Ginsenoside Rg5 promotes muscle regeneration via p38MAPK and Akt/mTOR signaling.

Background: Skeletal muscles play a key role in physical activity and energy metabolism. The loss of skeletal muscle mass can cause problems related to metabolism and physical activity. Studies are being conducted to prevent such diseases by increasing the mass and regeneration capacity of muscles. Ginsenoside Rg5 has been reported to exhibit a broad range of pharmacological activities. However, studies on the effects of Rg5 on muscle differentiation and growth are scarce. Methods: To investigate the effects of Rg5 on myogenesis, C2C12 myoblasts were induced to differentiate with Rg5, followed by immunoblotting, immunostaining, and qRT-PCR for myogenic markers and promyogenic signaling (p38MAPK). Immunoprecipitation confirmed that Rg5 increased the interaction between MyoD and E2A via p38MAPK. To investigate the effects of Rg5 on prevention of muscle mass loss, C2C12 myotubes were treated with dexamethasone to induce muscle atrophy. Immunoblotting, immunostaining, and qRT-PCR were performed for myogenic markers, Akt/mTOR signaling for protein synthesis, and atrophy-related genes (Atrogin-1 and MuRF1). Results: Rg5 promoted C2C12 myoblast differentiation through phosphorylation of p38MAPK and MyoD/E2A heterodimerization. Furthermore, Rg5 stimulated C2C12 myotube hypertrophy via phosphorylation of Akt/mTOR. Phosphorylation of Akt induces FoxO3a phosphorylation, which reduces the expression of Atrogin-1 and MuRF1. Conclusion: This study provides an understanding of how Rg5 promotes myogenesis and hypertrophy and prevents dexamethasone-induced muscle atrophy. The study is the first, to the best of our knowledge, to show that Rg5 promotes muscle regeneration and to suggest that Rg5 can be used for therapeutic intervention of muscle weakness and atrophy, including cancer cachexia.

Inonotus obliquus upregulates muscle regeneration and augments function through muscle oxidative metabolism.

Skeletal muscle wasting related to aging or pathological conditions is critically associated with the increased incidence and prevalence of secondary diseases including cardiovascular diseases, metabolic syndromes, and chronic inflammations. Much effort is made to develop agents to enhance muscle metabolism and function. Inonotus obliquus (I. obliquus; IO) is a mushroom popularly called chaga and has been widely employed as a folk medicine for inflammation, cardiovascular diseases, diabetes, and cancer in Eastern Europe and Asia. However, its effect on muscle health has not been explored. Here, we aimed to investigate the beneficial effect of IO extract in muscle regeneration and metabolism. The treatment of IO in C2C12 myoblasts led to increased myogenic differentiation and alleviation of dexamethasone-induced myotube atrophy. Network pharmacological analysis using the identified specific chemical constituents of IO extracts predicted protein kinase B (AKT)-dependent mechanisms to promote myogenesis and muscle regeneration. Consistently, IO treatment resulted in the activation of AKT, which suppressed muscle-specific ubiquitin E3 ligases induced by dexamethasone. IO treatment in mice improved the regeneration of cardiotoxin-injured muscles accompanied by elevated proliferation and differentiation of muscle stem cells. Furthermore, it elevated the mitochondrial content and muscle oxidative metabolism accompanied by the induction of peroxisome proliferator-activated receptor γ coactivator α (PGC-1α). Our current data suggest that IO is a promising natural agent in enhancing muscle regenerative capacity and oxidative metabolism thereby preventing muscle wasting.

Hornerin mediates phosphorylation of the polo-box domain in Plk1 by Chk1 to induce death in mitosis.

