bims-muscge Biomed News
on Muscle stem cells and gene therapy
Issue of 2023–11–12
23 papers selected by
Chance Bowman, Dartmouth College
  1. TAZ stimulates exercise-induced muscle satellite cell activation via Pard3-p38 MAPK-TAZ signalling axis.
  2. Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function.
  3. Role of Actin-Binding Proteins in Skeletal Myogenesis.
  4. Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology.
  5. Combinatorial activation of the WNT-dependent fibrogenic program by distinct complement subunits in dystrophic muscle.
  6. Tropomyosin 3 (TPM3) function in skeletal muscle and in myopathy.
  7. A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models.
  8. Mechanisms and effects of metformin on skeletal muscle disorders.
  9. Skeletal muscle regeneration failure in ischemic-damaged limbs is associated with pro-inflammatory macrophages and premature differentiation of satellite cells.
  10. Mitophagy Activation by Urolithin A to Target Muscle Aging.
  11. From workout to molecular switches: How does skeletal muscle produce, sense, and transduce subcellular redox signals?
  12. Considering the Promise of Vamorolone for Treating Duchenne Muscular Dystrophy.
  13. snRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis.
  14. Protocol for isolating mice skeletal muscle myoblasts and myotubes via differential antibody validation.
  15. Successes and challenges in clinical gene therapy.
  16. Development of a highly efficient prime editor system in mice and rabbits.
  17. Regulating Striated Muscle Contraction: Through Thick and Thin.
  18. Skeletal muscle-derived extracellular vesicles transport glycolytic enzymes to mediate muscle-to-bone crosstalk.
  19. Overcoming biological barriers by virus-like drug particles for drug delivery.
  20. 3D Printed Magneto-Active Microfiber Scaffolds for Remote Stimulation and Guided Organization of 3D In Vitro Skeletal Muscle Models.
  21. Adenine base editor-mediated splicing remodeling activates non-canonical splice sites.
  22. Single-Cell RNA Sequencing Technology Landscape in 2023.
  23. The State of Cell and Gene Therapy in 2023.
  1. J Cachexia Sarcopenia Muscle. 2023 Nov 03.
    TAZ stimulates exercise-induced muscle satellite cell activation via Pard3-p38 MAPK-TAZ signalling axis.
    Kyung Min Kim, Gi Don Yoo, Woong Heo, Ho Taek Oh, Jeekeon Park, Somin Shin, Youjin Do, Mi Gyeong Jeong, Eun Sook Hwang, Jeong-Ho Hong.
       BACKGROUND: Exercise stimulates the activation of muscle satellite cells, which facilitate the maintenance of stem cells and their myogenic conversion during muscle regeneration. However, the underlying mechanism is not yet fully understood. This study shows that the transcriptional co-activator with PDZ-binding motif (TAZ) stimulates muscle regeneration via satellite cell activation.
    METHODS: Tazf/f mice were crossed with the paired box gene 7 (Pax7)creERT2 mice to generate muscle satellite cell-specific TAZ knockout (sKO) mice. Mice were trained in an endurance exercise programme for 4 weeks. Regenerated muscles were harvested and analysed by haematoxylin and eosin staining. Muscle tissues were also analysed by immunofluorescence staining, immunoblot analysis and quantitative reverse transcription PCR (qRT-PCR). For the in vitro study, muscle satellite cells from wild-type and sKO mice were isolated and analysed. Mitochondrial DNA was quantified by qRT-PCR using primers that amplify the cyclooxygenase-2 region of mitochondrial DNA. Quiescent and activated satellite cells were stained with MitoTracker Red CMXRos to analyse mitochondria. To study the p38 mitogen-activated protein kinase (MAPK)-TAZ signalling axis, p38 MAPK was activated by introducing the MAPK kinase 6 plasmid into satellite cells and also inhibited by treatment with the p38 MAPK inhibitor, SB203580.
    RESULTS: TAZ interacts with Pax7 to induce Myf5 expression and stimulates mammalian target of rapamycin signalling for satellite cell activation. In sKO mice, TAZ depletion reduces muscle satellite cell number by 38% (0.29 ± 0.073 vs. 0.18 ± 0.034, P = 0.0082) and muscle regeneration. After muscle injury, TAZ levels (2.59-fold, P < 0.0001) increase in committed cells compared to self-renewing cells during asymmetric satellite cell division. Mechanistically, the polarity protein Pard3 induces TAZ (2.01-fold, P = 0.008) through p38 MAPK, demonstrating that the p38 MAPK-TAZ axis is important for muscle regeneration. Physiologically, endurance exercise training induces muscle satellite cell activation and increases muscle fibre diameter (1.33-fold, 43.21 ± 23.59 vs. 57.68 ± 23.26 μm, P = 0.0004) with increased TAZ levels (1.76-fold, P = 0.017). However, sKO mice had a 39% reduction in muscle satellite cell number (0.20 ± 0.03 vs. 0.12 ± 0.02, P = 0.0013) and 24% reduction in muscle fibre diameter compared to wild-type mice (61.07 ± 23.33 vs. 46.60 ± 24.29 μm, P = 0.0006).
