bims-muscge 2023-10-22 papers

bims-muscge

Biomed News

on Muscle stem cells and gene therapy

Issue of 2023–10–22
twenty-six papers selected by
Chance Bowman, Dartmouth College

Porous biomaterial scaffolds for skeletal muscle tissue engineering.
Myopathologic trajectory in Duchenne muscular dystrophy (DMD) reveals lack of regeneration due to senescence in satellite cells.
Cellular pathogenesis of Duchenne muscular dystrophy: progressive myofibre degeneration, chronic inflammation, reactive myofibrosis and satellite cell dysfunction.
Intravital microscopy of satellite cell dynamics and their interaction with myeloid cells during skeletal muscle regeneration.
Morpholino oligonucleotide-mediated exon skipping for DMD treatment: Past insights, present challenges and future perspectives.
The novel RNA-RNA activation of H19 on MyoD transcripts promoting myogenic differentiation of goat muscle satellite cells.
Impact of Human Recombinant Irisin on Tissue-Engineered Skeletal Muscle Structure and Function.
Targeting neuromuscular junction to treat neuromuscular disorders.
Exon 44 skipping in Duchenne muscular dystrophy: NS-089/NCNP-02, a dual-targeting antisense oligonucleotide.
Overcoming muscle stem cell aging.
Hypochlorous acid exposure impairs skeletal muscle function and Ca2+ signalling: implications for Duchenne muscular dystrophy pathology.
Klotho regulates the myogenic response of muscle to mechanical loading and exercise.
Matrix Nonlinear Viscoelasticity Regulates Skeletal Myogenesis through MRTF Nuclear Localization and Nuclear Mechanotransduction.
Inherited myogenic abilities in muscle precursor cells defined by the mitochondrial complex I-encoding protein.
Traumatic muscle injury.
Traumatic muscle injury.
Reprogramming of cis-regulatory networks during skeletal muscle atrophy in male mice.
Current and emerging targeted therapies for spinal muscular atrophy.
Modeling Human Paraxial Mesoderm Development with Pluripotent Stem Cells.
Mechanism of Skeletal Muscle Atrophy by Muscle Fiber Types in Male Rats under Long-term Fasting Stress.
Developing a cell-microcarrier tissue engineered product for muscle repair using a bioreactor system.
CRISPR-Cas9 delivery strategies with engineered extracellular vesicles.
FSHD muscle shows perturbation in fibroadipogenic progenitor cells, mitochondrial function and alternative splicing independently of inflammation.
Decreasing predictable DNA off-target effects and narrowing editing windows of adenine base editors by fusing human Rad18 protein variant.
TEAD4 antagonizes cellular senescence by remodeling chromatin accessibility at enhancer regions.
Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells.

Front Bioeng Biotechnol. 2023 ;11 1245897

Porous biomaterial scaffolds for skeletal muscle tissue engineering.

Natalie G Kozan, Mrunmayi Joshi, Sydnee T Sicherer, Jonathan M Grasman.

  Volumetric muscle loss is a traumatic injury which overwhelms the innate repair mechanisms of skeletal muscle and results in significant loss of muscle functionality. Tissue engineering seeks to regenerate these injuries through implantation of biomaterial scaffolds to encourage endogenous tissue formation and to restore mechanical function. Many types of scaffolds are currently being researched for this purpose. Scaffolds are typically made from either natural, synthetic, or conductive polymers, or any combination therein. A major criterion for the use of scaffolds for skeletal muscle is their porosity, which is essential for myoblast infiltration and myofiber ingrowth. In this review, we summarize the various methods of fabricating porous biomaterial scaffolds for skeletal muscle regeneration, as well as the various types of materials used to make these scaffolds. We provide guidelines for the fabrication of scaffolds based on functional requirements of skeletal muscle tissue, and discuss the general state of the field for skeletal muscle tissue engineering.

Keywords:  biomaterial; porosity; scaffold; skeletal muscle; skeletal muscle tissue engineering; tissue engineering; volumetric muscle loss

DOI:  https://doi.org/10.3389/fbioe.2023.1245897
Acta Neuropathol Commun. 2023 Oct 19. 11(1): 167

Myopathologic trajectory in Duchenne muscular dystrophy (DMD) reveals lack of regeneration due to senescence in satellite cells.

Nastasia Cardone, Valentina Taglietti, Serena Baratto, Kaouthar Kefi, Baptiste Periou, Ciryl Gitiaux, Christine Barnerias, Peggy Lafuste, France Leturcq Pharm, Juliette Nectoux Pharm, Chiara Panicucci, Isabelle Desguerre, Claudio Bruno, François-Jerome Authier, Chiara Fiorillo, Frederic Relaix, Edoardo Malfatti.

  Duchenne muscular dystrophy (DMD) is a devastating X-linked muscular disease, caused by mutations in the DMD gene encoding Dystrophin and affecting 1:5000 boys worldwide. Lack of Dystrophin leads to progressive muscle wasting and degeneration resulting in cardiorespiratory failure. Despite the absence of a definitive cure, innovative therapeutic avenues are emerging. Myopathologic studies are important to further understand the biological mechanisms of the disease and to identify histopathologic benchmarks for clinical evaluations. We conducted a myopathologic analysis on twenty-four muscle biopsies from DMD patients, with particular emphasis on regeneration, fibro-adipogenic progenitors and muscle stem cells behavior. We describe an increase in content of fibro-adipogenic progenitors, central orchestrators of fibrotic progression and lipid deposition, concurrently with a decline in muscle regenerative capacity. This regenerative impairment strongly correlates with compromised activation and expansion of muscle stem cells. Furthermore, our study uncovers an early acquisition of a senescence phenotype by DMD-afflicted muscle stem cells. Here we describe the myopathologic trajectory intrinsic to DMD and establish muscle stem cell senescence as a pivotal readout for future therapeutic interventions.