The centrosome assembles a bipolar spindle for faithful chromosome segregation during mitosis. To prevent the inheritance of DNA damage, the DNA damage response (DDR) triggers programmed spindle multipolarity and concomitant death in mitosis through a poorly understood mechanism. We identified hornerin, which forms a complex with checkpoint kinase 1 (Chk1) and polo-like kinase 1 (Plk1) to mediate phosphorylation at the polo-box domain (PBD) of Plk1, as the link between the DDR and death in mitosis. We demonstrate that hornerin mediates DDR-induced precocious centriole disengagement through a dichotomous mechanism that includes sequestration of Sgo1 and Plk1 in the cytoplasm through phosphorylation of the PBD in Plk1 by Chk1. Phosphorylation of the PBD in Plk1 abolishes the interaction with Sgo1 and phosphorylation-dependent Sgo1 translocation to the centrosome, leading to precocious centriole disengagement and spindle multipolarity. Mechanistically, hornerin traps phosphorylated Plk1 in the cytoplasm. Furthermore, PBD phosphorylation inactivates Plk1 and disrupts Cep192::Aurora A::Plk1 complex translocation to the centrosome and concurrent centrosome maturation. Remarkably, hornerin depletion leads to chemoresistance against DNA damaging agents by attenuating DDR-induced death in mitosis. These results reveal how the DDR eradicates mitotic cells harboring DNA damage to ensure genome integrity during cell division.

Cdon ablation in motor neurons causes age-related motor neuron degeneration and impaired sciatic nerve repair.

BACKGROUND: The functional deterioration and loss of motor neurons are tightly associated with degenerative motor neuron diseases and aging-related muscle wasting. Motor neuron diseases or aging-related muscle wasting in turn contribute to increased risk of adverse health outcomes in the elderly. Cdon (cell adhesion molecule-downregulated oncogene) belongs to the immunoglobulin superfamily of cell adhesion molecule and plays essential roles in multiple signalling pathways, including sonic hedgehog (Shh), netrin, and cadherin-mediated signalling. Cdon as a Shh coreceptor plays a critical role in motor neuron specification during embryonic development. However, its role in adult motor neuron function is unknown. METHODS: Hb9-Cre recombinase-driven motor neuron-specific Cdon deficient mice (mnKO) and a compound mutant mice (mnKO::SOD1G93A ) were generated to investigate the role of Cdon in motor neuron degeneration. Motor neuron regeneration was examined by using a sciatic nerve crush injury model. To investigate the phenotype, physical activity, compound muscle action potential, immunostaining, and transmission electron microscopy were carried out. In the mechanism study, RNA sequencing and RNA/protein analyses were employed. RESULTS: Mice lacking Cdon in motor neurons exhibited middle age onset lethality and aging-related decline in motor function. In the sciatic nerve crush injury model, mnKO mice exhibited an impairment in motor function recovery evident by prolonged compound muscle action potential duration (4.63 ± 0.35 vs. 3.93 ± 0.22 s for f/f, P < 0.01) and physical activity. Consistently, neuromuscular junctions of mnKO muscles were incompletely occupied (49.79 ± 5.74 vs. 79.39 ± 3.77% fully occupied neuromuscular junctions for f/f, P < 0.0001), suggesting an impaired reinnervation. The transmission electron microscopy analysis revealed that mnKO sciatic nerves had smaller axon diameter (0.88 ± 0.13 vs. 1.43 ± 0.48 µm for f/f, P < 0.0001) and myelination defects. RNA sequencing of mnKO lumbar spinal cords showed alteration in genes related to neurogenesis, inflammation and cell death. Among the altered genes, ErbB4 and FgfR expressions were significantly altered in mnKO as well as in Cdon-depleted NSC34 motor neuron cells. Consistently, Cdon-depleted NSC34 cells exhibited elevated levels of cleaved Caspase3 and γH2AX proteins, as well as Bax transcription. Cdon-depleted NSC34 cells also exhibited impaired activation of Akt in response to neuregulin-1 (NRG1) treatment. CONCLUSIONS: Our current data demonstrate the functional importance of Cdon in motor neuron function and nerve repair. Cdon ablation causes alterations in neurotrophin signalling that leads to motor neuron degeneration.

Protein Arginine Methyltransferase 1 Ablation in Motor Neurons Causes Mitochondrial Dysfunction Leading to Age-related Motor Neuron Degeneration with Muscle Loss.