    CONCLUSIONS: Our results demonstrate a novel mechanism of TAZ-induced satellite cell activation after muscle injury and exercise, suggesting that activation of TAZ in satellite cells may ameliorate the muscle ageing phenotype and may be an important target protein for the drug development in sarcopenia.
    Keywords:  exercise; muscle regeneration; muscle satellite cell; sarcopenia
    DOI:  https://doi.org/10.1002/jcsm.13348
  2. Commun Biol. 2023 11 04. 6(1): 1121
    Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function.
    Victor Martinez-Heredia, Danielle Blackwell, Sujith Sebastian, Timothy Pearson, Gi Fay Mok, Laura Mincarelli, Charlotte Utting, Leighton Folkes, Ernst Poeschl, Iain Macaulay, Ulrike Mayer, Andrea Münsterberg.
      Skeletal muscle stem cells (MuSC) are crucial for tissue homoeostasis and repair after injury. Following activation, they proliferate to generate differentiating myoblasts. A proportion of cells self-renew, re-enter the MuSC niche under the basal lamina outside the myofiber and become quiescent. Quiescent MuSC have a primary cilium, which is disassembled upon cell cycle entry. Ex vivo experiments suggest cilia are important for MuSC self-renewal, however, their requirement for muscle regeneration in vivo remains poorly understood. Talpid3 (TA3) is essential for primary cilia formation and Hedgehog (Hh) signalling. Here we use tamoxifen-inducible conditional deletion of TA3 in MuSC (iSC-KO) and show that regeneration is impaired in response to cytotoxic injury. Depletion of MuSC after regeneration suggests impaired self-renewal, also consistent with an exacerbated phenotype in TA3iSC-KO mice after repeat injury. Single cell transcriptomics of MuSC progeny isolated from myofibers identifies components of several signalling pathways, which are deregulated in absence of TA3, including Hh and Wnt. Pharmacological activation of Wnt restores muscle regeneration, while purmorphamine, an activator of the Smoothened (Smo) co-receptor in the Hh pathway, has no effect. Together, our data show that TA3 and primary cilia are important for MuSC self-renewal and pharmacological treatment can efficiently restore muscle regeneration.
    DOI:  https://doi.org/10.1038/s42003-023-05503-9
  3. Cells. 2023 Oct 25. pii: 2523. [Epub ahead of print]12(21):
    Role of Actin-Binding Proteins in Skeletal Myogenesis.
    Mai Thi Nguyen, Raju Dash, Kyuho Jeong, Wan Lee.
      Maintenance of skeletal muscle quantity and quality is essential to ensure various vital functions of the body. Muscle homeostasis is regulated by multiple cytoskeletal proteins and myogenic transcriptional programs responding to endogenous and exogenous signals influencing cell structure and function. Since actin is an essential component in cytoskeleton dynamics, actin-binding proteins (ABPs) have been recognized as crucial players in skeletal muscle health and diseases. Hence, dysregulation of ABPs leads to muscle atrophy characterized by loss of mass, strength, quality, and capacity for regeneration. This comprehensive review summarizes the recent studies that have unveiled the role of ABPs in actin cytoskeletal dynamics, with a particular focus on skeletal myogenesis and diseases. This provides insight into the molecular mechanisms that regulate skeletal myogenesis via ABPs as well as research avenues to identify potential therapeutic targets. Moreover, this review explores the implications of non-coding RNAs (ncRNAs) targeting ABPs in skeletal myogenesis and disorders based on recent achievements in ncRNA research. The studies presented here will enhance our understanding of the functional significance of ABPs and mechanotransduction-derived myogenic regulatory mechanisms. Furthermore, revealing how ncRNAs regulate ABPs will allow diverse therapeutic approaches for skeletal muscle disorders to be developed.
    Keywords:  actin dynamics; actin-binding proteins; differentiation; myogenesis; non-coding RNA; proliferation
    DOI:  https://doi.org/10.3390/cells12212523
  4. Cells. 2023 Nov 01. pii: 2560. [Epub ahead of print]12(21):
    Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology.
    Paul Dowling, Dieter Swandulla, Kay Ohlendieck.