Keywords:  Cellular senescence; Duchenne muscular dystrophy; FAPs; Fibrosis; Muscle regeneration

DOI:  https://doi.org/10.1186/s40478-023-01657-z
Eur J Transl Myol. 2023 10 16.

Cellular pathogenesis of Duchenne muscular dystrophy: progressive myofibre degeneration, chronic inflammation, reactive myofibrosis and satellite cell dysfunction.

Paul Dowling, Dieter Swandulla, Kay Ohlendieck.

Duchenne muscular dystrophy is a highly progressive muscle wasting disease of early childhood and characterized by complex pathophysiological and histopathological changes in the voluntary contractile system, including myonecrosis, chronic inflammation, fat substitution and reactive myofibrosis. The continued loss of functional myofibres and replacement with non-contractile cells, as well as extensive tissue scarring and decline in tissue elasticity, leads to severe skeletal muscle weakness. In addition, dystrophic muscles exhibit a greatly diminished regenerative capacity to counteract the ongoing process of fibre degeneration. In normal muscle tissues, an abundant stem cell pool consisting of satellite cells that are localized between the sarcolemma and basal lamina, provides a rich source for the production of activated myogenic progenitor cells that are involved in efficient myofibre repair and tissue regeneration. Interestingly, the self-renewal of satellite cells for maintaining an essential pool of stem cells in matured skeletal muscles is increased in dystrophin-deficient fibres. However, satellite cell hyperplasia does not result in efficient recovery of dystrophic muscles due to impaired asymmetric cell divisions. The lack of expression of the full-length dystrophin isoform Dp427-M, which is due to primary defects in the DMD gene, appears to affect key regulators of satellite cell polarity causing a reduced differentiation of myogenic progenitors, which are essential for myofibre regeneration. This review outlines the complexity of dystrophinopathy and describes the importance of the pathophysiological role of satellite cell dysfunction. A brief discussion of the bioanalytical usefulness of single cell proteomics for future studies of satellite cell biology is provided.

DOI: https://doi.org/10.4081/ejtm.2023.11856
Sci Adv. 2023 Oct 20. 9(42): eadi1891

Intravital microscopy of satellite cell dynamics and their interaction with myeloid cells during skeletal muscle regeneration.

Yingzhu He, Youshan Heng, Zhongya Qin, Xiuqing Wei, Zhenguo Wu, Jianan Qu.

Skeletal muscle regeneration requires the highly coordinated cooperation of muscle satellite cells (MuSCs) with other cellular components. Upon injury, myeloid cells populate the wound site, concomitant with MuSC activation. However, detailed analysis of MuSC-myeloid cell interaction is hindered by the lack of suitable live animal imaging technology. Here, we developed a dual-laser multimodal nonlinear optical microscope platform to study the dynamics of MuSCs and their interaction with nonmyogenic cells during muscle regeneration. Using three-dimensional time-lapse imaging on live reporter mice and taking advantages of the autofluorescence of reduced nicotinamide adenine dinucleotide (NADH), we studied the spatiotemporal interaction between nonmyogenic cells and muscle stem/progenitor cells during MuSC activation and proliferation. We discovered that their cell-cell contact was transient in nature. Moreover, MuSCs could activate with notably reduced infiltration of neutrophils and macrophages, and their proliferation, although dependent on macrophages, did not require constant contact with them. These findings provide a fresh perspective on myeloid cells' role during muscle regeneration.

DOI: https://doi.org/10.1126/sciadv.adi1891
J Biosci. 2023 ;pii: 38. [Epub ahead of print]48

Morpholino oligonucleotide-mediated exon skipping for DMD treatment: Past insights, present challenges and future perspectives.

Shalini Gupta, Swrajit Nath Sharma, Jayanta Kundu, Sankha Pattanayak, Surajit Sinha.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease primarily affecting boys causing loss of the dystrophin protein, ultimately leading to muscle wastage and death by cardiac or respiratory failure. The genetic mutation involved can be overcome with antisense oligonucleotides which bind to a pre-mRNA and results in reading frame restoration by exon skipping. Phosphorodiamidate morpholino oligonucleotides (PMOs) are a class of antisense agents with a neutral backbone derived from RNA which can induce effective exon skipping. In this review, the evolution of PMOs in exon skipping therapy for the last two decades has been detailed with the gradual structural and functional advancements. Even though the success rate of PMObased therapy has been high with four FDA approved drugs, several key challenges are yet to overcome, one being the dystrophin restoration in cardiac muscle. The current scenario in further improvement of PMOs has been discussed along with the future perspectives that have the potential to revolutionize the therapeutic benefits in DMD.
Int J Biol Macromol. 2023 Oct 16. pii: S0141-8130(23)04238-1. [Epub ahead of print]253(Pt 7): 127341

The novel RNA-RNA activation of H19 on MyoD transcripts promoting myogenic differentiation of goat muscle satellite cells.

