Group I Paks Promote Skeletal Myoblast Differentiation In Vivo and In Vitro

ABSTRACT Skeletal myogenesis is regulated by signal transduction, but the factors and mechanisms involved are not well understood. The group I Paks Pak1 and Pak2 are related protein kinases and direct effectors of Cdc42 and Rac1. Group I Paks are ubiquitously expressed and specifically required for myoblast fusion in Drosophila. We report that both Pak1 and Pak2 are activated during mammalian myoblast differentiation. One pathway of activation is initiated by N-cadherin ligation and involves the cadherin coreceptor Cdo with its downstream effector, Cdc42. Individual genetic deletion of Pak1 and Pak2 in mice has no overt effect on skeletal muscle development or regeneration. However, combined muscle-specific deletion of Pak1 and Pak2 results in reduced muscle mass and a higher proportion of myofibers with a smaller cross-sectional area. This phenotype is exacerbated after repair to acute injury. Furthermore, primary myoblasts lacking Pak1 and Pak2 display delayed expression of myogenic differentiation markers and myotube formation. These results identify Pak1 and Pak2 as redundant regulators of myoblast differentiation in vitro and in vivo and as components of the promyogenic Ncad/Cdo/Cdc42 signaling pathway.

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EphA7 promotes myogenic differentiation via cell-cell contact

eLife ◽

10.7554/elife.53689 ◽

2020 ◽

Vol 9 ◽

Author(s):

Laura L Arnold ◽

Alessandra Cecchini ◽

Danny A Stark ◽

Jacqueline Ihnat ◽

Rebecca N Craigg ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Muscle Development ◽

Myogenic Differentiation ◽

Cell Contact ◽

Postnatal Life ◽

Differentiation Markers ◽

Hindlimb Muscles

The conversion of proliferating skeletal muscle precursors (myoblasts) to terminally-differentiated myocytes is a critical step in skeletal muscle development and repair. We show that EphA7, a juxtacrine signaling receptor, is expressed on myocytes during embryonic and fetal myogenesis and on nascent myofibers during muscle regeneration in vivo. In EphA7-/- mice, hindlimb muscles possess fewer myofibers at birth, and those myofibers are reduced in size and have fewer myonuclei and reduced overall numbers of precursor cells throughout postnatal life. Adult EphA7-/- mice have reduced numbers of satellite cells and exhibit delayed and protracted muscle regeneration, and satellite cell-derived myogenic cells from EphA7-/- mice are delayed in their expression of differentiation markers in vitro. Exogenous EphA7 extracellular domain will rescue the null phenotype in vitro, and will also enhance commitment to differentiation in WT cells. We propose a model in which EphA7 expression on differentiated myocytes promotes commitment of adjacent myoblasts to terminal differentiation.

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MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation

Nature Communications ◽

10.1038/s41467-019-13598-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

Author(s):

Yu Zhao ◽

Jiajian Zhou ◽

Liangqiang He ◽

Yuying Li ◽

Jie Yuan ◽

...

Keyword(s):

Target Gene ◽

Myogenic Differentiation ◽

Myoblast Differentiation ◽

Enhancer Rna ◽

Super Enhancer ◽

And Function ◽

Nuclear Ribonucleoprotein ◽

Multiple Cells

AbstractEmerging evidence supports roles of enhancer RNAs (eRNAs) in regulating target gene. Here, we study eRNA regulation and function during skeletal myoblast differentiation. We provide a panoramic view of enhancer transcription and categorization of eRNAs. Master transcription factor MyoD is crucial in activating eRNA production. Super enhancer (se) generated seRNA-1 and -2 promote myogenic differentiation in vitro and in vivo. seRNA-1 regulates expression levels of two nearby genes, myoglobin (Mb) and apolipoprotein L6 (Apol6), by binding to heterogeneous nuclear ribonucleoprotein L (hnRNPL). A CAAA tract on seRNA-1 is essential in mediating seRNA-1/hnRNPL binding and function. Disruption of seRNA-1-hnRNPL interaction attenuates Pol II and H3K36me3 deposition at the Mb locus, in coincidence with the reduction of its transcription. Furthermore, analyses of hnRNPL binding transcriptome-wide reveal its association with eRNAs is a general phenomenon in multiple cells. Collectively, we propose that eRNA-hnRNPL interaction represents a mechanism contributing to target mRNA activation.

