. Collectively, our study demonstrates that Msi2 is a novel regulator of mammalian myogenesis and establishes a new functional link between muscular development and autophagy regulation.

Myosin heavy chain-perinatal regulates skeletal muscle differentiation, oxidative phenotype and regeneration.

Myosin heavy chain-perinatal (MyHC-perinatal) is one of two development-specific myosin heavy chains expressed exclusively during skeletal muscle development and regeneration. The specific functions of MyHC-perinatal are unclear, although mutations are known to lead to contracture syndromes such as Trismus-pseudocamptodactyly syndrome. Here, we characterize the functions of MyHC-perinatal during skeletal muscle differentiation and regeneration. Loss of MyHC-perinatal function leads to enhanced differentiation characterized by increased expression of myogenic regulatory factors and differentiation index as well as reduced reserve cell numbers in vitro. Proteomic analysis revealed that loss of MyHC-perinatal function results in a switch from oxidative to glycolytic metabolism in myofibers, suggesting a shift from slow type I to fast type IIb fiber type, also supported by reduced mitochondrial numbers. Paracrine signals mediate the effect of loss of MyHC-perinatal function on myogenic differentiation, possibly mediated by non-apoptotic caspase-3 signaling along with enhanced levels of the pro-survival apoptosis regulator Bcl2 and nuclear factor kappa-B (NF-κB). Knockdown of MyHC-perinatal during muscle regeneration in vivo results in increased expression of the differentiation marker myogenin (MyoG) and impaired differentiation, evidenced by smaller myofibers, elevated fibrosis and reduction in the number of satellite cells. Thus, we find that MyHC-perinatal is a crucial regulator of myogenic differentiation, myofiber oxidative phenotype and regeneration.

Tankyrase-1 regulates RBP-mediated mRNA turnover to promote muscle fiber formation.

Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.

Effect of nonsteroidal anti-inflammatory drugs on pelvic floor muscle regeneration in a preclinical birth injury rat model.