Li Li, Chenyu Qin, Yuan Chen, Wei Zhao, Qi Zhu, Dinghui Dai, Siyuan Zhan, Jiazhong Guo, Tao Zhong, Linjie Wang, Jiaxue Cao, Hongping Zhang.

  The elaborate interplay of coding and noncoding factors governs muscle growth and development. Here, we reported a mutual activation between long noncoding RNA (lncRNA) H19 and MyoD (myogenic determination gene number 1) in the muscle process. We successfully cloned the two isoforms of goat H19, which were significantly enriched and positively correlated with MyoD transcripts in skeletal muscles or differentiating muscle satellite cells (MuSCs). To systematically screen genes altered by H19, we performed RNA-seq using cDNA libraries of differentiating H19-deficiency MuSCs and consequently anchored MyoD as the critical genes in mediating H19 function. Intriguingly, some transcripts of MyoD and H19 overlapped in the cytoplasm, which was dramatically damaged when the core complementary nucleotides were mutated. Meanwhile, MyoD RNA successfully pulled down H19 in MS2-RIP experiments. Furthermore, HuR could bind both H19 and MyoD transcripts, while H19 or its truncated mutants successfully stabilized MyoD mRNA, with or without HuR deficiency. In turn, novel functional MyoD protein-binding sites were identified in the promoter and exons of the H19 gene. Our results suggest that MyoD activates H19 transcriptionally, and RNA-RNA hybridization is critical for H19-promoted MyoD expression, which extends our knowledge of the hierarchy of regulatory networks in muscle growth.

Keywords:  Long noncoding RNA H19; Myogenic differentiation 1 (MyoD); RNA-RNA interaction; Skeletal muscle satellite cells (MuSCs)

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.127341
Tissue Eng Part A. 2023 Oct 16.

Impact of Human Recombinant Irisin on Tissue-Engineered Skeletal Muscle Structure and Function.

Matthew Hung Nguyen, Christopher Stephen Kennedy, Olga Maria Wroblewski, Eileen Yuhan Su, Derek Hwang, Lisa Marie Larkin.

Tissue engineering of exogenous skeletal muscle units (SMUs) through isolation of muscle satellite cells from muscle biopsies is a potential treatment method for acute volumetric muscle loss (VML). A current issue with this treatment process is the limited capacity for muscle stem cell (satellite cell) expansion in cell culture, resulting in a decreased ability to obtain enough cells to fabricate SMUs of appropriate size and structural quality and that produce native levels of contractile force. This study determined the impact of human recombinant irisin on the growth and development of three-dimensional (3D) engineered skeletal muscle. Muscle satellite cells were cultured without irisin (control), or with either 50 ng/ml, 100 ng/ml, or 250 ng/ml of irisin supplementation. Light microscopy was used to analyze myotube formation with particular focus placed on the diameter and density of the monotubes during growth of the 3D SMU. Following the formation of 3D constructs, SMUs underwent measurement of maximum tetanic force to analyze contractile function as well as immunohistochemical staining to characterize muscle structure. The results indicate that irisin supplementation with 250 ng/ml significantly increased the average diameter of myotubes and increased the proliferation and differentiation of myoblasts in culture but did not have a consistent significant impact on force production. In conclusion, supplementation with 250 ng/ml of human recombinant irisin promotes the proliferation and differentiation of myotubes and has the potential for impacting contractile force production in scaffold-free tissue-engineered skeletal muscle .

DOI: https://doi.org/10.1089/ten.TEA.2023.0187
Life Sci. 2023 Oct 17. pii: S0024-3205(23)00821-4. [Epub ahead of print]333 122186

Targeting neuromuscular junction to treat neuromuscular disorders.

Rizwan Qaisar.

  The integrity and preservation of the neuromuscular junction (NMJ), the interface between the motor neuron and skeletal muscle, is critical for maintaining a healthy skeletal muscle. The structural and/or functional defects in the three cellular components of NMJ, namely the pre-synaptic terminal, synaptic cleft, and post-synaptic region, negatively affect skeletal muscle mass and/or strength. Therefore, NMJ repair appears to be an appropriate therapy for muscle disorders. Mouse models provide a detailed molecular characterization of various cellular components of NMJ with relevance to human diseases. This review discusses different molecular targets on the three cellular components of NMJ for treating muscle diseases. The potential effects of these therapies on NMJ morphology and motor performance, their therapeutic efficacy, and clinical relevance are discussed. Collectively, the available data supports targeting NMJ alone or as an adjunct therapy in treating muscle disorders. However, the potential impact of such interventions on human patients with muscle disorders requires further investigation.

Keywords:  Chronic obstructive pulmonary disease; Muscle wasting; Neuromuscular junction

DOI:  https://doi.org/10.1016/j.lfs.2023.122186
Mol Ther Nucleic Acids. 2023 Dec 12. 34 102034

Exon 44 skipping in Duchenne muscular dystrophy: NS-089/NCNP-02, a dual-targeting antisense oligonucleotide.

Naoki Watanabe, Yuichiro Tone, Tetsuya Nagata, Satoru Masuda, Takashi Saito, Norio Motohashi, Kazuchika Takagaki, Yoshitsugu Aoki, Shin'ichi Takeda.