      Voluntary striated muscles are characterized by a highly complex and dynamic proteome that efficiently adapts to changed physiological demands or alters considerably during pathophysiological dysfunction. The skeletal muscle proteome has been extensively studied in relation to myogenesis, fiber type specification, muscle transitions, the effects of physical exercise, disuse atrophy, neuromuscular disorders, muscle co-morbidities and sarcopenia of old age. Since muscle tissue accounts for approximately 40% of body mass in humans, alterations in the skeletal muscle proteome have considerable influence on whole-body physiology. This review outlines the main bioanalytical avenues taken in the proteomic characterization of skeletal muscle tissues, including top-down proteomics focusing on the characterization of intact proteoforms and their post-translational modifications, bottom-up proteomics, which is a peptide-centric method concerned with the large-scale detection of proteins in complex mixtures, and subproteomics that examines the protein composition of distinct subcellular fractions. Mass spectrometric studies over the last two decades have decisively improved our general cell biological understanding of protein diversity and the heterogeneous composition of individual myofibers in skeletal muscles. This detailed proteomic knowledge can now be integrated with findings from other omics-type methodologies to establish a systems biological view of skeletal muscle function.
    Keywords:  mass spectrometry; muscle cell biology; muscle proteomics; myology; organelle proteomics
    DOI:  https://doi.org/10.3390/cells12212560
  5. EMBO Mol Med. 2023 Nov 06. e17405
    Combinatorial activation of the WNT-dependent fibrogenic program by distinct complement subunits in dystrophic muscle.
    Francesca Florio, Sara Vencato, Filomena T Papa, Michela Libergoli, Eyemen Kheir, Imen Ghzaiel, Thomas A Rando, Yvan Torrente, Stefano Biressi.
      Fibrosis is associated with compromised muscle functionality in Duchenne muscular dystrophy (DMD). We report observations with tissues from dystrophic patients and mice supporting a model to explain fibrosis in DMD, which relies on the crosstalk between the complement and the WNT signaling pathways and the functional interactions of two cellular types. Fibro-adipogenic progenitors and macrophages, which populate the inflamed dystrophic muscles, act as a combinatorial source of WNT activity by secreting distinct subunits of the C1 complement complex. The resulting aberrant activation of the WNT signaling in responsive cells, such as fibro-adipogenic progenitors, contributes to fibrosis. Indeed, pharmacological inhibition of the C1r/s subunits in a murine model of DMD mitigated the activation of the WNT signaling pathway, reduced the fibrogenic characteristics of the fibro-adipogenic progenitors, and ameliorated the dystrophic phenotype. These studies shed new light on the molecular and cellular mechanisms responsible for fibrosis in muscular dystrophy and open to new therapeutic strategies.
    Keywords:  Duchenne muscular dystrophy; complement C1 complex; fibro-adipogenic progenitors; fibrosis; skeletal muscle regeneration
    DOI:  https://doi.org/10.15252/emmm.202317405
  6. Skelet Muscle. 2023 Nov 07. 13(1): 18
    Tropomyosin 3 (TPM3) function in skeletal muscle and in myopathy.
    Matthias R Lambert, Emanuela Gussoni.
      The tropomyosin genes (TPM1-4) contribute to the functional diversity of skeletal muscle fibers. Since its discovery in 1988, the TPM3 gene has been recognized as an indispensable regulator of muscle contraction in slow muscle fibers. Recent advances suggest that TPM3 isoforms hold more extensive functions during skeletal muscle development and in postnatal muscle. Additionally, mutations in the TPM3 gene have been associated with the features of congenital myopathies. The use of different in vitro and in vivo model systems has leveraged the discovery of several disease mechanisms associated with TPM3-related myopathy. Yet, the precise mechanisms by which TPM3 mutations lead to muscle dysfunction remain unclear. This review consolidates over three decades of research about the role of TPM3 in skeletal muscle. Overall, the progress made has led to a better understanding of the phenotypic spectrum in patients affected by mutations in this gene. The comprehensive body of work generated over these decades has also laid robust groundwork for capturing the multiple functions this protein plays in muscle fibers.
    Keywords:  Congenital myopathy; Rare diseases; Skeletal muscle; TPM3; Thin filaments; Tropomyosin
    DOI:  https://doi.org/10.1186/s13395-023-00327-x
  7. Hum Mol Genet. 2023 Nov 08. pii: ddad192. [Epub ahead of print]
    A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models.
    Joseph M Hoolachan, Eve McCallion, Emma R Sutton, Özge Çetin, Paloma Pacheco-Torres, Maria Dimitriadi, Suat Sari, Gavin J Miller, Magnus Okoh, Lisa M Walter, Peter Claus, Matthew J A Wood, Daniel P Tonge, Melissa Bowerman.
      Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolone's activities. Using an RNA-sequencing, bioinformatics, and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone-treated and untreated Smn-/-; SMN2 SMA and Smn+/-; SMN2 healthy mice, we identified molecular targets linked to prednisolone's ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolone's potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.
    Keywords:  animal models; drug repurposing; skeletal muscle; spinal muscular atrophy; transcriptomics
    DOI:  https://doi.org/10.1093/hmg/ddad192
  8. Front Neurol. 2023 ;14 1275266
    Mechanisms and effects of metformin on skeletal muscle disorders.
    Ren Shang, Jing Miao.