BACKGROUND: Pelvic floor muscle injury is a common consequence of vaginal childbirth. Nonsteroidal anti-inflammatory drugs are widely used postpartum analgesics. Multiple studies have reported negative effects of these drugs on limb muscle regeneration, but their impact on pelvic floor muscle recovery following birth injury has not been explored. OBJECTIVE: Using a validated rat model, we assessed the effects of nonsteroidal anti-inflammatory drug on acute and longer-term pelvic floor muscle recovery following simulated birth injury. STUDY DESIGN: Three-month old Sprague Dawley rats were randomly assigned to the following groups: (1) controls, (2) simulated birth injury, (3) simulated birth injury+nonsteroidal anti-inflammatory drug, or (4) nonsteroidal anti-inflammatory drug. Simulated birth injury was induced using a well-established vaginal balloon distension protocol. Ibuprofen was administered in drinking water (0.2 mg/mL), which was consumed by the animals ad libitum. Animals were euthanized at 1, 3, 5, 7, 10, and 28 days after birth injury/ibuprofen administration. The pubocaudalis portion of the rat levator ani, which, like the human pubococcygeus, undergoes greater parturition-associated strains, was harvested (N=3-9/time point/group). The cross-sectional areas of regenerating (embryonic myosin heavy chain+) and mature myofibers were assessed at the acute and 28-day time points, respectively. The intramuscular collagen content was assessed at the 28-day time point. Myogenesis was evaluated using anti-Pax7 and anti-myogenin antibodies to identify activated and differentiated muscle stem cells, respectively. The overall immune infiltrate was assessed using anti-CD45 antibody. Expression of genes coding for pro- and anti-inflammatory cytokines was assessed by quantitative reverse transcriptase polymerase chain reaction at 3, 5, and 10 days after injury. RESULTS: The pubocaudalis fiber size was significantly smaller in the simulated birth injury+nonsteroidal anti-inflammatory drug compared with the simulated birth injury group at 28 days after injury (P<.0001). The median size of embryonic myosin heavy chain+ fibers was also significantly reduced, with the fiber area distribution enriched with smaller fibers in the simulated birth injury+nonsteroidal anti-inflammatory drug group relative to the simulated birth injury group at 3 days after injury (P<.0001), suggesting a delay in the onset of regeneration in the presence of nonsteroidal anti-inflammatory drugs. By 10 days after injury, the median embryonic myosin heavy chain+ fiber size in the simulated birth injury group decreased from 7 days after injury (P<.0001) with a tight cross-sectional area distribution, indicating nearing completion of this state of regeneration. However, in the simulated birth injury+nonsteroidal anti-inflammatory drug group, the size of embryonic myosin heavy chain+ fibers continued to increase (P<.0001) with expansion of the cross-sectional area distribution, signifying a delay in regeneration in these animals. Nonsteroidal anti-inflammatory drugs decreased the muscle stem cell pool at 7 days after injury (P<.0001) and delayed muscle stem cell differentiation, as indicated by persistently elevated number of myogenin+ cells 7 days after injury (P<.05). In contrast, a proportion of myogenin+ cells returned to baseline by 5 days after injury in the simulated birth injury group. The analysis of expression of genes coding for pro- and anti-inflammatory cytokines demonstrated only transient elevation of Tgfb1 in the simulated birth injury+nonsteroidal anti-inflammatory drug group at 5 but not at 10 days after injury. Consistently with previous studies, nonsteroidal anti-inflammatory drug administration following simulated birth injury resulted in increased deposition of intramuscular collagen relative to uninjured animals. There were no significant differences in any outcomes of interest between the nonsteroidal anti-inflammatory drug group and the unperturbed controls. CONCLUSION: Nonsteroidal anti-inflammatory drugs negatively impacted pelvic floor muscle regeneration in a preclinical simulated birth injury model. This appears to be driven by the negative impact of these drugs on pelvic muscle stem cell function, resulting in delayed temporal progression of pelvic floor muscle regeneration following birth injury. These findings provide impetus to investigate the impact of postpartum nonsteroidal anti-inflammatory drug administration on muscle regeneration in women at high risk for pelvic floor muscle injury.

Klotho regulates the myogenic response of muscle to mechanical loading and exercise.

NEW FINDINGS: What is the central question of this study? Does the hormone Klotho affect the myogenic response of muscle cells to mechanical loading or exercise? What is the main finding and its importance? Klotho prevents direct, mechanical activation of genes that regulate muscle differentiation, including genes that encode the myogenic regulatory factor myogenin and proteins in the canonical Wnt signalling pathway. Similarly, elevated levels of klotho expression in vivo prevent the exercise-induced increase in myogenin-expressing cells and reduce exercise-induced activation of the Wnt pathway. These findings demonstrate a new mechanism through which the responses of muscle to the mechanical environment are regulated. ABSTRACT: Muscle growth is influenced by changes in the mechanical environment that affect the expression of genes that regulate myogenesis. We tested whether the hormone Klotho could influence the response of muscle to mechanical loading. Applying mechanical loads to myoblasts in vitro increased RNA encoding transcription factors that are expressed in activated myoblasts (Myod) and in myogenic cells that have initiated terminal differentiation (Myog). However, application of Klotho to myoblasts prevented the loading-induced activation of Myog without affecting loading-induced activation of Myod. This indicates that elevated Klotho inhibits mechanically-induced differentiation of myogenic cells. Elevated Klotho also reduced the transcription of genes encoding proteins involved in the canonical Wnt pathway or their target genes (Wnt9a, Wnt10a, Ccnd1). Because the canonical Wnt pathway promotes differentiation of myogenic cells, these findings indicate that Klotho inhibits the differentiation of myogenic cells experiencing mechanical loading. We then tested whether these effects of Klotho occurred in muscles of mice experiencing high-intensity interval training (HIIT) by comparing wild-type mice and klotho transgenic mice. The expression of a klotho transgene combined with HIIT synergized to tremendously elevate numbers of Pax7+ satellite cells and activated MyoD+ cells. However, transgene expression prevented the increase in myogenin+ cells caused by HIIT in wild-type mice. Furthermore, transgene expression diminished the HIIT-induced activation of the canonical Wnt pathway in Pax7+ satellite cells. Collectively, these findings show that Klotho inhibits loading- or exercise-induced activation of muscle differentiation and indicate a new mechanism through which the responses of muscle to the mechanical environment are regulated.