  Exon-skipping therapy mediated by antisense oligonucleotides is expected to provide a therapeutic option for Duchenne muscular dystrophy. Antisense oligonucleotides for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated antisense oligonucleotides for exon 44 skipping (applicable to approximately 6% of all Duchenne muscular dystrophy patients) to improve activity by using a novel antisense oligonucleotide design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to target two splicing regulators in exon 44 simultaneously, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a Duchenne muscular dystrophy patient to whom exon 44 skipping is applicable. We also assessed the in vivo activity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an antisense oligonucleotide with a novel connected-sequence design, showed highly efficient exon skipping both in vitro and in vivo.

Keywords:  Duchenne muscular dystrophy; MT: Oligonucleotides: Therapies and Applications; NS-089/NCNP-02; antisense oligonucleotide; antisense therapeutics; dual-targeting; dystrophin; exon 44; exon skipping; morpholino; splice modulation

DOI:  https://doi.org/10.1016/j.omtn.2023.102034
Curr Opin Genet Dev. 2023 Oct 13. pii: S0959-437X(23)00107-7. [Epub ahead of print]83 102127

Overcoming muscle stem cell aging.

Sebastian Memczak, Juan Ci Belmonte.

Reduced muscle strength and mass is one of the hallmarks of physiological aging in humans and can result in severe impairment of the quality of life. In part this is caused by a functional loss of the highly specialized muscle stem cells (MuSCs), which in healthy conditions provide maintenance, growth, and regeneration. Recent progress in understanding of the stem cell niche and results from single cell technologies reveal exciting insights at unprecedented detail into MuSCs and muscle biology during aging. Here, we review this field and discuss the implications of current findings with a focus on cellular reprogramming approaches as a novel therapeutic avenue for age-related muscle decline.

DOI: https://doi.org/10.1016/j.gde.2023.102127
J Physiol. 2023 Oct 21.

Hypochlorous acid exposure impairs skeletal muscle function and Ca2+ signalling: implications for Duchenne muscular dystrophy pathology.

Thomas A Lea, Peter M Panizza, Peter G Arthur, Anthony J Bakker, Gavin J Pinniger.

  Duchenne muscular dystrophy (DMD) is a fatal X-linked disease characterised by severe muscle wasting. The mechanisms underlying the DMD pathology likely involve the interaction between inflammation, oxidative stress and impaired Ca2+ signalling. Hypochlorous acid (HOCl) is a highly reactive oxidant produced endogenously via myeloperoxidase; an enzyme secreted by neutrophils that is significantly elevated in dystrophic muscle. Oxidation of Ca2+ -handling proteins by HOCl may impair Ca2+ signalling. This study aimed to determine the effects of HOCl on skeletal muscle function and its potential contribution to the dystrophic pathology. Extensor digitorum longus (EDL), soleus and interosseous muscles were surgically isolated from anaesthetised C57 (wild-type) and mdx (dystrophic) mice for measurement of ex vivo force production and intracellular Ca2+ concentration. In whole EDL muscle, HOCl (200 μM) significantly decreased maximal force and increased resting muscle tension which was only partially reversible by dithiothreitol. The effects of HOCl (200 μM) on maximal force in slow-twitch soleus were lower than found in the fast-twitch EDL muscle. In single interosseous myofibres, HOCl (10 μM) significantly increased resting intracellular Ca2+ concentration and decreased Ca2+ transient amplitude. These effects of HOCl were reduced by the application of tetracaine, Gd3+ or streptomycin, implicating involvement of ryanodine receptors and transient receptor potential channels. These results demonstrate the potent effects of HOCl on skeletal muscle function potentially mediated by HOCl-induced oxidation to Ca2+ signalling proteins. Hence, HOCl may provide a link between chronic inflammation, oxidative stress and impaired Ca2+ handling that is characteristic of DMD and presents a potential therapeutic target for DMD. KEY POINTS: Duchenne muscular dystrophy is a fatal genetic disease with pathological mechanisms which involve the complex interaction of chronic inflammation, increased reactive oxygen species production and increased cytosolic Ca2+ concentrations. Hypochlorous acid can be endogenously produced by neutrophils via the enzyme myeloperoxidase. Both neutrophil and myeloperoxidase activity are increased in dystrophic mice. This study found that hypochlorous acid decreased muscle force production and increased cytosolic Ca2+ concentrations in isolated muscles from wild-type and dystrophic mice at relatively low concentrations of hypochlorous acid. These results indicate that hypochlorous acid may be key in the Duchenne muscular dystrophy disease pathology and may provide a unifying link between the chronic inflammation, increased reactive oxygen species production and increased cytosolic Ca2+ concentrations observed in Duchenne muscular dystrophy. Hypochlorous acid production may be a potential target for therapeutic treatments of Duchenne muscular dystrophy.

Keywords:  Ca2+; Duchenne muscular dystrophy; hypochlorous acid; inflammation; myeloperoxidase; neutrophils; reactive oxygen species; skeletal muscle

DOI:  https://doi.org/10.1113/JP285263
Exp Physiol. 2023 Oct 20.

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

Eisuke Ochi, Alice Barrington, Michelle Wehling-Henricks, Marcus Avila, Makoto Kuro-O, James G Tidball.

   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.

Keywords:  Klotho; Wnt; exercise; myogenesis; satellite cell; skeletal muscle

DOI:  https://doi.org/10.1113/EP091263
Small. 2023 Oct 17. e2305218

Matrix Nonlinear Viscoelasticity Regulates Skeletal Myogenesis through MRTF Nuclear Localization and Nuclear Mechanotransduction.