      Skeletal muscle disorders are mostly genetic and include several rare diseases. With disease progression, muscle fibrosis and adiposis occur, resulting in limited mobility. The long course of these diseases combined with limited treatment options affect patients both psychologically and economically, hence the development of novel treatments for neuromuscular diseases is crucial to obtain a better quality of life. As a widely used hypoglycemic drug in clinical practice, metformin not only has anti-inflammatory, autophagy-regulating, and mitochondrial biogenesis-regulating effects, but it has also been reported to improve the symptoms of neuromuscular diseases, delay hypokinesia, and regulate skeletal muscle mass. However, metformin's specific mechanism of action in neuromuscular diseases requires further elucidation. This review summarizes the evidence showing that metformin can regulate inflammation, autophagy, and mitochondrial biogenesis through different pathways, and further explores its mechanism of action in Duchenne muscular dystrophy, statin-associated muscle disorders, and age-related sarcopenia. This review clarifies the directions of future research on therapy for neuromuscular diseases.
    Keywords:  autophagy; inflammation; metformin; mitochondrial biogenesis; skeletal muscle disorders
    DOI:  https://doi.org/10.3389/fneur.2023.1275266
  9. Genome Med. 2023 Nov 10. 15(1): 95
    Skeletal muscle regeneration failure in ischemic-damaged limbs is associated with pro-inflammatory macrophages and premature differentiation of satellite cells.
    Kevin W Southerland, Yueyuan Xu, Derek T Peters, Xin Lin, Xiaolin Wei, Yu Xiang, Kaileen Fei, Lindsey A Olivere, Jeremy M Morowitz, James Otto, Qunsheng Dai, Christopher D Kontos, Yarui Diao.
       BACKGROUND: Chronic limb-threatening ischemia (CLTI), a severe manifestation of peripheral arterial disease (PAD), is associated with a 1-year limb amputation rate of approximately 15-20% and substantial mortality. A key feature of CLTI is the compromised regenerative ability of skeletal muscle; however, the mechanisms responsible for this impairment are not yet fully understood. In this study, we aim to delineate pathological changes at both the cellular and transcriptomic levels, as well as in cell-cell signaling pathways, associated with compromised muscle regeneration in limb ischemia in both human tissue samples and murine models of CLTI.
    METHODS: We performed single-cell transcriptome analysis of ischemic and non-ischemic muscle from the same CLTI patients and from a murine model of CLTI. In both datasets, we analyzed gene expression changes in macrophage and muscle satellite cell (MuSC) populations as well as differential cell-cell signaling interactions and differentiation trajectories.
    RESULTS: Single-cell transcriptomic profiling and immunofluorescence analysis of CLTI patient skeletal muscle demonstrated that ischemic-damaged tissue displays a pro-inflammatory macrophage signature. Comparable results were observed in a murine CLTI model. Moreover, integrated analyses of both human and murine datasets revealed premature differentiation of MuSCs to be a key feature of failed muscle regeneration in the ischemic limb. Furthermore, in silico inferences of intercellular communication and in vitro assays highlight the importance of macrophage-MuSC signaling in ischemia induced muscle injuries.
    CONCLUSIONS: Collectively, our research provides the first single-cell transcriptome atlases of skeletal muscle from CLTI patients and a murine CLTI model, emphasizing the crucial role of macrophages and inflammation in regulating muscle regeneration in CLTI through interactions with MuSCs.
    Keywords:  Chronic limb-threatening ischemia; Macrophage polarization; Murine hindlimb ischemia; Muscle satellite cells; Peripheral arterial disease; Single-cell transcriptome analysis; Skeletal muscle regeneration
    DOI:  https://doi.org/10.1186/s13073-023-01250-y
  10. Calcif Tissue Int. 2023 Nov 05.
    Mitophagy Activation by Urolithin A to Target Muscle Aging.
    Julie Faitg, Davide D'Amico, Chris Rinsch, Anurag Singh.
      The age-related loss of skeletal muscle function starts from midlife and if left unaddressed can lead to an impaired quality of life. A growing body of evidence indicates that mitochondrial dysfunction is causally involved with muscle aging. Muscles are tissues with high metabolic requirements, and contain rich mitochondria supply to support their continual energy needs. Cellular mitochondrial health is maintained by expansing of the mitochondrial pool though mitochondrial biogenesis, by preserving the natural mitochondrial dynamic process, via fusion and fission, and by ensuring the removal of damaged mitochondria through mitophagy. During aging, mitophagy levels decline and negatively impact skeletal muscle performance. Nutritional and pharmacological approaches have been proposed to manage the decline in muscle function due to impaired mitochondria bioenergetics. The natural postbiotic Urolithin A has been shown to promote mitophagy, mitochondrial function and improved muscle function across species in different experimental models and across multiple clinical studies. In this review, we explore the biology of Urolithin A and the clinical evidence of its impact on promoting healthy skeletal muscles during age-associated muscle decline.