Neuromuscular dysfunction is tightly associated with muscle wasting that occurs with age or due to degenerative diseases. However, the molecular mechanisms underlying neuromuscular dysfunction are currently unclear. Recent studies have proposed important roles of Protein arginine methyltransferase 1 (Prmt1) in muscle stem cell function and muscle maintenance. In the current study, we set out to determine the role of Prmt1 in neuromuscular function by generating mice with motor neuron-specific ablation of Prmt1 (mnKO) using Hb9-Cre. mnKO exhibited age-related motor neuron degeneration and neuromuscular dysfunction leading to premature muscle loss and lethality. Prmt1 deficiency also impaired motor function recovery and muscle reinnervation after sciatic nerve injury. The transcriptome analysis of aged mnKO lumbar spinal cords revealed alterations in genes related to inflammation, cell death, oxidative stress, and mitochondria. Consistently, mnKO lumbar spinal cords of sciatic nerve injury model or aged mice exhibited elevated cellular stress response in motor neurons. Furthermore, Prmt1 inhibition in motor neurons elicited mitochondrial dysfunction. Our findings demonstrate that Prmt1 ablation in motor neurons causes age-related motor neuron degeneration attributing to muscle loss. Thus, Prmt1 is a potential target for the prevention or intervention of sarcopenia and neuromuscular dysfunction related to aging.

Distribution of Neuroglobin in Pericytes is Associated with Blood-Brain Barrier Leakage against Cerebral Ischemia in Mice.

With emerging data on the various functions of neuroglobin (Ngb), such as neuroprotection and neurogenesis, we investigated the role of Ngb in the neurovascular unit (NVU) of the brain. To study the distribution and function of Ngb after cerebral ischemia, transient middle cerebral artery occlusion (tMCAO) was performed in mice. Brain immunostaining and fluorescence-activated cell sorting were used to analyze the role of Ngb according to the location and cell type. In normal brain tissue, it was observed that Ngb was distributed not only in neurons but also around the brain's blood vessels. Interestingly, Ngb was largely expressed in platelet-derived growth factor receptor ß (PDGFRß)-positive pericytes in the NVU. After tMCAO, Ngb levels were significantly decreased in the core of the infarct, and Ngb and PDGFRß-positive pericytes were detached from the vasculature. In contrast, in the penumbra of the infarct, PDGFRß-positive pericytes expressing Ngb were increased compared with that in the core of the infarct. Moreover, the cerebral blood vessels, which have Ngb-positive PDGFRß pericytes, showed reduced blood-brain barrier (BBB) leakage after tMCAO. It showed that Ngb-positive PDGFRß pericytes stayed around the endothelial cells and reduced the BBB leakage in the NVU. Our results indicate that Ngb may play a role in attenuating BBB leakage in part by its association with PDGFRß. In this study, the distribution and function of Ngb in the pericytes of the cerebrovascular system have been elucidated, which contributes to the treatment of stroke through a new function of Ngb.

PRMT7 Inhibitor SGC8158 Enhances Doxorubicin-Induced DNA Damage and Its Cytotoxicity.

Protein arginine methyltransferase 7 (PRMT7) regulates various cellular responses, including gene expression, cell migration, stress responses, and stemness. In this study, we investigated the biological role of PRMT7 in cell cycle progression and DNA damage response (DDR) by inhibiting PRMT7 activity with either SGC8158 treatment or its specific siRNA transfection. Suppression of PRMT7 caused cell cycle arrest at the G1 phase, resulting from the stabilization and subsequent accumulation of p21 protein. In addition, PRMT7 activity is closely associated with DNA repair pathways, including both homologous recombination and non-homologous end-joining. Interestingly, SGC8158, in combination with doxorubicin, led to a synergistic increase in both DNA damage and cytotoxicity in MCF7 cells. Taken together, our data demonstrate that PRMT7 is a critical modulator of cell growth and DDR, indicating that it is a promising target for cancer treatment.

A novel telomerase-derived peptide GV1001-mediated inhibition of angiogenesis: Regulation of VEGF/VEGFR-2 signaling pathways.

GV1001, a human telomerase reverse transcriptase catalytic subunit-derived 16-mer peptide, has been developed as a novel anticancer vaccine against various cancers including pancreatic cancer. In the current study, we demonstrate the regulatory roles and mechanisms of GV1001 in endothelial cell responses in vitro and microvessel sprouting ex vivo. GV1001 markedly inhibits vascular endothelial growth factor-A (VEGF-A)-stimulated endothelial cell permeability, proliferation, migration, invasion, tube formation as well as microvessel outgrowth from rat aortic rings. These anti-angiogenic effects of GV1001 were associated with the inhibition of VEGF-A/VEGFR-2 signaling pathways, redistribution of vascular endothelial-cadherin to cell-cell contacts, and down-regulation of VEGFR-2 and matrix metalloproteinase-2. Furthermore, GV1001 suppresses the proliferation and invasion of non-small cell lung cancer cells, and the release of VEGF from the cells, suggesting the regulatory role of GV1001 in tumor-derived angiogenesis as well as cancer cell growth and progression. Collectively, our study reports the pharmacological potential of GV1001 in the regulation of angiogenesis, and warrants further evaluation and development of GV1001 as a promising therapeutic agent for a variety of angiogenesis-related diseases including cancer.

