Regulation Of Myogenesis Research Articles

A Novel Nanobody Directed against Ovine Myostatin to Enhance Muscle Growth in Mouse.

Simple SummaryMyostatin (MSTN) is a negative regulator of myogenesis, and various strategies have been used to improve livestock production by inhibiting MSTN. In this study, we developed a single-variable domain of the heavy chain antibody-recombinant MSTN nanobody (RMN) against MSTN. The selected RMN was expressed, purified, and assessed for its cytotoxicity, affinity, specificity, and the ability to inhibit MSTN. The results demonstrated that RMN could specifically detect and bind MSTN, further inhibit myostatin activity, as well as enhance muscle growth in mice.Myostatin (MSTN) is a member of the transforming growth factor beta superfamily and is a negative regulator of myogenesis. It has been shown to function by controlling the proliferation of myoblasts. MSTN inhibition is considered as a promising treatment for promoting animal growth in livestock. Nanobodies, a special antibody discovered in camel, have arisen as an alternative to conventional antibodies and have shown great potential when used as tools in different biotechnology fields, such as diagnostics and therapy. In this study, we examined the effect of MSTN inhibition by RMN on the muscle growth of mice. The results showed that RMN could specifically detect and bind MSTN, as well as inhibit MSTN activity. A significant increase in skeletal muscle mass was observed after intramuscular injection of RMN into mice. Enhanced muscle growth occurred because of myofiber hypertrophy. These results offer a promising approach to enhance muscle growth that warrants further investigation in domestic animals.

Regulation of the Mammalian SWI/SNF Family of Chromatin Remodeling Enzymes by Phosphorylation during Myogenesis.

Myogenesis is the biological process by which skeletal muscle tissue forms. Regulation of myogenesis involves a variety of conventional, epigenetic, and epigenomic mechanisms that control chromatin remodeling, DNA methylation, histone modification, and activation of transcription factors. Chromatin remodeling enzymes utilize ATP hydrolysis to alter nucleosome structure and/or positioning. The mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) family of chromatin remodeling enzymes is essential for myogenesis. Here we review diverse and novel mechanisms of regulation of mSWI/SNF enzymes by kinases and phosphatases. The integration of classic signaling pathways with chromatin remodeling enzyme function impacts myoblast viability and proliferation as well as differentiation. Regulated processes include the assembly of the mSWI/SNF enzyme complex, choice of subunits to be incorporated into the complex, and sub-nuclear localization of enzyme subunits. Together these processes influence the chromatin remodeling and gene expression events that control myoblast function and the induction of tissue-specific genes during differentiation.

Single Cell RNA Sequencing Of Regenerating Skeletal Muscle Reveals A Senescence Response Which Is Necessary For Optimal Muscle Repair

Satellite cells drive skeletal muscle regeneration, a process regulated by factors released into the local muscle environment; however the source of this trophic support is poorly defined. In this regard, recent work has identified a supportive role for cells commonly associated with aging and pathology, termed “senescent cells”. The PURPOSE of this study was to determine the function of cellular senescence in normal skeletal muscle repair in rodents. METHODS: The tibialis anterior (TA) of C57BL6 mice was injured with cardiotoxin (CTX) and collected across a time-course. To examine senescent cell function during muscle repair, mice where treated with the senolytic compound (ABT-263) to selectively ablate senescent cells. RESULTS: The number of senescent cells (SA-β-gal+ cells) rapidly increased following injury (p <0.05) which returned to baseline by 21 days post-CTX. SA-β-gal+ cells displayed other markers consistent with senescence such as a lack of proliferation (EDU-) and the presence of DNA damage (γH2AX). qPCR analysis of putative senescence pathways including p16, p21 and p53 as well as factors commonly secreted by senescent cells were significantly upregulated in CTX-injected muscle in comparison to uninjured muscle (p <0.05). To identify the cell types which become senescent, single-cell RNA sequencing (scRNAseq) was performed on 5-day post CTX skeletal muscle which revealed that fibrogenic-adipogenic progenitors (FAPs), endothelial cells and macrophages demonstrated increased expression of the senescence markers Glb1, CDKN1A and Trp53 while no satellite cells become senescent. These findings were confirmed in vivo through IHC analysis of SA-β-gal and marker specific analysis of FAPs (PDGFRα), endothelial cells (CD31) and macrophages (F4/80). Importantly, senolytic treatment during regenerative myogenesis in vivo reduced the number of SA-β-gal+ cells by 44% which coincided with significant reductions in muscle fibre cross-sectional area (25%) and the number of nuclei/fibre (12%). CONCLUSION: A transient wave of cellular senescence contributes to endogenous muscle repair to influence muscle fibre size following injury.