    Keywords:  Aging; Mitochondria; Mitophagy; Muscle health; Urolithin A
    DOI:  https://doi.org/10.1007/s00223-023-01145-5
  11. Free Radic Biol Med. 2023 Nov 02. pii: S0891-5849(23)01076-6. [Epub ahead of print]209(Pt 2): 355-365
    From workout to molecular switches: How does skeletal muscle produce, sense, and transduce subcellular redox signals?
    Carlos Henriquez-Olguin, Roberto Meneses-Valdes, Paraskevi Kritsiligkou, Eduardo Fuentes-Lemus.
      Skeletal muscle is crucial for maintaining human health and overall quality of life. Acute exercise introduces a multifaceted intracellular stress, with numerous post-translational modifications believed to underpin the health benefits of sustained exercise training. Reactive oxygen species (ROS) are posited to serve as second messengers, triggering cytoprotective adaptations such as the upregulation of enzymatic scavenger systems. However, a significant knowledge gap exists between the generation of oxidants in muscle and the exact mechanisms driving muscle adaptations. This review delves into the current research on subcellular redox biochemistry and its role in the physiological adaptations to exercise. We propose that the subcellular regulation of specific redox modifications is key to ensuring specificity in the intracellular response.
    Keywords:  Exercise training; Hydrogen peroxide; Mitochondria; Redox signaling; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.10.404
  12. J Neuromuscul Dis. 2023 Oct 27.
    Considering the Promise of Vamorolone for Treating Duchenne Muscular Dystrophy.
    Miranda D Grounds, Erin M Lloyd.
      This commentary provides an independent consideration of data related to the drug vamorolone (VBP15) as an alternative steroid proposed for treatment of Duchenne muscular dystrophy (DMD). Glucocorticoids such as prednisone and deflazacort have powerful anti-inflammatory benefits and are the standard of care for DMD, but their long-term use can result in severe adverse side effects; thus, vamorolone was designed as a unique dissociative steroidal anti-inflammatory drug, to retain efficacy and minimise these adverse effects. Extensive clinical trials (ongoing) have investigated the use of vamorolone for DMD, with two trials also for limb-girdle muscular dystrophies including dysferlinopathy (current), plus a variety of pre-clinical trials published. Vamorolone looks very promising, with similar efficacy and some reduced adverse effects (e.g., related to height) compared with other glucocorticoids, specifically prednisone/prednisolone, although it has not yet been directly compared with deflazacort. Of particular interest to clarify is the optimal clinical dose and other aspects of vamorolone that are proposed to provide additional benefits for membranes of dystrophic muscle: to stabilise and protect the sarcolemma from damage and enhance repair. The use of vamorolone (and other glucocorticoids) needs to be evaluated in terms of overall long-term efficacy and cost, and also in comparison with many candidate non-steroidal drugs with anti-inflammatory and other benefits for DMD.
    Keywords:  DMD; VBP15; Vamorolone; clinical trials; dysferlinopathy; dystropathology; glucocorticoids; muscle necrosis; muscular dystrophies; pre-clinical studies
    DOI:  https://doi.org/10.3233/JND-230161
  13. Hum Mol Genet. 2023 Nov 02. pii: ddad186. [Epub ahead of print]
    snRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis.
    Dongxu Zheng, Annelot Wondergem, Susan Kloet, Iris Willemsen, Judit Balog, Stephen J Tapscott, Ahmed Mahfouz, Anita van den Heuvel, Silvère M van der Maarel.
      The sporadic nature of DUX4 expression in FSHD muscle challenges comparative transcriptome analyses between FSHD and control samples. A variety of DUX4 and FSHD-associated transcriptional changes have been identified, but bulk RNA-seq strategies prohibit comprehensive analysis of their spatiotemporal relation, interdependence and role in the disease process. In this study, we used single-nucleus RNA-sequencing of nuclei isolated from patient- and control-derived multinucleated primary myotubes to investigate the cellular heterogeneity in FSHD. Taking advantage of the increased resolution in snRNA-sequencing of fully differentiated myotubes, two distinct populations of DUX4-affected nuclei could be defined by their transcriptional profiles. Our data provides insights into the differences between these two populations and suggests heterogeneity in two well-known FSHD-associated transcriptional aberrations: increased oxidative stress and inhibition of myogenic differentiation. Additionally, we provide evidence that DUX4-affected nuclei share transcriptome features with early embryonic cells beyond the well-described cleavage stage, progressing into the 8-cell and blastocyst stages. Altogether, our data suggests that the FSHD transcriptional profile is defined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses and cellular state-dependent downstream effects.
    Keywords:  DUX4; FSHD; cellular heterogeneity; muscular dystrophy; single-nucleus RNA-sequencing
    DOI:  https://doi.org/10.1093/hmg/ddad186
  14. STAR Protoc. 2023 Nov 07. pii: S2666-1667(23)00558-0. [Epub ahead of print]4(4): 102591
    Protocol for isolating mice skeletal muscle myoblasts and myotubes via differential antibody validation.