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The LIM-only protein FHL2 interacts with β-catenin and promotes differentiation of mouse myoblasts

The Journal of Cell Biology ◽

10.1083/jcb.200202075 ◽

2002 ◽

Vol 159 (1) ◽

pp. 113-122 ◽

Cited By ~ 98

Author(s):

Bernd Martin ◽

Richard Schneider ◽

Stefanie Janetzky ◽

Zoe Waibler ◽

Petra Pandur ◽

...

Keyword(s):

Mammalian Cells ◽

Muscle Development ◽

Target Genes ◽

Myogenic Differentiation ◽

Myoblast Differentiation ◽

Lim Domains ◽

Vitro Binding ◽

Specific Proteins

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified β-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of β-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts β-catenin–mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell–specific manner. After injection into Xenopus embryos, FHL2 inhibited the β-catenin–induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with β-catenin.

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Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System

Cells Tissues Organs ◽

10.1159/000443634 ◽

2016 ◽

Vol 202 (3-4) ◽

pp. 143-158 ◽

Cited By ~ 21

Author(s):

Alec S.T. Smith ◽

Samantha L. Passey ◽

Neil R.W. Martin ◽

Darren J. Player ◽

Vivek Mudera ◽

...

Keyword(s):

Skeletal Muscle ◽

Motor Neurons ◽

Muscle Development ◽

Functional Performance ◽

Cell Types ◽

Original Data ◽

Synaptic Proteins ◽

Myoblast Differentiation

Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field.

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KDM4A regulates myogenesis by demethylating H3K9me3 of myogenic regulatory factors

Cell Death and Disease ◽

10.1038/s41419-021-03799-1 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Qi Zhu ◽

Feng Liang ◽

Shufang Cai ◽

Xiaorong Luo ◽

Tianqi Duo ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Proliferation ◽

Satellite Cells ◽

Muscle Development ◽

Kinase Inhibitor ◽

Myogenic Differentiation ◽

Myoblast Differentiation ◽

Proliferation And Differentiation ◽

H3k9me3 Level

AbstractHistone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.

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Hes6 regulates myogenic differentiation

Development ◽

10.1242/dev.129.9.2195 ◽

2002 ◽

Vol 129 (9) ◽

pp. 2195-2207

Author(s):

Judy Cossins ◽

Ann E. Vernon ◽

Yun Zhang ◽

Anna Philpott ◽

Philip H. Jones

Keyword(s):

Protein Interactions ◽

Kinase Inhibitor ◽

Myogenic Differentiation ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

C2c12 Myoblast ◽

Xenopus Embryos ◽

Enhancer Of Split

Hes6 is a basic helix-loop-helix transcription factor homologous to Drosophila Enhancer of Split (EoS) proteins. It is known to promote neural differentiation and to bind to Hes1, a related protein that is part of the Notch signalling pathway, affecting Hes1-regulated transcription. We show that Hes6 is expressed in the murine embryonic myotome and is induced on C2C12 myoblast differentiation in vitro. Hes6 binds DNA containing the Enhancer of Split E box (ESE) motif, the preferred binding site of Drosophila EoS proteins, and represses transcription of an ESE box reporter. When overexpressed in C2C12 cells, Hes6 impairs normal differentiation, causing a decrease in the induction of the cyclin-dependent kinase inhibitor, p21Cip1, and an increase in the number of cells that can be recruited back into the cell cycle after differentiation in culture. In Xenopus embryos, Hes6 is co-expressed with MyoD in early myogenic development. Microinjection of Hes6 RNA in vivo in Xenopus embryos results in an expansion of the myotome, but suppression of terminal muscle differentiation and disruption of somite formation at the tailbud stage. Analysis of Hes6 mutants indicates that the DNA-binding activity of Hes6 is not essential for its myogenic phenotype, but that protein-protein interactions are. Thus, we demonstrate a novel role for Hes6 in multiple stages of muscle formation.