Transcriptome Characterization of Repressed Embryonic Myogenesis Due to Maternal Calorie Restriction.

Fetal malnutrition decreases skeletal myofiber number and muscle mass in neonatal mammals, which increases the risk of developing obesity and diabetes in adult life. However, the associated molecular mechanisms remain unclear. Here, we investigated how the nutrient (calorie) availability affects embryonic myogenesis using a porcine model. Sows were given a normal or calorie restricted diet, following which skeletal muscle was harvested from the fetuses at 35, 55, and 90 days of gestation (dg) and used for histochemical analysis and high-throughput sequencing. We observed abrupt repression of primary myofiber formation following maternal calorie restriction (MCR). Transcriptome profiling of prenatal muscles revealed that critical genes and muscle-specific miRNAs associated with increased proliferation and myoblast differentiation were downregulated during MCR-induced repression of myogenesis. Moreover, we identified several novel miRNA-mRNA interactions through an integrative analysis of their expression profiles, devising a putative molecular network involved in the regulation of myogenesis. Interestingly, NC_010454.3_1179 was identified as a novel myogenic miRNA that can base-pair with sequences in the 3′-UTR of myogenic differentiation protein 1 (MyoD1). And we found that this UTR inhibited the expression of a linked reporter gene encoding a key myogenic regulatory factor, resulting in suppression of myogenesis. Our results greatly increase our understanding of the mechanisms underlying the nutrient-modulated myogenesis, and may also serve as a valuable resource for further investigation of fundamental developmental processes or assist in rational target selection ameliorating repressed myogenesis under fetal malnutrition.

Disruption of mstna and mstnb gene through CRISPR/Cas9 leads to elevated muscle mass in blunt snout bream (Megalobrama amblycephala)

Myostatin (mstn) belongs to the TGF-β family, which is mainly involved in the negative regulation of myogenesis in vertebrates. Upon implementation of several gene-editing techniques (ZFN, TALEN, RNAi and CRISPR/Cas9), disruption of mstn gene locus in several animal species has been shown to result in elevated muscle growth. As there are numerous reports on gene editing of mstn in animals (common carp, yellow catfish, channel catfish, etc.), we aimed to study the complete disruption of mstn in economy-farmed fish. Here, we analyzed and characterized mstna and mstnb in blunt snout bream. In blunt snout bream, both mstna and mstnb peptides were found to possess unique structural characteristics of the TGF-β family. We designed three target sites (targets 1, 2, and 3) to generate knock out mutants within the mature mRNA sequence of mstna or mstnb that were located in exon 1, exon 3, and exon 3, respectively. The fertilized eggs (1–2 cell stage) were injected with 1 nL mixture of gRNA (100 pg) and Cas9 protein (900 pg). The mutation efficiency of mstna targets 1, 2, and 3 was 5.1%, 8.2%, and 10.0% with mutation types 5, 4, and 12, respectively. Similarly, the mutation efficiency of mstnb targets 1, 2, and 3 was 15.3%, 14.7%, and 26.0% with mutation types 2, 3, and 5, respectively. At the age of six months, the body weight, height, and thickness of both mstna- (mstna+/−) and mstnb- (mstnb+/−) deficient fish were found to be increased significantly (p < .01) when compared to those of wild-type control fish. Moreover, the average muscle fiber sizes of both mstna+/− and mstnb+/− fish were significantly larger (p < .01) than that of control fish. Our results provide valuable data associated with the regulation of muscle development and growth in blunt snout bream. These results also provide a basis and reference for the application of CRISPR/Cas9 (robust and efficient gene-engineering tool) in other aquaculture fish species.