Broussonin A- and B-mediated inhibition of angiogenesis by blockade of VEGFR-2 signalling pathways and integrin ß1 expression.

In the present study, we demonstrate the regulatory effects and mechanism of broussonin A and B, diphenylpropane derivatives isolated from Broussonetia kazinoki, on vascular endothelial growth factor-A (VEGF-A)-stimulated endothelial cell responses in vitro and microvessel sprouting ex vivo. Treatment with broussonin A or B suppressed VEGF-A-stimulated endothelial cell proliferation by regulating the expression of cell cycle-related proteins and the phosphorylation status of retinoblastoma protein. In addition, treatment with broussonin A or B abrogated VEGF-A-stimulated angiogenic responses including endothelial cell migration, invasion, tube formation and microvessel formation from rat aortic rings. These anti-angiogenic activities of broussonin A and B were mediated through inactivation of VEGF-A-stimulated downstream signalling pathways, localization of vascular endothelial-cadherin at cell-cell contacts, and down-regulation of integrin ß1 and integrin-liked kinase. Furthermore, treatment with broussonin A or B inhibited proliferation and invasion of non-small cell lung cancer and ovarian cancer cells. Taken together, our findings suggest the pharmacological potential of broussonin A and B in the regulation of angiogenesis, cancer cell growth and progression.

PRMT7 ablation in cardiomyocytes causes cardiac hypertrophy and fibrosis through ß-catenin dysregulation.

Angiotensin II (AngII) has potent cardiac hypertrophic effects mediated through activation of hypertrophic signaling like Wnt/ß-Catenin signaling. In the current study, we examined the role of protein arginine methyltransferase 7 (PRMT7) in cardiac function. PRMT7 was greatly decreased in hypertrophic hearts chronically infused with AngII and cardiomyocytes treated with AngII. PRMT7 depletion in rat cardiomyocytes resulted in hypertrophic responses. Consistently, mice lacking PRMT7 exhibited the cardiac hypertrophy and fibrosis. PRMT7 overexpression abrogated the cellular hypertrophy elicited by AngII, while PRMT7 depletion exacerbated the hypertrophic response caused by AngII. Similar with AngII treatment, the cardiac transcriptome analysis of PRMT7-deficient hearts revealed the alteration in gene expression profile related to Wnt signaling pathway. Inhibition of PRMT7 by gene deletion or an inhibitor treatment enhanced the activity of ß-catenin. PRMT7 deficiency decreases symmetric dimethylation of ß-catenin. Mechanistic studies reveal that methylation of arginine residue 93 in ß-catenin decreases the activity of ß-catenin. Taken together, our data suggest that PRMT7 is important for normal cardiac function through suppression of ß-catenin activity.

Ginsenoside Rg3 protects glucocorticoid­induced muscle atrophy in vitro through improving mitochondrial biogenesis and myotube growth.

Ginsenoside Rg3 (Rg3), amplified by iterative heating processing with fresh ginseng, has a broad range of pharmacological activities and improves mitochondrial biogenesis in skeletal muscle. However, thus far no study has examined how Rg3 affects myotube growth or muscle atrophy, to the best of the authors' knowledge. The present study was conducted to examine the myogenic effect of Rg3 on dexamethasone (DEX)­induced myotube atrophy and the underlying molecular mechanisms. Rg3 activated Akt/mammalian target of rapamycin signaling to prevent DEX­induced myotube atrophy thereby stimulating the expression of muscle­specific genes, including myosin heavy chain and myogenin, and suppressing muscle­specific ubiquitin ligases as demonstrated by immunoblotting and immunostaining assays. Furthermore, Rg3 efficiently prevented DEX­triggered mitochondrial dysfunction of myotubes through peroxisome proliferator­activated receptor­Î³ coactivator1α activities and its mitochondrial biogenetic transcription factors, nuclear respiratory factor­1 and mitochondrial transcription factor A. These were confirmed by immunoblotting, luciferase assays, RT­qPCR and mitochondrial analysis measuring the levels of ROS, ATP and membrane potential. By providing a mechanistic insight into the effect of Rg3 on myotube atrophy, the present study suggested that Rg3 has potential as a therapeutic or nutraceutical remedy to intervene in muscle aging or diseases including cancer cachexia.