Nianyuan Shi, Jing Wang, Shaoxin Tang, Hui Zhang, Zhao Wei, Ang Li, Yufei Ma, Feng Xu.

  Mechanically sensitive tissues (e.g., skeletal muscles) greatly need mechanical stimuli during the development and maturation. The extracellular matrix (ECM) mediates these signals through nonlinear viscoelasticity of collagen networks that are predominant components of the ECM. However, the interactions between cells and ECM form a feedback loop, and it has not yet been possible to determine the degree to which, if any, of the features of matrix nonlinear viscoelasticity affect skeletal muscle development and regeneration. In this study, a nonlinear viscoelastic feature (i.e., strain-enhanced stress relaxation (SESR)) in normal skeletal muscles is observed, which however is almost absent in diseased muscles from Duchenne muscular dystrophy mice. It is recapitulated such SESR feature in vitro and separated the effects of mechanical strain and ECM viscoelasticity on myoblast response by developing a collagen-based hydrogel platform. Both strain and stress relaxation induce myogenic differentiation and myotube formation by C2C12 myoblasts, and myogenesis is more promoted by applying SESR. This promotion can be explained by the effects of SESR on actin polymerization-mediated myocardin related transcription factor (MRTF) nuclear localization and nuclear mechanotransduction. This study represents the first attempt to investigate the SESR phenomenon in skeletal muscles and reveal underlying mechanobiology, which will provide new opportunities for the tissue injury treatments.

Keywords:  collagen; myogenesis; nuclear mechanotransduction; strain-dependent viscoelasticity

DOI:  https://doi.org/10.1002/smll.202305218
Cell Death Dis. 2023 Oct 19. 14(10): 689

Inherited myogenic abilities in muscle precursor cells defined by the mitochondrial complex I-encoding protein.

Norio Motohashi, Katsura Minegishi, Yoshitsugu Aoki.

Skeletal muscle comprises different muscle fibers, including slow- and fast-type muscles, and satellite cells (SCs), which exist in individual muscle fibers and possess different myogenic properties. Previously, we reported that myoblasts (MBs) from slow-type enriched soleus (SOL) had a high potential to self-renew compared with cells derived from fast-type enriched tibialis anterior (TA). However, whether the functionality of myogenic cells in adult muscles is attributed to the muscle fiber in which they reside and whether the characteristics of myogenic cells derived from slow- and fast-type fibers can be distinguished at the genetic level remain unknown. Global gene expression analysis revealed that the myogenic potential of MBs was independent of the muscle fiber type they reside in but dependent on the region of muscles they are derived from. Thus, in this study, proteomic analysis was conducted to clarify the molecular differences between MBs derived from TA and SOL. NADH dehydrogenase (ubiquinone) iron-sulfur protein 8 (Ndufs8), a subunit of NADH dehydrogenase in mitochondrial complex I, significantly increased in SOL-derived MBs compared with that in TA-derived cells. Moreover, the expression level of Ndufs8 in MBs significantly decreased with age. Gain- and loss-of-function experiments revealed that Ndufs8 expression in MBs promoted differentiation, self-renewal, and apoptosis resistance. In particular, Ndufs8 suppression in MBs increased p53 acetylation, followed by a decline in NAD/NADH ratio. Nicotinamide mononucleotide treatment, which restores the intracellular NAD+ level, could decrease p53 acetylation and increase myogenic cell self-renewal ability in vivo. These results suggested that the functional differences in MBs derived from SOL and TA governed by the mitochondrial complex I-encoding gene reflect the magnitude of the decline in SC number observed with aging, indicating that the replenishment of NAD+ is a possible approach for improving impaired cellular functions caused by aging or diseases.

DOI: https://doi.org/10.1038/s41419-023-06192-2
Nat Rev Dis Primers. 2023 Oct 19. 9(1): 57

Traumatic muscle injury.

DOI: https://doi.org/10.1038/s41572-023-00473-y
Nat Rev Dis Primers. 2023 Oct 19. 9(1): 56

Traumatic muscle injury.

Pascal Edouard, Gustaaf Reurink, Abigail L Mackey, Richard L Lieber, Tania Pizzari, Tero A H Järvinen, Thomas Gronwald, Karsten Hollander.

Traumatic muscle injury represents a collection of skeletal muscle pathologies caused by trauma to the muscle tissue and is defined as damage to the muscle tissue that can result in a functional deficit. Traumatic muscle injury can affect people across the lifespan and can result from high stresses and strains to skeletal muscle tissue, often due to muscle activation while the muscle is lengthening, resulting in indirect and non-contact muscle injuries (strains or ruptures), or from external impact, resulting in direct muscle injuries (contusion or laceration). At a microscopic level, muscle fibres can repair focal damage but must be completely regenerated after full myofibre necrosis. The diagnosis of muscle injury is based on patient history and physical examination. Imaging may be indicated to eliminate differential diagnoses. The management of muscle injury has changed within the past 5 years from initial rest, immobilization and (over)protection to early activation and progressive loading using an active approach. One challenge of muscle injury management is that numerous medical treatment options, such as medications and injections, are often used or proposed to try to accelerate muscle recovery despite very limited efficacy evidence. Another challenge is the prevention of muscle injury owing to the multifactorial and complex nature of this injury.

DOI: https://doi.org/10.1038/s41572-023-00469-8
Nat Commun. 2023 Oct 18. 14(1): 6581

Reprogramming of cis-regulatory networks during skeletal muscle atrophy in male mice.