Muscle Regenerative Capacity May Be Reduced When Aerobic Exercise Is Initiated with Low Glycogen Content

Abstract Objectives Maintaining low muscle glycogen content during recovery from aerobic exercise with low carbohydrate, high fat feeding has been shown to reduce insulin-mediated anabolic signaling compared to high carbohydrate feeding. The effects of low muscle glycogen content on intracellular regulators of muscle mass before and after aerobic exercise with carbohydrate ingestion is unclear. This study examined the effect of initiating aerobic exercise with low muscle glycogen content on postprandial insulin-dependent muscle anabolic signaling and myogenesis. Methods Twelve men (mean ± SD, age: 21 ± 4 y; body mass: 83 ± 11 kg; VO2peak: 44 ± 3 mL/kg/min) completed 2 cycle ergometry glycogen depletion trials separated by 7 d, followed by a 24-h period of isocaloric high fat (1.5 g/kg carbohydrate, 3.0 g/kg fat) or high carbohydrate (6.0 g/kg carbohydrate, 1.0 g/kg fat) refeeding to elicit low (LOW; 217 ± 103 mmol/kg dry wt) or adequate (AD; 396 ± 70 mmol/kg dry wt) glycogen content in randomized order. Participants then performed 80 min of cycle ergometry (64 ± 3% VO2peak) while ingesting 146 g of carbohydrate. Protein signaling (Western blotting) and gene transcription (RT-qPCR) were determined from vastus lateralis biopsies obtained before glycogen depletion (baseline, BASE), and before (PRE) and after (POST) exercise. Data presented as fold change relative to BASE for LOW and AD. Results Independent of time, carbohydrate sensing p-AMPKThr172 was higher (P &amp;lt; 0.05) in LOW compared to AD, while p-p38MAPKThr180/Tyr182 was higher (P &amp;lt; 0.05) in LOW at POST, but not different PRE. Insulin sensitive p-AKTThr473 was higher (P &amp;lt; 0.05) in AD compared to LOW, regardless of time. Anabolic regulators, p-mTORC1Ser2448, p-p70S6KSer424/421, and p-rpS6Ser235/236 were higher (P &amp;lt; 0.05) POST compared to PRE and BASE, independent of group. Regulators of myogenesis, MYOD and MYOGENIN were lower (P &amp;lt; 0.05) in LOW compared to AD, regardless of time, while PAX7 was lower (P &amp;lt; 0.05) in LOW compared to AD at PRE, but not different POST. Conclusions Initiating aerobic exercise with low muscle glycogen content does not appear to affect downstream insulin-dependent anabolic signaling, yet reductions in myogenic regulator factors suggest muscle repair and remodeling in recovery from exercise may be impaired. Funding Sources Work supported by DHP JPC-5/MOMRP; authors’ views not official U.S. Army or DoD policy.

HRP2-DPF3a-BAF complex coordinates histone modification and chromatin remodeling to regulate myogenic gene transcription.

Functional crosstalk between histone modifications and chromatin remodeling has emerged as a key regulatory mode of transcriptional control during cell fate decisions, but the underlying mechanisms are not fully understood. Here we discover an HRP2–DPF3a–BAF epigenetic pathway that coordinates methylated histone H3 lysine 36 (H3K36me) and ATP-dependent chromatin remodeling to regulate chromatin dynamics and gene transcription during myogenic differentiation. Using siRNA screening targeting epigenetic modifiers, we identify hepatoma-derived growth factor-related protein 2 (HRP2) as a key regulator of myogenesis. Knockout of HRP2 in mice leads to impaired muscle regeneration. Mechanistically, through its HIV integrase binding domain (IBD), HRP2 associates with the BRG1/BRM-associated factor (BAF) chromatin remodeling complex by interacting directly with the BAF45c (DPF3a) subunit. Through its Pro-Trp-Trp-Pro (PWWP) domain, HRP2 preferentially binds to H3K36me2. Consistent with the biochemical studies, ChIP-seq analyses show that HRP2 colocalizes with DPF3a across the genome and that the recruitment of HRP2/DPF3a to chromatin is dependent on H3K36me2. Integrative transcriptomic and cistromic analyses, coupled with ATAC-seq, reveal that HRP2 and DPF3a activate myogenic genes by increasing chromatin accessibility through recruitment of BRG1, the ATPase subunit of the BAF complex. Taken together, these results illuminate a key role for the HRP2-DPF3a-BAF complex in the epigenetic coordination of gene transcription during myogenic differentiation.