    Dominique C Stephens, Margaret Mungai, Amber Crabtree, Heather K Beasley, Edgar Garza-Lopez, Larry Vang, Kit Neikirk, Zer Vue, Neng Vue, Andrea G Marshall, Kyrin Turner, Jian-Qiang Shao, Bishnu Sarker, Sandra Murray, Jennifer A Gaddy, Jamaine Davis, Steven M Damo, Antentor O Hinton.
      Isolation of skeletal muscles allows for the exploration of many complex diseases. Here, we present a protocol for isolating mice skeletal muscle myoblasts and myotubes that have been differentiated through antibody validation. We describe steps for collecting and preparing murine skeletal tissue, myoblast cell maintenance, plating, and cell differentiation. We then detail procedures for cell incubation, immunostaining, slide preparation and storage, and imaging for immunofluorescence validation.
    Keywords:  Cell Culture; Cell Isolation; Model Organisms
    DOI:  https://doi.org/10.1016/j.xpro.2023.102591
  15. Gene Ther. 2023 Nov 08.
    Successes and challenges in clinical gene therapy.
    Donald B Kohn, Yvonne Y Chen, Melissa J Spencer.
      Despite the ups and downs in the field over three decades, the science of gene therapy has continued to advance and provide enduring treatments for increasing number of diseases. There are active clinical trials approaching a variety of inherited and acquired disorders of different organ systems. Approaches include ex vivo modification of hematologic stem cells (HSC), T lymphocytes and other immune cells, as well as in vivo delivery of genes or gene editing reagents to the relevant target cells by either local or systemic administration. In this article, we highlight success and ongoing challenges in three areas of high activity in gene therapy: inherited blood cell diseases by targeting hematopoietic stem cells, malignant disorders using immune effector cells genetically modified with chimeric antigen receptors, and ophthalmologic, neurologic, and coagulation disorders using in vivo administration of adeno-associated virus (AAV) vectors. In recent years, there have been true cures for many of these diseases, with sustained clinical benefit that exceed those from other medical approaches. Each of these treatments faces ongoing challenges, namely their high one-time costs and the complexity of manufacturing the therapeutic agents, which are biological viruses and cell products, at pharmacologic standards of quality and consistency. New models of reimbursement are needed to make these innovative treatments widely available to patients in need.
    DOI:  https://doi.org/10.1038/s41434-023-00390-5
  16. Cell Mol Life Sci. 2023 Nov 04. 80(11): 346
    Development of a highly efficient prime editor system in mice and rabbits.
    Yuqiang Qian, Di Wang, Wenchao Niu, Zheng Shi, Mao Wu, Ding Zhao, Jinze Li, Xun Gao, Zhongtian Zhang, Liangxue Lai, Zhanjun Li.
      The recently developed prime-editing (PE) technique is more precise than previously available techniques and permits base-to-base conversion, replacement, and insertions and deletions in the genome. However, previous reports show that the efficiency of prime editing is insufficient to produce genome-edited animals. In fact, prime-guide RNA (pegRNA) designs have posed a challenge in achieving favorable editing efficiency. Here, we designed prime binding sites (PBS) with a melting temperature (Tm) of 42 °C, leading to optimal performance in cells, and we found that the optimal Tm was affected by the culture temperature. In addition, the ePE3max system was developed by updating the PE architecture to PEmax and expressing engineered pegRNA (epegRNA) based on the original PE3 system. The updated ePE3max system can efficiently induce gene editing in mouse and rabbit embryos. Furthermore, we successfully generated a Hoxd13 (c. 671 G > T) mutation in mice and a Tyr (c. 572 del) mutation in rabbits by ePE3max. Overall, the editing efficiency of modified ePE3max systems is superior to that of the original PE3 system in producing genome-edited animals, which can serve as an effective and versatile genome-editing tool for precise genome modification in animal models.
    Keywords:  Editing efficiency; Mouse; Precise editing; Prime editing; Rabbit
    DOI:  https://doi.org/10.1007/s00018-023-05003-3
  17. Annu Rev Physiol. 2023 Nov 06.
    Regulating Striated Muscle Contraction: Through Thick and Thin.
    Elisabetta Brunello, Luca Fusi.