Unilateral Hypofunction of the Masseter Leads to Molecular and 3D Morphometric Signs of Atrophy in Ipsilateral Agonist Masticatory Muscles in Adult Mice.

Mice are commonly used to study mandibular dynamics due to their similarity in chewing cycle patterns with humans. Adult mice treated unilaterally with botulinum toxin type A (BoNTA) in the masseter exhibit atrophy of this muscle characterized by an increase in the gene expression of atrophy-related molecular markers, and a reduction in both muscle fiber diameter and muscle mass at 14d. However, the impact of this muscle imbalance on the non-treated masticatory muscles remains unexplored. Here, we hypothesize that the unilateral masseter hypofunction leads to molecular and 3D morphometric signs of atrophy of the masseter and its agonist masticatory muscles in adult mice. Twenty-three 8-week-old male BALB/c mice received a single injection of BoNTA in the right masseter, whereas the left masseter received the same volume of saline solution (control side). Animals were euthanized at 2d, 7d, and 14d, and the masticatory muscles were analyzed for mRNA expression. Five heads were harvested at 14d, fixed, stained with a contrast-enhanced agent, and scanned using X-ray microtomography. The three-dimensional morphometric parameters (the volume and thickness) from muscles in situ were obtained. Atrogin-1/MAFbx, MuRF-1, and Myogenin mRNA gene expression were significantly increased at 2 and 7d for both the masseter and temporalis from the BoNTA side. For medial pterygoid, increased mRNA gene expression was found at 7d for Atrogin-1/MAFbx and at 2d-7d for Myogenin. Both the volume and thickness of the masseter, temporalis, and medial pterygoid muscles from the BoNTA side were significantly reduced at 14d. In contrast, the lateral pterygoid from the BoNTA side showed a significant increase in volume at 14d. Therefore, the unilateral hypofunction of the masseter leads to molecular and morphological signs of atrophy in both the BoNTA-injected muscle and its agonistic non-injected masticatory muscles. The generalized effect on the mouse masticatory apparatus when one of its components is intervened suggests the need for more clinical studies to determine the safety of BoNTA usage in clinical dentistry.

Establishment and Characterization of Continuous Satellite Muscle Cells from Olive Flounder (Paralichthys olivaceus): Isolation, Culture Conditions, and Myogenic Protein Expression.

Olive flounder (Paralichthys olivaceus) muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl2 and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs.

Nonsense-mediated mRNA decay suppresses injury-induced muscle regeneration via inhibiting MyoD transcriptional activity.

Skeletal muscle regeneration is a crucial physiological process that occurs in response to injury or disease. As an important transcriptome surveillance system that regulates tissue development, the role of nonsense-mediated mRNA decay (NMD) in muscle regeneration remains unclear. Here, we found that NMD inhibits myoblast differentiation by targeting the phosphoinositide-3-kinase regulatory subunit 5 gene, which leads to the suppression of the transcriptional activity of myogenic differentiation (MyoD), a key regulator of myoblast differentiation. This disruption of MyoD transcriptional activity subsequently affects the expression levels of myogenin and myosin heavy chain, crucial markers of myoblast differentiation. Additionally, through up-frameshift protein 1 knockdown experiments, we observed that inhibiting NMD can accelerate muscle regeneration in vivo. These findings highlight the potential of NMD as a novel therapeutic target for the treatment of muscle-related injuries and diseases.