Biochemical characterization of skeletal muscle cells differentiated from tonsil-derived stem cell

Background & Aim The ease of human tonsil-derived stem cell (TMSC) source is useful for stem cell therapy in various skeletal muscle disorder (SKMD), and previously we reported that TMSCs can differentiate into skeletal muscle cell (SKMC)s, which are therefore a promising candidate in cell therapies for the SKMD. The skeletal muscles which account for the majority of insulin-mediated glucose uptake in the post-prandial state play an important role in maintaining glucose homeostasis. In skeletal muscles, insulin promotes glucose uptake by activating phosphatidylinositol-3 kinase (PI3K)/Akt signaling, leading to increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Therefore, we investigated the biochemical characterization of SKMCs differentiated from TMSCs for the use of clinical purposes. Methods, Results & Conclusion After the differentiating in our own methods, we confirmed that the expressions of acetylcholine receptor (AchR) and GLUT4 in TMSC-SKMCs have been increased compared to undifferentiated TMSCs. Also, to test their insulin responsiveness, by treating TMSC-SKMCs with 100 nM insulin for 30 minutes, the changes of the GLUT4 expression and the PI3K/Akt signaling under the low or high glucose medium in the TMSC-SKMCs were analyzed. Furthermore, the mitochondria of TMSC-SKMCs were observed during insulin responsiveness, which was play a role in the regulation of myogenesis. Collectively, we report the biochemical characterization of TMSC-SKMCs is associated with GLUT4 and PI3K/Akt signaling, likely to human SKMCs.

Regulation of Myogenesis by a Caveolin Binding Motif in Na/K‐ATPase

The caveolin binding motif (CBM) in Na/K‐ATPase (NKA) is highly conserved in metazoan animals and plays essential roles in cell signaling. The genetic mutation of this CBM sequence (F97A;F100A) in mice gave rise to the diminished somite and open neural tube formation, and eventually mutant embryos died at around E12.5. Subsequent analyses with RNA‐seq and real‐time RT‐PCR demonstrated that this CBM mutation drastically inhibited gene expression of transcription factors Myogenin and NeuroD, as well as component members in signaling pathways essential for embryogenesis, such as Wnt, Notch and Sonic Hedgehog. In order to further dissect the molecular mechanisms behind this genetic control, we used human induced pluripotent stem cells (iPSC) and exemplified the regulation of CBM‐NKA on stem cell differentiation using skeletal muscle cells (Skm). Through CRISPR/Cas9 genome editing, we created the mutant CBM iPSC and further differentiated them into skeletal muscle cells with sequential induction system. Compared to the parental human iPSC as the control, the CBM mutant stem cells lost their ability to differentiate into skeletal muscle cells. Moreover, the differentiated cells from CBM mutants did not contain the banding structures shown in the EM imaging and immunofluorescence staining with the antibody against Actinin. As expected, these differentiated mutant cells dramatically reduced mRNA expression of marker genes at both early differentiation stages (PAX3, MYOD and MYOGENIN) and late stages (myosin heavy chain 3, MYH3; myosin heavy chain 8, MYH8; Troponin T1, TNNT1; and Caveolin 3, CAV3). Exogenous expression of a mouse MyoD gene via the lentiviral gene delivery regained the ability of CBM mutant iPSC cells to differentiate into muscle cells, reflected at the level of both marker gene expression and cell structure imaging. Finally, we assessed one of the essential signaling pathway in vertebrate myogenesis, beta‐catenin/Wnt. The TOPFlash assay consisting of 7XTCF binding sequences as the Wnt reporter demonstrated a significant reduction of luciferase activity in CBM mutant iPSC. This reduced Wnt activity was further proven to associate with the disrupted cell membrane localization of the beta‐catenin protein, which was demonstrated through an immunostaining and confocal imaging, as well as the cell fractionation assay. Altogether, our study has revealed a novel pathway by which NKA controls myogenesis through Wnt signaling.