      Force generation in striated muscle is primarily controlled by structural changes in the actin-containing thin filaments triggered by an increase in intracellular calcium concentration. However, recent studies have elucidated a new class of regulatory mechanisms, based on the myosin-containing thick filament, that control the strength and speed of contraction by modulating the availability of myosin motors for the interaction with actin. This review summarizes the mechanisms of thin and thick filament activation that regulate the contractility of skeletal and cardiac muscle. A novel dual-filament paradigm of muscle regulation is emerging, in which the dynamics of force generation depends on the coordinated activation of thin and thick filaments. We highlight the interfilament signaling pathways based on titin and myosin-binding protein-C that couple thin and thick filament regulatory mechanisms. This dual-filament regulation mediates the length-dependent activation of cardiac muscle that underlies the control of the cardiac output in each heartbeat. Expected final online publication date for the Annual Review of Physiology, Volume 86 is February 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-physiol-042222-022728
  18. Cell Metab. 2023 Nov 07. pii: S1550-4131(23)00382-0. [Epub ahead of print]35(11): 2028-2043.e7
    Skeletal muscle-derived extracellular vesicles transport glycolytic enzymes to mediate muscle-to-bone crosstalk.
    Shixing Ma, Xiaotao Xing, Haisen Huang, Xin Gao, Xun Xu, Jian Yang, Chengcheng Liao, Xuanhao Zhang, Jinglun Liu, Weidong Tian, Li Liao.
      Identification of cues originating from skeletal muscle that govern bone formation is essential for understanding the crosstalk between muscle and bone and for developing therapies for degenerative bone diseases. Here, we identified that skeletal muscle secreted multiple extracellular vesicles (Mu-EVs). These Mu-EVs traveled through the bloodstream to reach bone, where they were phagocytized by bone marrow mesenchymal stem/stromal cells (BMSCs). Mu-EVs promoted osteogenic differentiation of BMSCs and protected against disuse osteoporosis in mice. The quantity and bioactivity of Mu-EVs were tightly correlated with the function of skeletal muscle. Proteomic analysis revealed numerous proteins in Mu-EVs, some potentially regulating bone metabolism, especially glycolysis. Subsequent investigations indicated that Mu-EVs promoted the glycolysis of BMSCs by delivering lactate dehydrogenase A into these cells. In summary, these findings reveal that Mu-EVs play a vital role in BMSC metabolism regulation and bone formation stimulation, offering a promising approach for treating disuse osteoporosis.
    Keywords:  aerobic glycolysis; bone marrow mesenchymal stem/stromal cells; extracellular vesicles; glycolytic enzyme; lactate dehydrogenase A; skeletal muscle
    DOI:  https://doi.org/10.1016/j.cmet.2023.10.013
  19. Adv Drug Deliv Rev. 2023 Nov 03. pii: S0169-409X(23)00449-0. [Epub ahead of print] 115134
    Overcoming biological barriers by virus-like drug particles for drug delivery.
    Yu-Ling Chen, Chun-Jie Bao, Jia-Lun Duan, Ying Xie, Wan-Liang Lu.
      Virus-like particles (VLPs) have natural structural antigens similar to those found in viruses, making them valuable in vaccine immunization. Furthermore, VLPs have demonstrated significant potential in drug delivery, and emerged as promising vectors for transporting chemical drug, genetic drug, peptide/protein, and even nanoparticle drug. With virus-like permeability and strong retention, they can effectively target specific organs, tissues or cells, facilitating efficient intracellular drug release. Further modifications allow VLPs to transfer across various physiological barriers, thus acting the purpose of efficient drug delivery and accurate therapy. This article provides an overview of VLPs, covering their structural classifications, deliverable drugs, potential physiological barriers in drug delivery, strategies for overcoming these barriers, and future prospects.
    Keywords:  Virus-like particles (VLPs); drug delivery; physiological barriers; strategies
    DOI:  https://doi.org/10.1016/j.addr.2023.115134
  20. Small. 2023 Nov 10. e2307178
    3D Printed Magneto-Active Microfiber Scaffolds for Remote Stimulation and Guided Organization of 3D In Vitro Skeletal Muscle Models.
    Gerardo Cedillo-Servin, Ouafa Dahri, João Meneses, Joost van Duijn, Harrison Moon, Fanny Sage, Joana Silva, André Pereira, Fernão D Magalhães, Jos Malda, Niels Geijsen, Artur M Pinto, Miguel Castilho.
      This work reports the rational design and fabrication of magneto-active microfiber meshes with controlled hexagonal microstructures via melt electrowriting (MEW) of a magnetized polycaprolactone-based composite. In situ iron oxide nanoparticle deposition on oxidized graphene yields homogeneously dispersed magnetic particles with sizes above 0.5 µm and low aspect ratio, preventing cellular internalization and toxicity. With these fillers, homogeneous magnetic composites with high magnetic content (up to 20 weight %) are obtained and processed in a solvent-free manner for the first time. MEW of magnetic composites enabled the creation of skeletal muscle-inspired design of hexagonal scaffolds with tunable fiber diameter, reconfigurable modularity, and zonal distribution of magneto-active and nonactive material, with elastic tensile deformability. External magnetic fields below 300 mT are sufficient to trigger out-of-plane reversible deformation. In vitro culture of C2C12 myoblasts on three-dimensional (3D) Matrigel/collagen/MEW scaffolds showed that microfibers guided the formation of 3D myotube architectures, and the presence of magnetic particles does not significantly affect viability or differentiation rates after 8 days. Centimeter-sized skeletal muscle constructs allowed for reversible, continued, and dynamic magneto-mechanical stimulation. Overall, these innovative microfiber scaffolds provide magnetically deformable platforms suitable for dynamic culture of skeletal muscle, offering potential for in vitro disease modeling.