Aronia Upregulates Myogenic Differentiation and Augments Muscle Mass and Function Through Muscle Metabolism.

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.

Inducible Prmt1 ablation in adult vascular smooth muscle leads to contractile dysfunction and aortic dissection.

Vascular smooth muscle cells (VSMCs) have remarkable plasticity in response to diverse environmental cues. Although these cells are versatile, chronic stress can trigger VSMC dysfunction, which ultimately leads to vascular diseases such as aortic aneurysm and atherosclerosis. Protein arginine methyltransferase 1 (Prmt1) is a major enzyme catalyzing asymmetric arginine dimethylation of proteins that are sources of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase. Although a potential role of Prmt1 in vascular pathogenesis has been proposed, its role in vascular function has yet to be clarified. Here, we investigated the role and underlying mechanism of Prmt1 in vascular smooth muscle contractility and function. The expression of PRMT1 and contractile-related genes was significantly decreased in the aortas of elderly humans and patients with aortic aneurysms. Mice with VSMC-specific Prmt1 ablation (smKO) exhibited partial lethality, low blood pressure and aortic dilation. The Prmt1-ablated aortas showed aortic dissection with elastic fiber degeneration and cell death. Ex vivo and in vitro analyses indicated that Prmt1 ablation significantly decreased the contractility of the aorta and traction forces of VSMCs. Prmt1 ablation downregulated the expression of contractile genes such as myocardin while upregulating the expression of synthetic genes, thus causing the contractile to synthetic phenotypic switch of VSMCs. In addition, mechanistic studies demonstrated that Prmt1 directly regulates myocardin gene activation by modulating epigenetic histone modifications in the myocardin promoter region. Thus, our study demonstrates that VSMC Prmt1 is essential for vascular homeostasis and that its ablation causes aortic dilation/dissection through impaired myocardin expression.

Understanding of sarcopenia: from definition to therapeutic strategies.

Sarcopenia refers to the gradual loss of skeletal muscle mass and function along with aging and is a social burden due to growing healthcare cost associated with a super-aging society. Therefore, researchers have established guidelines and tests to diagnose sarcopenia. Several studies have been conducted actively to reveal the cause of sarcopenia and find an economic therapy to improve the quality of life in elderly individuals. Sarcopenia is caused by multiple factors such as reduced regenerative capacity, imbalance in protein turnover, alteration of fat and fibrotic composition in muscle, increased reactive oxygen species, dysfunction of mitochondria and increased inflammation. Based on these mechanisms, nonpharmacological and pharmacological strategies have been developed to prevent and treat sarcopenia. Although several studies are currently in progress, no treatment is available yet. This review presents the definition of sarcopenia and summarizes recent understanding on the detailed mechanisms, diagnostic criteria, and strategies for prevention and treatment.

Protein arginine methyltransferases: promising targets for cancer therapy.

Protein methylation, a post-translational modification (PTM), is observed in a wide variety of cell types from prokaryotes to eukaryotes. With recent and rapid advancements in epigenetic research, the importance of protein methylation has been highlighted. The methylation of histone proteins that contributes to the epigenetic histone code is not only dynamic but is also finely controlled by histone methyltransferases and demethylases, which are essential for the transcriptional regulation of genes. In addition, many nonhistone proteins are methylated, and these modifications govern a variety of cellular functions, including RNA processing, translation, signal transduction, DNA damage response, and the cell cycle. Recently, the importance of protein arginine methylation, especially in cell cycle regulation and DNA repair processes, has been noted. Since the dysregulation of protein arginine methylation is closely associated with cancer development, protein arginine methyltransferases (PRMTs) have garnered significant interest as novel targets for anticancer drug development. Indeed, several PRMT inhibitors are in phase 1/2 clinical trials. In this review, we discuss the biological functions of PRMTs in cancer and the current development status of PRMT inhibitors in cancer therapy.

BST204 Protects Dexamethasone-Induced Myotube Atrophy through the Upregulation of Myotube Formation and Mitochondrial Function.