TGF-β-driven muscle degeneration and failed regeneration underlie disease onset in a DMD mouse model.

Duchenne muscular dystrophy (DMD) is a chronic muscle disease characterized by poor myogenesis and replacement of muscle by extracellular matrix. Despite the shared genetic basis, severity of these deficits varies among patients. One source of these variations is the genetic modifier that leads to increased TGF-β activity. While anti-TGF-β therapies are being developed to target muscle fibrosis, their effect on the myogenic deficit is underexplored. Our analysis of in vivo myogenesis in mild (C57BL/10ScSn-mdx/J and C57BL/6J-mdxΔ52) and severe DBA/2J-mdx (D2-mdx) dystrophic models reveals no defects in developmental myogenesis in these mice. However, muscle damage at the onset of disease pathology, or by experimental injury, drives up TGF-β activity in the severe, but not in the mild, dystrophic models. Increased TGF-β activity is accompanied by increased accumulation of fibroadipogenic progenitors (FAPs) leading to fibro-calcification of muscle, together with failure of regenerative myogenesis. Inhibition of TGF-β signaling reduces muscle degeneration by blocking FAP accumulation without rescuing regenerative myogenesis. These findings provide in vivo evidence of early-stage deficit in regenerative myogenesis in D2-mdx mice and implicates TGF-β as a major component of a pathogenic positive feedback loop in this model, identifying this feedback loop as a therapeutic target.

Genetic variants in MYF5 affected growth traits and beef quality traits in Chinese Qinchuan cattle

Myogenic factor 5 plays actively roles in the regulation of myogenesis. The aims of this study are to identify the evolution information of MYF5 protein among 10 domestic and mammalian animals, to uncover the expression patterns of MYF5 gene in calves and adults of Qinchuan cattle, and to expose the genetic variants of the MYF5 gene and explore its effect on cattle growth traits and beef quality traits in Qinchuan cattle. The bioinformatics results showed that the MYF5 proteins highly conserved in different mammalian or domestic animals apart from chicken. The expression level of MYF5 gene in the heart, muscle, lung, large intestine and liver was greater than that of other tissues. PCR amplicons sequencing identified four novel SNPs at g.5738A>G, g.5785C>T and g.5816A>G in the 3rd exon region and g.6535A>G in the 3’ UTR. Genotypic frequencies of g.5785C>T was harshly deviated from the HWE (P < .05). Genetic diversity was low or intermediate for the four SNPs and those SNPs were in the weak linkage disequilibrium. Association analysis results indicated g.5785C>T, g.5816A>G and g.6535A>G significant effect on growth performance and beef quality traits of Qinchuan cattle. H1H3 diplotype had greater body size and better beef quality. All the results implicate that the MYF5 gene might be applied as a promising candidate gene in Qinchuan cattle breeding.

Treatment of Dystrophic mdx Mice with an ADAMTS-5 Specific Monoclonal Antibody Increases the Ex Vivo Strength of Isolated Fast Twitch Hindlimb Muscles