    Keywords:  fiber scaffolds; magnetic actuation; melt electrowriting; skeletal muscle; stimuli responsive biomaterials
    DOI:  https://doi.org/10.1002/smll.202307178
  21. J Biol Chem. 2023 Nov 08. pii: S0021-9258(23)02470-5. [Epub ahead of print] 105442
    Adenine base editor-mediated splicing remodeling activates non-canonical splice sites.
    Yuanyuan Liu, Qing Li, Tong Yan, Haoran Chen, Jiahua Wang, Yingyi Wang, Yeqin Yang, Lue Xiang, Zailong Chi, Kaiqun Ren, Bin Lin, Ge Lin, Jinsong Li, Yong Liu, Feng Gu.
      Adenine base editors (ABEs) are genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion. The discovery of split genes revealed that all introns contain two highly conserved dinucleotides, canonical "AG" (acceptor) and "GT" (donor) splice sites. ABE can directly edit splice acceptor sites of the adenine (A) base, leading to aberrant gene splicing, which may be further adopted to remodel splicing. However, spliced isoforms triggered with ABE have not been well explored. To address it, we initially generated a cell line harboring C-terminal enhanced Green Fluorescent Protein (eGFP)-tagged β-actin (ACTB), in which the eGFP signal can track endogenous β-actin expression. Expectedly, after the editing of splice acceptor sites, we observed a dramatical decrease in the percentage of eGFP-positive cells and generation of splicing products with the non-canonical splice site. Furthermore, we manipulated Peroxidasin in mouse embryos with ABE, in which a non-canonical acceptor was activated to remodel splicing, successfully generating a mouse disease model of anophthalmia and severely malformed microphthalmia. Collectively, we demonstrate that ABE-mediated splicing remodeling (ABE-SR) can activate a non-canonical acceptor to manipulate human and mouse genomes, which will facilitate the investigation of basic and translational medicine studies.
    Keywords:  Adenine base editors (ABEs); Gene editing; Non-canonical acceptor; Splice sites; Splicing acceptor
    DOI:  https://doi.org/10.1016/j.jbc.2023.105442
  22. Stem Cells. 2023 Nov 02. pii: sxad077. [Epub ahead of print]
    Single-Cell RNA Sequencing Technology Landscape in 2023.
    Hui-Qi Qu, Charlly Kao, Hakon Hakonarson.
      Single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of cellular heterogeneity and the dynamics of gene expression, bearing profound significance in stem cell research. Depending on the starting materials used for analysis, scRNA-seq encompasses scRNA-seq and single-nucleus RNA sequencing (snRNA-seq). scRNA-seq excels in capturing cellular heterogeneity and characterizing rare cell populations within complex tissues, while snRNA-seq is advantageous in situations where intact cell dissociation is challenging or undesirable (eg, epigenomic studies). A number of scRNA-seq technologies have been developed as of late, including but not limited to droplet-based, plate-based, hydrogel-based, and spatial transcriptomics. The number of cells, sequencing depth, and sequencing length in scRNA-seq can vary across different studies. Addressing current technical challenges will drive the future of scRNA-seq, leading to more comprehensive and precise insights into cellular biology and disease mechanisms informing therapeutic interventions.
    Keywords:  cellular heterogeneity; gene expression; single-cell RNA sequencing; single-nucleus RNA sequencing; transcriptomics
    DOI:  https://doi.org/10.1093/stmcls/sxad077
  23. Mol Ther. 2023 Nov 04. pii: S1525-0016(23)00602-0. [Epub ahead of print]
    The State of Cell and Gene Therapy in 2023.
    Daniel Chancellor, David Barrett, Ly Nguyen-Jatkoe, Shardha Millington, Fenwick Eckhardt.
      Progress in the understanding of human diseases has coincided with the advent of precision medicine, whereby the underlying genetic and molecular contributors can be used as diagnostic and therapeutic biomarkers. To address these, drug developers have designed a range of different treatment strategies, including gene therapy, which the American Society of Gene and Cell Therapy defines as the use of genetic material to treat or prevent disease. A number of approaches exist, including the delivery of genetic material in vivo or ex vivo, as well as the use of RNA species to alter gene expression in particular disease states. Through the end of the first quarter of 2023, there are more than 100 different approved gene, cell, and RNA therapies throughout the world, with over 3,700 more in clinical and preclinical development. This review comprehensively captures the landscape for such advanced therapies, including the different genetic technologies employed and diseases targeted in clinical trials.
    DOI:  https://doi.org/10.1016/j.ymthe.2023.11.001
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