BST204 is a purified ginseng dry extract that has an inhibitory effect on lipopolysaccharide-induced inflammatory responses, but its effect on muscle atrophy is yet to be investigated. In this study, C2C12 myoblasts were induced to differentiate for three days followed by the treatment of dexamethasone (DEX), a corticosteroid drug, with vehicle or BST204 for one day and subjected to immunoblotting, immunocytochemistry, qRT-PCR and biochemical analysis for mitochondrial function. BST204 alleviates the myotube atrophic effect mediated by DEX via the activation of protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling. Through this pathway, BST204 suppresses the expression of muscle-specific E3 ubiquitin ligases contributing to the enhanced myotube formation and enlarged myotube diameter in DEX-treated myotubes. In addition, BST204 treatment significantly decreases the mitochondrial reactive oxygen species production in DEX-treated myotubes. Furthermore, BST204 improves mitochondrial function by upregulating the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) in DEX-induced myotube atrophy. This study provides a mechanistic insight into the effect of BST204 on DEX-induced myotube atrophy, suggesting that BST204 has protective effects against the toxicity of a corticosteroid drug in muscle and promising potential as a nutraceutical remedy for the treatment of muscle weakness and atrophy.

PRMT5 promotes DNA repair through methylation of 53BP1 and is regulated by Src-mediated phosphorylation.

PRMT5 participates in various cellular processes, including transcription regulation, signal transduction, mRNA splicing, and DNA repair; however, its mechanism of regulation is poorly understood. Here, we demonstrate that PRMT5 is phosphorylated at residue Y324 by Src kinase, a negative regulator of its activity. Either phosphorylation or substitution of the Y324 residue suppresses PRMT5 activity by preventing its binding with the methyl donor S-adenosyl-L-methionine. Additionally, we show that PRMT5 activity is associated with non-homologous end joining (NHEJ) repair by methylating and stabilizing p53-binding protein 1 (53BP1), which promotes cellular survival after DNA damage. Src-mediated phosphorylation of PRMT5 and the subsequent inhibition of its activity during the DNA damage process blocks NHEJ repair, leading to apoptotic cell death. Altogether, our findings suggest that PRMT5 regulates DNA repair through Src-mediated Y324 phosphorylation in response to DNA damage.

Ginsenoside Rb1 and Rb2 upregulate Akt/mTOR signaling-mediated muscular hypertrophy and myoblast differentiation.

BACKGROUND: As a process of aging, skeletal muscle mass and function gradually decrease. It is reported that ginsenoside Rb1 and Rb2 play a role as AMP-activated protein kinase activator, resulting in regulating glucose homeostasis, and Rb1 reduces oxidative stress in aged skeletal muscles through activating the phosphatidylinositol 3-kinase/Akt/Nrf2 pathway. We examined the effects of Rb1 and Rb2 on differentiation of the muscle stem cells and myotube formation. METHODS: C2C12 myoblasts treated with Rb1 and/or Rb2 were differentiated and induced to myotube formation, followed by immunoblotting for myogenic marker proteins, such as myosin heavy chain, MyoD, and myogenin, or immunostaining for myosin heavy chain or immunoprecipitation analysis for heterodimerization of MyoD/E-proteins. RESULTS: Rb1 and Rb2 enhanced myoblast differentiation through accelerating MyoD/E-protein heterodimerization and increased myotube hypertrophy, accompanied by activation of Akt/mammalian target of rapamycin signaling. In addition, Rb1 and Rb2 induced the MyoD-mediated transdifferentiation of the rhabdomyosarcoma cells into myoblasts. Furthermore, co-treatment with Rb1 and Rb2 had synergistically enhanced myoblast differentiation through Akt activation. CONCLUSION: Rb1 and Rb2 upregulate myotube growth and myogenic differentiation through activating Akt/mammalian target of rapamycin signaling and inducing myogenic conversion of fibroblasts. Thus, our first finding indicates that Rb1 and Rb2 have strong potential as a helpful remedy to prevent and treat muscle atrophy, such as age-related muscular dystrophy.

BST204, a Rg3 and Rh2 Enriched Ginseng Extract, Upregulates Myotube Formation and Mitochondrial Function in TNF-α-Induced Atrophic Myotubes.