Aberrant extracellular matrix synthesis and remodeling contributes to muscle degeneration and weakness in Duchenne muscular dystrophy (DMD). ADAMTS-5, a secreted metalloproteinase with catalytic activity against versican, is implicated in myogenesis and inflammation. Here, using the mdx mouse model of DMD, we report increased ADAMTS-5 expression in dystrophic hindlimb muscles, localized to regions of regeneration and inflammation. To investigate the pathophysiological significance of this, 4-week-old mdx mice were treated with an ADAMTS-5 monoclonal antibody (mAb) or IgG2c (IgG) isotype control for 3 weeks. ADAMTS-5 mAb treatment did not reduce versican processing, as protein levels of the cleaved versikine fragment did not differ between hindlimb muscles from ADAMTS-5 mAb or IgG treated mdx mice. Nonetheless, ADAMTS-5 blockade improved ex vivo strength of isolated fast extensor digitorum longus, but not slow soleus, muscles. The underpinning mechanism may include modulation of regenerative myogenesis, as ADAMTS-5 blockade reduced the number of recently repaired desmin positive myofibers without affecting the number of desmin positive muscle progenitor cells. Treatment with the ADAMTS-5 mAb did not significantly affect markers of muscle damage, inflammation, nor fiber size. Altogether, the positive effects of ADAMTS-5 blockade in dystrophic muscles are fiber-type-specific and independent of versican processing.

Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts.

Estrogen (E2) and polyunsaturated fatty acids (n-3PUFA) supplements independently support general wellbeing and enhance muscle regeneration in-vivo and myotube formation in-vitro. However, the combined effect of E2 and n-3PUFA on myoblast differentiation is not known. The purpose of the study was to identify whether E2 and n-3PUFA possess a synergistic effect on in-vitro myogenesis. Mouse C2C12 myoblasts, a reliable model to reiterate myogenic events in-vitro, were treated with 10nM E2 and 50μM eicosapentaenoic acid (EPA) independently or combined, for 0–24 h or 0–120 h during differentiation. Immunofluorescence, targeted qPCR and next generation sequencing (NGS) were used to characterize morphological changes and differential expression of key genes involved in the regulation of myogenesis and muscle function pathways. E2 increased estrogen receptor α (Erα) and the expression of the mitogen-activated protein kinase 11 (Mapk11) within 1 h of treatment and improved myoblast differentiation and myotube formation. A significant reduction (p < 0.001) in myotube formation and in the expression of myogenic regulatory factors Mrfs (MyoD, Myog and Myh1) and the myoblast fusion related gene, Tmem8c, was observed in the presence of EPA and the combined E2/EPA treatment. Additionally, EPA treatment at 48 h of differentiation inhibited the majority of genes associated with the myogenic and striated muscle contraction pathways. In conclusion, EPA and E2 had no synergistic effect on myotube formation in-vitro. Independently, EPA inhibited myoblast differentiation and overrides the stimulatory effect of E2 when used in combination with E2.

Isolation, Culture, and Differentiation of Primary Myoblasts Derived from Muscle Satellite Cells

The skeletal muscle is key for body mobility and motor performance, but aging and diseases often lead to progressive loss of muscle mass due to wasting or degeneration of muscle cells. Muscle satellite cells (MuSCs) represent a population of tissue stem cells residing in the skeletal muscles and are responsible for homeostatic maintenance and regeneration of skeletal muscles. Growth, injury, and degenerative signals activate MuSCs, which then proliferate (proliferating MuSCs are called myoblasts), differentiate and fuse with existing multinuclear muscle cells (myofibers) to mediate muscle growth and repair. Here, we describe a protocol for isolating MuSCs from skeletal muscles of mice for in vitro analysis. In addition, we provide a detailed protocol on how to culture and differentiate primary myoblasts into myotubes and an immunofluorescent staining procedure to characterize the cells. These methods are essential for modeling regenerative myogenesis in vitro to understand the dynamics, function and molecular regulation of MuSCs.

Role of Mutant TBP in Regulation of Myogenesis on Muscle Satellite Cells.