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Abstract 17273: Long Non-Coding RNA Ppp1r1b Regulates Myogenic Differentiation Through Modulating Histone 3 Methylation

Circulation ◽

10.1161/circ.138.suppl_1.17273 ◽

2018 ◽

Vol 138 (Suppl_1) ◽

Author(s):

Xuedong Kang ◽

Yan Zhao ◽

Marlin Touma

Keyword(s):

Histone Modification ◽

Protein Complex ◽

Myogenic Differentiation ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

Mouse Heart ◽

Histone 3

Introduction: Long noncoding RNAs (lncRNAs), emerged as critical epigenetic regulators of transcriptome, play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction. In our work, we identified a novel lncRNA that regulates myogenesis. Hypothesis: LncRNA Ppp1r1b regulates myogenesis by modulating Histone 3 methylation Methods: After treated with antisense oligonucleotides (GapmeR) or siRNA against Ppp1r1b-LncRNA, real time PCR and Western blot analyses were performed to examine the expression of myogenic and sarcomere genes. Chromatin immunoprecipitation (CHIP) was used to comparatively analyze gene specific histone modification level. RNA pull-down was employed to identify the protein molecules that interact with Ppp1r1b-LncRNA. Results: By silencing Ppp1r1b-LncRNA with GapmeR, C2C12, a skeletal myoblast cell line, did not develop fully differentiated myotubes, but tend to remain in a quiescent mono-nucleated status. In vivo analysis of GapmeR injected neonatal mouse heart and in vitro siRNA silenced human skeletal myoblasts further confirmed the important role of Ppp1r1b-LncRNA on myogenesis. Members of the MyoD family of muscle-specific transcription factors (MyoD and myogenin) failed to be up-regulated during myogenic differentiation when treated with Ppp1r1b-LncRNA specific GapmeR or siRNA. Key proteins essential for establishing and maintaining normal skeletal muscle architecture, including Tcap and Dystropnin, are also suppressed in Ppp1r1b LncRNA- deficient heart. Analysis of histone modification levels at Myogenin, MyoD1 and Tcap in C2C12 cells revealed more histone tri-methylation at these myogenic and sarcomere structural genes compared to untreated cells. Additional lncRNA- protein complex isolation has further revealed insight into the biological roles of Ppp1r1b-LncRNA. Conclusions: Our results support the role of Ppp1r1b-LncRNA in promoting myogenic differentiation. Ppp1r1b-lncRNA function is mediated by inhibiting histone methylation on promoters of multiple myogenic and sarcomere genes. In particular, the identification of EZH2 in pulled Pp1r1b LncRNA: protein complex implies that Polycomb repressive complex 2 (PRC2) is involved in Ppp1r1b-LncRNA modulated myoblast differentiation.

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Lactate Promotes Myoblast Differentiation and Myotube Hypertrophy via a Pathway Involving MyoD In Vitro and Enhances Muscle Regeneration In Vivo

International Journal of Molecular Sciences ◽

10.3390/ijms19113649 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3649 ◽

Cited By ~ 14

Author(s):

Sakuka Tsukamoto ◽

Ayako Shibasaki ◽

Ayano Naka ◽

Hazuki Saito ◽

Kaoruko Iida

Keyword(s):

Gene Expression ◽

Myogenic Differentiation ◽

Underlying Mechanism ◽

Myoblast Differentiation ◽

Dependent Manner ◽

Gene And Protein Expression ◽

Myotube Hypertrophy ◽

Myod Gene

Lactate is a metabolic substrate mainly produced in muscles, especially during exercise. Recently, it was reported that lactate affects myoblast differentiation; however, the obtained results are inconsistent and the in vivo effect of lactate remains unclear. Our study thus aimed to evaluate the effects of lactate on myogenic differentiation and its underlying mechanism. The differentiation of C2C12 murine myogenic cells was accelerated in the presence of lactate and, consequently, myotube hypertrophy was achieved. Gene expression analysis of myogenic regulatory factors showed significantly increased myogenic determination protein (MyoD) gene expression in lactate-treated cells compared with that in untreated ones. Moreover, lactate enhanced gene and protein expression of myosin heavy chain (MHC). In particular, lactate increased gene expression of specific MHC isotypes, MHCIIb and IId/x, in a dose-dependent manner. Using a reporter assay, we showed that lactate increased promoter activity of the MHCIIb gene and that a MyoD binding site in the promoter region was necessary for the lactate-induced increase in activity. Finally, peritoneal injection of lactate in mice resulted in enhanced regeneration and fiber hypertrophy in glycerol-induced regenerating muscles. In conclusion, physiologically high lactate concentrations modulated muscle differentiation by regulating MyoD-associated networks, thereby enhancing MHC expression and myotube hypertrophy in vitro and, potentially, in vivo.