Foxo3 Knockdown Mediates Decline of Myod1 and Myog Reducing Myoblast Conversion to Myotubes.

Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization, subsequently increasing healthcare costs while limiting quality of life. In sarcopenic muscle fibers, the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32) is expressed at substantially higher levels, driving ubiquitin-proteasomal muscle protein degradation. As one of the key regulators of muscular equilibrium, the transcription factor Forkhead Box O3 (FOXO3) can increase the expression of Fbxo32, making it a possible target for the regulation of this detrimental pathway. To test this hypothesis, murine C2C12 myoblasts were transduced with AAVs carrying a plasmid for four specific siRNAs against Foxo3. Successfully transduced myoblasts were selected via FACS cell sorting to establish single clone cell lines. Sorted myoblasts were further differentiated into myotubes and stained for myosin heavy chain (MHC) by immunofluorescence. The resulting area was calculated. Myotube contractions were induced by electrical stimulation and quantified. We found an increased Foxo3 expression in satellite cells in human skeletal muscle and an age-related increase in Foxo3 expression in older mice in silico. We established an in vitro AAV-mediated FOXO3 knockdown on protein level. Surprisingly, the myotubes with FOXO3 knockdown displayed a smaller myotube size and a lower number of nuclei per myotube compared to the control myotubes (AAV-transduced with a functionless control plasmid). During differentiation, a lower level of FOXO3 reduced the expression Fbxo32 within the first three days. Moreover, the expression of Myod1 and Myog via ATM and Tp53 was reduced. Functionally, the Foxo3 knockdown myotubes showed a higher contraction duration and time to peak. Early Foxo3 knockdown seems to terminate the initiation of differentiation due to lack of Myod1 expression, and mediates the inhibition of Myog. Subsequently, the myotube size is reduced and the excitability to electrical stimulation is altered.

The Functional Role of Myogenin in Cardiomyoblast H9c2 Cells Treated with High Glucose and Palmitic Acid: Insights into No-Rejection Heart Transplantation.

Various pathological alterations, including lipid-deposition-induced comparative cardiac lipotoxicity, contribute to cardiac aging in the failing heart. A decline in endogenous myogenin proteins can lead to the reversal of muscle cell differentiation and the creation of mononucleated muscle cells. Myogenin may be a specific regulator of adaptive responses to avoid pathological hypertrophy in the heart. Hence, it is important to understand the regulation of myogenin expression and functions in response to exposure to varied stresses. In this study, we first examined and verified the cytotoxic effect of palmitic acid on H9c2 cells. The reduction in myogenin mRNA and protein expression by palmitic acid was independent of the effect of glucose. Meanwhile, the induction of cyclooxygenase 2 and activating transcription factor 3 mRNAs and proteins by palmitic acid was dependent on the presence of glucose. In addition, palmitic acid failed to disrupt cell cycle progression when H9c2 cells were treated with no glucose. Next, we examined the functional role of myogenin in palmitic-acid-treated H9c2 cells and found that myogenin may be involved in palmitic-acid-induced mitochondrial and cytosolic ROS generation, cellular senescence, and mitochondrial membrane potential. Finally, the GSE150059 dataset was deposited in the Gene Expression Omnibus website and the dataset was further analyzed via the molecular microscope diagnostic system (MMDx), demonstrating that many heart transplant biopsies currently diagnosed as no rejection have mild molecular-antibody-mediated rejection-related changes. Our data show that the expression levels of myogenin were lower than the average level in the studied population. Combining these results, we uncover part of the functional role of myogenin in lipid- and glucose-induced cardiac cell stresses. This finding provides valuable insight into the differential role of fatty-acid-associated gene expression in cardiovascular tissues. Additionally, the question of whether this gene expression is regulated by myogenin also highlights the usefulness of a platform such as MMDx-Heart and can help elucidate the functional role of myogenin in heart transplantation.