In polyglutamine (PolyQ) diseases, mutant proteins cause not only neurological problems but also peripheral tissue abnormalities. Among all systemic damages, skeletal muscle dystrophy is the severest. Previously by studying knock-in (KI) mouse models of spinal cerebellar ataxia 17 (SCA17), it was found that mutant TATA box binding protein (TBP) decreases its interaction with myogenic differentiation antigen, thus reducing the expression of skeletal muscle structural proteins and resulting in muscle degeneration. In this paper, the role of mutant TBP in myogenesis was investigated. Single myofibers were isolated from tibialis anterior muscles of wild type (WT) and SCA17KI mice. The 1TBP18 staining confirmed the expression of mutant TBP in muscle satellite cells in SCA17KI mice. In the BaCl2-induced TA muscle injury, H&E cross-section staining showed no significant change in myofibril size before and after BaCl2 treatment, and there was no significant difference in centralized nuclei between WT and SCA17KI mice, suggesting that mutant TBP had no significant effect on muscle regeneration. In the cultured primary myoblasts from WT and SCA17KI mice in vitro, representative BrdU immunostaining showed no significant difference in proliferation of muscle satellite cells. The primary myoblasts were then induced to differentiate and immunostained for eMyHC, and the staining showed there was no significant difference in differentiation of primary myoblasts between WT and SCA1KI mice. Our findings confirmed that mutant TBP had no significant effect on myogenesis.

P.127KER-050, a novel muscle anabolic, functions as a ligand trap that binds myo-catabolic TGFβ ligands and has reduced binding affinity for BMP9, a critical vascular remodeling ligand

Muscular dystrophy (MD) is a group of over 30 inherited diseases caused by mutations that result in progressive muscle weakness and loss. One therapeutic strategy being explored in MD is the use of muscle anabolic agents to increase or maintain lean mass. Multiple TGFβ superfamily ligands signal through the receptors ActRIIA and ActRIIB to negatively regulate muscle mass, including myostatin along with activins A and B. Inhibition of these ligands has been demonstrated, through various therapeutic modalities, to increase muscle mass and strength. Inhibition of these negative regulators of myogenesis with a soluble form of ActRIIB (ActRIIB-Fc) increased lean mass in healthy volunteers and increased muscle mass and muscle function in Duchenne muscular dystrophy patients; however, clinical studies were halted due to adverse events of nose and gum bleeding. This effect was attributed to ActRIIB-Fc inhibition of BMP9, a ligand involved in vascular remodeling and not in the regulation of muscle mass. Supporting this, a soluble form of ActRIIA, a homologous receptor to ActRIIB that lacks BMP9 binding, does not cause these adverse effects on vasculature. KER-050, a novel muscle anabolic, is comprised of a modified ActRIIA extracellular domain fused to the Fc of a human IgG. In vitro data demonstrate that KER-050 binds and inhibits the TGFβ ligands involved in the negative regulation of muscle. In vivo KER-050 has the muscle anabolic properties of ActRIIB-Fc, and increases lean mass and strength in both wild-type and dystrophic mice. In vitro data demonstrate that KER-050 retained the ActRIIA property of low BMP9 binding. Additionally, a neonatal mouse retinal vascularization model was used to assess BMP9 inhibition in vivo. Treatment with ActRIIB-Fc, a potent BMP9 inhibitor, substantially disrupted retinal vascularization whereas KER-050, a low BMP9 binder, did not. Therefore, KER-050 is a muscle anabolic agent that lacks the BMP9-mediated vascular safety liabilities.

Circular RNA Regulation of Myogenesis.

Circular RNA (circRNA) is a novel class of non-coding RNA generated by pre-mRNA back splicing, which is characterized by a closed-loop structure. Although circRNAs were firstly reported decades ago, their regulatory roles have not been discovered until recently. In this review, we discussed the putative biogenesis pathways and regulatory functions of circRNAs. Recent studies showed that circRNAs are abundant in skeletal muscle tissue, and their expression levels are regulated during muscle development and aging. We, thus, characterized the expression profile of circRNAs in skeletal muscle and discussed regulatory functions and mechanism-of-action of specific circRNAs in myogenesis. The future investigation into the roles of circRNAs in both physiological and pathological conditions may provide novel insights in skeletal muscle development and provide new therapeutic strategies for muscular diseases.

A requirement of Polo-like kinase 1 in murine embryonic myogenesis and adult muscle regeneration.