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Exogenous Expression of an Alternative Splicing Variant of Myostatin Prompts Leg Muscle Fiber Hyperplasia in Japanese Quail

International Journal of Molecular Sciences ◽

10.3390/ijms20184617 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4617 ◽

Cited By ~ 1

Author(s):

Paula Renee Chen ◽

Yeunsu Suh ◽

Sangsu Shin ◽

Rachel Marie Woodfint ◽

Seongsoo Hwang ◽

...

Keyword(s):

Skeletal Muscle ◽

Alternative Splicing ◽

Japanese Quail ◽

Muscle Development ◽

Pectoralis Major Muscle ◽

Muscle Growth ◽

Cross Sectional ◽

Proliferation And Differentiation

Myostatin (MSTN) negatively regulates muscle growth and development through inhibiting myoblast proliferation and differentiation. Five alternative splicing isoforms of MSTN (MSTN-A to MSTN-E) have been discovered in domestic avian species. MSTN-A has high expression in skeletal muscle and encodes the full-length peptide with anti-myogenic activity. Another isoform, MSTN-B, is also highly expressed in skeletal muscle and encodes a truncated peptide that has pro-myogenic capabilities in vitro, which include promoting the proliferation and differentiation of quail muscle precursor cells. The objective of this study was to investigate overexpression of MSTN-B in vivo by using two independent lines of transgenic Japanese quail with expression directed in the skeletal muscle. Unexpectedly, the chicken skeletal muscle alpha actin 1 (cACTA1) promoter resulted in restricted exogenous MSTN-B protein expression to certain skeletal muscles, such as the gastrocnemius and tibialis anterior, but not the pectoralis major muscle. Gastrocnemius weight as a percentage of body weight in transgenic quail was increased compared to non-transgenic quail at posthatch day 21 (D21) and posthatch D42. An increase in the size of the gastrocnemius in transgenic quail was attributed to an increase in fiber number but not fiber cross-sectional area (CSA). During embryonic development, paired box 7 (PAX7) expression was prolonged in the transgenic embryos, but other myogenic regulatory factors (MRFs) were unchanged after MSTN-B overexpression. Taken together, these data provide novel insights into the regulation of skeletal muscle development by alternative splicing mechanisms in avians.

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Selection of a Dominant Negative Retinoblastoma Protein (RB) Inhibiting Satellite Myoblast Differentiation Implies an Indirect Interaction between MyoD and RB

Molecular and Cellular Biology ◽

10.1128/mcb.20.14.5129-5139.2000 ◽

2000 ◽

Vol 20 (14) ◽

pp. 5129-5139 ◽

Cited By ~ 20

Author(s):

Feng-Qian Li ◽

Archie Coonrod ◽

Marshall Horwitz

Keyword(s):

Myogenic Differentiation ◽

Retinoblastoma Protein ◽

Dominant Negative ◽

Myoblast Differentiation ◽

Loss Of Function ◽

Lysosomal Protease ◽

Dominant Negative Form ◽

Genes Encoding

ABSTRACT Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but the genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interrupting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosomal protease cathepsin B (CB), such that the fusion protein intracellularly diverts interacting factors to the lysosome. Among other gene fragments selected in this screen, including those of known and novel sequence, is the retinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indistinguishable from recessive loss of function RB mutations. The fact that the dominant negative RB inhibits myogenic differentiation in the presence of nonlimiting concentrations of either RB or MyoD suggests that these two proteins do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore, E2F1 is a potential mediator of the dominant negative inhibition of MyoD by CB-RB during satellite cell differentiation. We propose this approach to be generally suited to the investigation of gene function, even when little is known about the pathway being studied.

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