Hongchun Lin, Hui Peng, Yuxiang Sun, Meijun Si, Jiao Wu, Yanlin Wang, Sandhya S Thomas, Zheng Sun, Zhaoyong Hu.

A comprehensive atlas of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of cellular structure maintenance, metabolism, and responses to the environment. Here we show, using matched single-nucleus chromatin accessibility and RNA-sequencing from juvenile male C57BL6 mice, an atlas of accessible chromatin regions in both normal and denervated skeletal muscles. We identified cell-type-specific cis-regulatory networks, highlighting the dynamic regulatory circuits mediating transitions between myonuclear types. Through comparison of normal and perturbed muscle, we delineated the reprogramming of cis-regulatory networks in response to denervation, described the interplay of promoters/enhancers and target genes. We further unveil a hierarchical structure of transcription factors that delineate a regulatory network in atrophic muscle, identifying ELK4 as a key atrophy-related transcription factor that instigates muscle atrophy through TGF-β1 regulation. This study furnishes a rich genomic resource, essential for decoding the regulatory dynamics of skeletal muscle in both physiological and pathological states.

DOI: https://doi.org/10.1038/s41467-023-42313-3
Expert Rev Neurother. 2023 Oct 16. 1-11

Current and emerging targeted therapies for spinal muscular atrophy.

Yuliya Lakhina, Nicholas M Boulis, Anthony Donsante.

   INTRODUCTION: Spinal muscular atrophy (SMA) is a progressive neurodegenerative disorder caused by insufficiency or total absence of the survival motor neuron protein due to a mutation in the SMN1 gene. The copy number of its paralog, SMN2, influences disease onset and phenotype severity. Current therapeutic approaches include viral and non-viral modalities affecting gene expression. Regulatory-approved drugs Spinraza (Nusinersen), Zolgensma (Onasemnogene abeparvovec), and Evrysdi (Risdiplam) are still being investigated during clinical trials and show benefits in the long-term for symptomatic and pre-symptomatic patients. However, some ongoing interventions require repeated drug administration.
AREAS COVERED: In this review, the authors describe the existing therapy based on point of application, focusing on recent clinical trials of antisense oligonucleotides, viral gene therapy, and splice modulators and thepotential routes for correcting the mutation to provide therapeutic levels of SMN protein.
EXPERT OPINION: In the opinion of the authors, multiple treatment options for patients with SMA shifted the treatment paradigm from palliative supportive care to improvedmotor function, increased survival, and greater quality of life for such patients. They further believe that the future in SMA treatment development lies incombining existing treatment options, targeting aspects of the disease refractory to these treatments, and using gene editing technologies.

Keywords:  Base editors; SMN1; nusinersen; onasmogene abeparvovec; risdiplam; spinal muscular atrophy

DOI:  https://doi.org/10.1080/14737175.2023.2268276
Methods Mol Biol. 2023 Oct 17.

Modeling Human Paraxial Mesoderm Development with Pluripotent Stem Cells.

Yuchuan Miao, Margarete Diaz-Cuadros, Olivier Pourquié.

  Paraxial mesoderm in the early embryo is segmented into epithelial blocks called somites that establish the metameric organization of the vertebrate body plan. Somites are sequentially formed from head to tail in a rhythmic manner controlled by an oscillating gene regulatory network known as the segmentation clock. We know very little about this important process during human development due to limited access to human embryos and ethical concerns. To bypass these difficulties, model systems derived from human pluripotent stem cells have been established. Here, we detail three protocols modeling different aspects of human paraxial mesoderm development in vitro: a 2D cell monolayer system recapitulating dynamics of the human segmentation clock, a 3D organoid system called "somitoid" supporting the simultaneous formation of somite-like structures, and another organoid system called "segmentoid" reconstituting in vivo-like hallmarks of somitogenesis. Together, these complementary model systems provide an excellent platform to decode somitogenesis and advance human developmental biology.

Keywords:  Directed differentiation; Human development; Organoid; Paraxial mesoderm; Pluripotent stem cell; Somite; Somitogenesis; The segmentation clock

DOI:  https://doi.org/10.1007/7651_2023_507
Steroids. 2023 Oct 18. pii: S0039-128X(23)00156-3. [Epub ahead of print] 109328

Mechanism of Skeletal Muscle Atrophy by Muscle Fiber Types in Male Rats under Long-term Fasting Stress.

Takahiro Ieko, Jumpei Fujiki, Yasuhiro Hasegawa, Tomohito Iwasaki, Hidetomo Iwano, Naoyuki Maeda.

  Fasting induces metabolic changes in muscles, which are differentiated by muscle fiber type. In this study, the mechanism of fasting-induced muscle atrophy in rats was examined to determine the differences between muscle fiber types in energy production. Fasting for 96 h did not alter the weight of the soleus (SOL), a fiber type I muscle, but did significantly reduce the weight of gastrocnemius (GM), a fiber type II muscle. GM, SOL and blood pregnenolone and testosterone levels decreased under fasting, which induced energy deprivation, whereas corticosterone (CORT) levels significantly increased. However, the expression of 3β-HSD and P45011β in GM was unaffected by fasting. The decrease in GM weight may be due to decreased levels of testosterone and reduced synthesis of mammalian target of rapamycin (mTOR). Significant increases in CORT both GM and SOL were associated with increases in the amount of branched-chain amino acids available for energy production. However, decreased levels of mTOR and IGF1 and increased levels of CORT and IL-6 in SOL suggest that GM proteolysis was followed by SOL proteolysis for additional energy production. In conclusion, IGF1 levels decreased significantly in SOL, whereas those of IL-6 significantly increased in SOL and blood but decreased in GM. Blood branched-chain amino acids (BCAA) levels were unaffected due to fasting, whereas an increase was noted in the levels of BCAA in GM and SOL. These results show that fasting for 96 h restricts energy supply, producing fast-twitch muscle atrophy followed by slow-twitch muscle atrophy.