Muscle development and regeneration require delicate cell cycle regulation of embryonic myoblasts and adult muscle satellite cells (MuSCs). Through analysis of the Polo-like kinase (Plk) family cell-cycle regulators in mice, we show that Plk1's expression closely mirrors myoblast dynamics during embryonic and postnatal myogenesis. Cell-specific deletion of Plk1 in embryonic myoblasts leads to depletion of myoblasts, developmental failure and prenatal lethality. Postnatal deletion of Plk1 in MuSCs does not perturb their quiescence but depletes activated MuSCs as they enter the cell cycle, leading to regenerative failure. The Plk1-null MuSCs are arrested at the M-phase, accumulate DNA damage, and apoptose. Mechanistically, Plk1 deletion upregulates p53, and inhibition of p53 promotes survival of the Plk1-null myoblasts. Pharmacological inhibition of Plk1 similarly inhibits proliferation but promotes differentiation of myoblasts in vitro, and blocks muscle regeneration in vivo. These results reveal for the first time an indispensable role of Plk1 in developmental and regenerative myogenesis.

Local Vibration Stimuli Induce Mechanical Stress-Induced Factors and Facilitate Recovery From Immobilization-Induced Oxidative Myofiber Atrophy in Rats.

Muscle atrophy can be caused by unloading stress such as microgravity environments or cast immobilization. Therapies for such disuse muscle atrophy and their underlying mechanisms are incompletely understood. Here, we investigated the therapeutic effects of local vibration stimulation on immobilization-induced skeletal muscle atrophy. A rat model was made by placing the left hindlimb in a cast for 1 week, leading to oxidative myofiber atrophy without myopathic changes in soleus skeletal muscle. Vibration stimulus (90 Hz, 15 min) to the plantar fascia of the atrophic hindlimb was performed once a day using a hand-held vibration massager after removal of a cast at the end of the immobilization period. After 2 weeks, rats were dissected, and quantitative analysis of the cross-sectional areas of soleus myofibers was performed. The results revealed that vibration induced significant recovery from disuse muscle atrophy, compared with untreated immobilized samples. Furthermore, vibration treatment suppressed the fiber transition from slow to fast fiber types compared with vibration-untreated immobilized samples. Western blotting analyses of mechanical stress-induced factors revealed that the expression of mechano-growth factor (MGF), systemic insulin-like growth factor I, and the mechanotransduction protein, Yes-associated protein 1 (YAP1), was decreased in untreated immobilized soleus muscle, whereas vibration stimulation restored their expression. No change in the level of phosphorylation of YAP1Ser127 was observed, leading to no change in p-YAP1/YAP1 ratio in vibration-treated immobilized soleus muscle. The results indicate that vibration stimulus is effective to restore immobilization-induced inactivation of YAP1 pathway. Phosphorylation of ERK 1/2, but not AKT, was enhanced in vibration-treated immobilized soleus muscle. Furthermore, vibration stimuli restored immobilization-induced downregulation of the paired box transcription factor, PAX7, a critical factor for regenerative myogenesis in muscle satellite cells. Our results indicate that cyclic vibration stimuli are effective in activating satellite cells and facilitate recovery from immobilization-induced oxidative myofiber atrophy through upregulation of MGF and YAP1.

MicroRNA-323-3p promotes myogenesis by targeting Smad2.

Skeletal muscle is an important and complex organ with multiple biological functions in humans and animals. Proliferation and differentiation of myoblasts are the key steps during the development of skeletal muscle. MicroRNA (miRNA) is a class of 21-nucleotide noncoding RNAs regulating gene expression by combining with the 3'-untranslated region of target messenger RNA. Many studies in recent years have suggested that miRNAs play a critical role in myogenesis. Through high-throughput sequencing, we found that miR-323-3p showed significant changes in the longissimus dorsi muscle of Rongchang pigs in different age groups. In this study, we discovered that overexpression of miR-323-3p repressed myoblast proliferation and promoted differentiation, whereas the inhibitor of miR-323-3p displayed the opposite results. Furthermore, we predicted Smad2 as the target gene of miR-323-3p and found that miR-323-3p directly modulated the expression level of Smad2. Then luciferase reporter assays verified that Smad2 was a target gene of miR-323-3p during the differentiation of myoblasts. These findings reveal that miR-323-3p is a positive regulator of myogenesis by targeting Smad2. This provides a novel mechanism of miRNAs in myogenesis.