The loss of skeletal muscle mass and function is a serious consequence of chronic diseases and aging. BST204 is a purified ginseng (the root of Panax ginseng) extract that has been processed using ginsenoside-ß-glucosidase and acid hydrolysis to enrich ginsenosides Rg3 and Rh2 from the crude ginseng. BST204 has a broad range of health benefits, but its effects and mechanism on muscle atrophy are currently unknown. In this study, we have examined the effects and underlying mechanisms of BST204 on myotube formation and myotube atrophy induced by tumor necrosis factor-α (TNF-α). BST204 promotes myogenic differentiation and multinucleated myotube formation through Akt activation. BST204 prevents myotube atrophy induced by TNF-α through the activation of Akt/mTOR signaling and down-regulation of muscle-specific ubiquitin ligases, MuRF1, and Atrogin-1. Furthermore, BST204 treatment in atrophic myotubes suppresses mitochondrial reactive oxygen species (ROS) production and regulates mitochondrial transcription factors such as NRF1 and Tfam, through enhancing the activity and expression of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Collectively, our findings indicate that BST204 improves myotube formation and PGC1α-mediated mitochondrial function, suggesting that BST204 is a potential therapeutic or neutraceutical remedy to intervene muscle weakness and atrophy.

Satellite cell-specific ablation of Cdon impairs integrin activation, FGF signalling, and muscle regeneration.

BACKGROUND: Perturbation in cell adhesion and growth factor signalling in satellite cells results in decreased muscle regenerative capacity. Cdon (also called Cdo) is a component of cell adhesion complexes implicated in myogenic differentiation, but its role in muscle regeneration remains to be determined. METHODS: We generated inducible satellite cell-specific Cdon ablation in mice by utilizing a conditional Cdon allele and Pax7 CreERT2 . To induce Cdon ablation, mice were intraperitoneally injected with tamoxifen (tmx). Using cardiotoxin-induced muscle injury, the effect of Cdon depletion on satellite cell function was examined by histochemistry, immunostaining, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Isolated myofibers or myoblasts were utilized to determine stem cell function and senescence. To determine pathways related to Cdon deletion, injured muscles were subjected to RNA sequencing analysis. RESULTS: Satellite cell-specific Cdon ablation causes impaired muscle regeneration with fibrosis, likely attributable to decreased proliferation, and senescence, of satellite cells. Cultured Cdon-depleted myofibers exhibited 32 ± 9.6% of EdU-positive satellite cells compared with 58 ± 4.4% satellite cells in control myofibers (P < 0.05). About 32.5 ± 3.7% Cdon-ablated myoblasts were positive for senescence-associated ß-galactosidase (SA-ß-gal) while only 3.6 ± 0.5% of control satellite cells were positive (P < 0.001). Transcriptome analysis of muscles at post-injury Day 4 revealed alterations in genes related to mitogen-activated protein kinase signalling (P < 8.29 e-5 ) and extracellular matrix (P < 2.65 e-24 ). Consistent with this, Cdon-depleted tibialis anterior muscles had reduced phosphorylated extracellular signal-regulated kinase (p-ERK) protein levels and expression of ERK targets, such as Fos (0.23-fold) and Egr1 (0.31-fold), relative to mock-treated control muscles (P < 0.001). Cdon-depleted myoblasts exhibited impaired ERK activation in response to basic fibroblast growth factor. Cdon ablation resulted in decreased and/or mislocalized integrin ß1 activation in satellite cells (weak or mislocalized integrin1 in tmx = 38.7 ± 1.9%, mock = 21.5 ± 6%, P < 0.05), previously linked with reduced fibroblast growth factor (FGF) responsiveness in aged satellite cells. In mechanistic studies, Cdon interacted with and regulated cell surface localization of FGFR1 and FGFR4, likely contributing to FGF responsiveness of satellite cells. Satellite cells from a progeria model, Zmpste24-/- myofibers, showed decreased Cdon levels (Cdon-positive cells in Zmpste24-/- = 63.3 ± 11%, wild type = 90 ± 7.7%, P < 0.05) and integrin ß1 activation (weak or mislocalized integrin ß1 in Zmpste24-/- = 64 ± 6.9%, wild type = 17.4 ± 5.9%, P < 0.01). CONCLUSIONS: Cdon deficiency in satellite cells causes impaired proliferation of satellite cells and muscle regeneration via aberrant integrin and FGFR signalling.