Keywords:  and fasting stress; branched-chain amino acids; corticosterone; interleukin-6; mammalian target of rapamycin

DOI:  https://doi.org/10.1016/j.steroids.2023.109328
Tissue Eng Part C Methods. 2023 Oct 16.

Developing a cell-microcarrier tissue engineered product for muscle repair using a bioreactor system.

Ana Luísa Cartaxo, Ana Fernandes, Carlos Rodrigues, Ana M Melo, Katja Tecklenburg, Eva Margreiter, Richard M Day, Cláudia L da Silva, Joaquim M S Cabral.

Fecal incontinence, although not life-threatening, has a high impact in the economy and in patient quality of life. So far, available treatments are based on both surgical and non-surgical approaches. These can range from changes in diet, to bowel training, or sacral nerve stimulation, but none of which provide a long-term solution. New regenerative medicine-based therapies are emerging, which aim at regenerating the sphincter muscle and restore continence. Usually, these consist of the administration of a suspension of expanded skeletal-derived muscle cells (SkMDCs) to the damaged site. However, this strategy often results in a reduced cell viability due to the need for cell harvesting from the expansion platform, as well as the non-native use of a cell suspension to deliver the anchorage-dependent cells. Here, we propose the proof-of-concept for the bioprocessing of a new cell delivery method for the treatment of fecal incontinence, obtained by a scalable two-step process. Firstly, patient isolated SkMDCs were expanded using planar static culture systems. Secondly, by using a single-use PBS-mini Vertical-Wheel®bioreactor, the expanded SkMDCs were combined with biocompatible and biodegradable (i.e., directly implantable) poly(lactic-co-glycolic acid) (PLGA) microcarriers, previously prepared by thermally induced phase separation (TIPS). This process allowed for up to 80% efficiency of the SkMDCs to attach to the microcarriers. Importantly, SkMDCs were viable during all the process and maintained their myogenic features (e.g., expression of the CD56 marker) after adhesion and culture on the microcarriers. When SKMDCs-containing microcarriers were placed on a culture dish, cells were able to migrate from the microcarriers onto the culture surface and differentiate into multinucleated myotubes, which highlights their potential to regenerate the damaged sphincter muscle after administration into the patient. Overall, this study proposes an innovative method to attach SkMDCs to biodegradable microcarriers, which can provide a new treatment for fecal incontinence.

DOI: https://doi.org/10.1089/ten.TEC.2023.0122
Mol Ther Nucleic Acids. 2023 Dec 12. 34 102040

CRISPR-Cas9 delivery strategies with engineered extracellular vesicles.

Yaoyao Lu, Kelly Godbout, Gabriel Lamothe, Jacques P Tremblay.

  Therapeutic genome editing has the potential to cure diseases by directly correcting genetic mutations in tissues and cells. Recent progress in the CRISPR-Cas9 systems has led to breakthroughs in gene editing tools because of its high orthogonality, versatility, and efficiency. However, its safe and effective administration to target organs in patients is a major hurdle. Extracellular vesicles (EVs) are endogenous membranous particles secreted spontaneously by all cells. They are key actors in cell-to-cell communication, allowing the exchange of select molecules such as proteins, lipids, and RNAs to induce functional changes in the recipient cells. Recently, EVs have displayed their potential for trafficking the CRISPR-Cas9 system during or after their formation. In this review, we highlight recent developments in EV loading, surface functionalization, and strategies for increasing the efficiency of delivering CRISPR-Cas9 to tissues, organs, and cells for eventual use in gene therapies.

Keywords:  CRISPR-Cas9; MT: Exploiting Extracellular Vesicles as Therapeutic Agents Special Issue; delivery; extracellular vesicles; gene therapy; tissue targeting

DOI:  https://doi.org/10.1016/j.omtn.2023.102040
Hum Mol Genet. 2023 Oct 19. pii: ddad175. [Epub ahead of print]

FSHD muscle shows perturbation in fibroadipogenic progenitor cells, mitochondrial function and alternative splicing independently of inflammation.

Elise N Engquist, Anna Greco, Leo A B Joosten, Baziel G M van Engelen, Peter S Zammit, Christopher R S Banerji.

  Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy. FSHD is highly heterogeneous, with patients following a variety of clinical trajectories, complicating clinical trials. Skeletal muscle in FSHD undergoes fibrosis and fatty replacement that can be accelerated by inflammation, adding to heterogeneity. Well controlled molecular studies are thus essential to both categorise FSHD patients into distinct subtypes and understand pathomechanisms. Here, we further analysed RNA-sequencing data from 24 FSHD patients, each of whom donated a biopsy from both a non-inflamed (TIRM-) and inflamed (TIRM+) muscle, and 15 FSHD patients who donated peripheral blood mononucleated cells (PBMCs), alongside non-affected control individuals. Differential gene expression analysis identified suppression of mitochondrial biogenesis and up-regulation of fibroadipogenic progenitor (FAP) gene expression in FSHD muscle, which was particularly marked on inflamed samples. PBMCs demonstrated suppression of antigen presentation in FSHD. Gene expression deconvolution revealed FAP expansion as a consistent feature of FSHD muscle, via meta-analysis of 7 independent transcriptomic datasets. Clustering of muscle biopsies separated patients in an unbiased manner into clinically mild and severe subtypes, independently of known disease modifiers (age, sex, D4Z4 repeat length). Lastly, the first genome-wide analysis of alternative splicing in FSHD muscle revealed perturbation of autophagy, BMP2 and HMGB1 signalling. Overall, our findings reveal molecular subtypes of FSHD with clinical relevance and identify novel pathomechanisms for this highly heterogeneous condition.

Keywords:  alterrnative splicing; facioscapulohumeral muscular dystropy (FSHD); fibroadipogenic progenitor cells (FAPs); mitochondrial function; transcriptomics

DOI:  https://doi.org/10.1093/hmg/ddad175
Int J Biol Macromol. 2023 Oct 15. pii: S0141-8130(23)04315-5. [Epub ahead of print] 127418

Decreasing predictable DNA off-target effects and narrowing editing windows of adenine base editors by fusing human Rad18 protein variant.

Ziru Wang, Hongming Yuan, Lin Yang, Lerong Ma, Yuanzhu Zhang, Jiacheng Deng, Xueyuan Li, Wenyu Xiao, Zhanjun Li, Jiazhang Qiu, Hongsheng Ouyang, Daxin Pang.

  Adenine base editors, enabling targeted A-to-G conversion in genomic DNA, have enormous potential in therapeutic applications. However, the currently used adenine base editors are limited by wide editing windows and off-target effects in genetic therapy. Here, we report human e18 protein, a RING type E3 ubiquitin ligase variant, fusing with adenine base editors can significantly improve the preciseness and narrow the editing windows compared with ABEmax and ABE8e by diminishing the abundance of base editor protein. As a proof of concept, ABEmax-e18 and ABE8e-e18 dramatically decrease Cas9-dependent and Cas9-independent off-target effects than traditional adenine base editors. Moreover, we utilized ABEmax-e18 to establish syndactyly mouse models and achieve accurate base conversion at human PCSK9 locus in HepG2 cells which exhibited its potential in genetic therapy. Furthermore, a truncated version of base editors-RING (ABEmax-RING or AncBE4max-RING), which fusing the 63 amino acids of e18 protein RING domain to the C terminal of ABEmax or AncBE4max, exhibited similar effect compared to ABEmax-e18 or AncBE4max-e18.In summary, the e18 or RING protein fused with base editors strengthens the precise toolbox in gene modification and maybe works well with various base editing tools with a more applicable to precise genetic therapies in the future.

Keywords:  Base editors; Editing windows; Off-target; RING type E3 ubiquitin ligase

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.127418
Cell Mol Life Sci. 2023 Oct 19. 80(11): 330

TEAD4 antagonizes cellular senescence by remodeling chromatin accessibility at enhancer regions.

Donghui Zhang, Yanmei Zhu, Yanmin Ju, Hongyong Zhang, Xiaopeng Zou, Shangrong She, Danping Zhu, Yiting Guan.

  Dramatic alterations in epigenetic landscapes are known to impact genome accessibility and transcription. Extensive evidence demonstrates that senescent cells undergo significant changes in chromatin structure; however, the mechanisms underlying the crosstalk between epigenetic parameters and gene expression profiles have not been fully elucidated. In the present study, we delineate the genome-wide redistribution of accessible chromatin regions that lead to broad transcriptome effects during senescence. We report that distinct senescence-activated accessibility regions (SAAs) are always distributed in H3K27ac-occupied enhancer regions, where they are responsible for elevated flanking senescence-associated secretory phenotype (SASP) expression and aberrant cellular signaling relevant to SASP secretion. Mechanistically, a single transcription factor, TEAD4, moves away from H3K27ac-labled SAAs to allow for prominent chromatin accessibility reconstruction during senescence. The enhanced SAAs signal driven by TEAD4 suppression subsequently induces a robust increase in the expression of adjacent SASP genes and the secretion of downstream factors, which contribute to the progression of senescence. Our findings illustrate a dynamic landscape of chromatin accessibility following senescence entry, and further reveal an insightful function for TEAD4 in regulating the broad chromatin state that modulates the overall transcriptional program of SASP genes.

Keywords:  Chromatin accessibility; Enhancer; SASP; Senescence; TEAD4

DOI:  https://doi.org/10.1007/s00018-023-04980-9
Clin Epigenetics. 2023 Oct 19. 15(1): 167

Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells.

Eleanor O'Brien, Carmen Tse, Ian Tracy, Ian Reddin, Joanna Selfe, Jane Gibson, William Tapper, Reuben J Pengelly, Jinhui Gao, Ewa Aladowicz, Gemma Petts, Khin Thway, Sergey Popov, Anna Kelsey, Timothy J Underwood, Janet Shipley, Zoë S Walters.

   BACKGROUND: Rhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours.
RESULTS: We demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS.
CONCLUSIONS: The results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

Keywords:  All-trans retinoic acid; Differentiation therapy; EZH2; Epigenetic therapy; Rhabdomyosarcoma

DOI:  https://doi.org/10.1186/s13148-023-01583-w