bims-muscge 2023-10-01 papers

bims-muscge

Biomed News

on Muscle stem cells and gene therapy

Issue of 2023–10–01
27 papers selected by
Chance Bowman, Dartmouth College

METTL3 Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Regulating MEF2C mRNA Stability in a m6A-Dependent Manner.
DOCK3 regulates normal skeletal muscle regeneration and glucose metabolism.
Deficiency of phosphatidylethanolamine synthesis: consequences for skeletal muscle.
Extracellular matrix contribution to disease progression and dysfunction in myopathy.
Highly contractile 3D tissue engineered skeletal muscles from human iPSCs reveal similarities with primary myoblast-derived tissues.
Evaluation of an AAV9-mini-dystrophin gene therapy candidate in a rat model of Duchenne muscular dystrophy.
mRNA in situ hybridization exhibits unbalanced nuclear/cytoplasmic dystrophin transcript repartition in Duchenne myogenic cells and skeletal muscle biopsies.
Reconstructing vascular networks promotes the repair of skeletal muscle following volumetric muscle loss by pre-vascularized tissue constructs.
A Systemically Administered Unconjugated Antisense Oligonucleotide Targeting DUX4 Improves Muscular Injury and Motor Function in FSHD Model Mice.
Bleomycin-treated myoblasts undergo p21-associated cellular senescence and have severely impaired differentiation.
Bioprocessing Considerations towards the Manufacturing of Therapeutic Skeletal and Smooth Muscle Cells.
Creation of an iPSC-Based Skeletal Muscle Model of McArdle Disease Harbouring the Mutation c.2392T>C (p.Trp798Arg) in the PYGM Gene.
The long non-coding RNA lnc-H19 is important for endurance exercise by maintaining slow muscle fiber types.
Dimethyl fumarate modulates the dystrophic disease program following short-term treatment.
Tenascin-C-EGFR activation induces functional human satellite cell proliferation and promotes wound-healing of skeletal muscles via oleanic acid.
Cryo-EM structures of Myomaker reveal a molecular basis for myoblast fusion.
Therapeutic Myogenesis Induced by Ultrasound Exposure in a Volumetric Skeletal Muscle Loss Injury Model.
A-Band assembly in avian skeletal muscles observed with super-resolution microscopy.
Regulation of adult stem cell function by ketone bodies.
Redox Profile of Skeletal Muscles: Implications for Research Design and Interpretation.
FLII Modulates the Myogenic Differentiation of Progenitor Cells via Actin Remodeling-Mediated YAP1 Regulation.
Advances in Dystrophinopathy Diagnosis and Therapy.
Evolution of CRISPR/Cas Systems for Precise Genome Editing.
A Novel Imaging Method (FIM-ID) Reveals that Myofibrillogenesis Plays a Major Role in the Mechanically Induced Growth of Skeletal Muscle.
Generation of two induced pluripotent stem cell lines from Duchenne muscular dystrophy patients.
Protein-based delivery systems for RNA delivery.
The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs.

Int J Mol Sci. 2023 Sep 14. pii: 14115. [Epub ahead of print]24(18):

METTL3 Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Regulating MEF2C mRNA Stability in a m6A-Dependent Manner.

Sen Zhao, Jiaxue Cao, Yanjin Sun, Helin Zhou, Qi Zhu, Dinghui Dai, Siyuan Zhan, Jiazhong Guo, Tao Zhong, Linjie Wang, Li Li, Hongping Zhang.

  The development of mammalian skeletal muscle is a highly complex process involving multiple molecular interactions. As a prevalent RNA modification, N6-methyladenosine (m6A) regulates the expression of target genes to affect mammalian development. Nevertheless, it remains unclear how m6A participates in the development of goat muscle. In this study, methyltransferase 3 (METTL3) was significantly enriched in goat longissimus dorsi (LD) tissue. In addition, the global m6A modification level and differentiation of skeletal muscle satellite cells (MuSCs) were regulated by METTL3. By performing mRNA-seq analysis, 8050 candidate genes exhibited significant changes in expression level after the knockdown of METTL3 in MuSCs. Additionally, methylated RNA immunoprecipitation sequencing (MeRIP-seq) illustrated that myocyte enhancer factor 2c (MEF2C) mRNA contained m6A modification. Further experiments demonstrated that METTL3 enhanced the differentiation of MuSCs by upregulating m6A levels and expression of MEF2C. Moreover, the m6A reader YTH N6-methyladenosine RNA binding protein C1 (YTHDC1) was bound and stabilized to MEF2C mRNA. The present study reveals that METTL3 enhances myogenic differentiation in MuSCs by regulating MEF2C and provides evidence of a post-transcriptional mechanism in the development of goat skeletal muscle.

Keywords:  MEF2C; METTL3; MuSCs; RNA m6A methylation; goat

DOI:  https://doi.org/10.3390/ijms241814115
FASEB J. 2023 10;37(10): e23198

DOCK3 regulates normal skeletal muscle regeneration and glucose metabolism.

Adrienne Samani, Muthukumar Karuppasamy, Katherine G English, Colin A Siler, Yimin Wang, Jeffrey J Widrick, Matthew S Alexander.

  DOCK (dedicator of cytokinesis) is an 11-member family of typical guanine nucleotide exchange factors (GEFs) expressed in the brain, spinal cord, and skeletal muscle. Several DOCK proteins have been implicated in maintaining several myogenic processes such as fusion. We previously identified DOCK3 as being strongly upregulated in Duchenne muscular dystrophy (DMD), specifically in the skeletal muscles of DMD patients and dystrophic mice. Dock3 ubiquitous KO mice on the dystrophin-deficient background exacerbated skeletal muscle and cardiac phenotypes. We generated Dock3 conditional skeletal muscle knockout mice (Dock3 mKO) to characterize the role of DOCK3 protein exclusively in the adult muscle lineage. Dock3 mKO mice presented with significant hyperglycemia and increased fat mass, indicating a metabolic role in the maintenance of skeletal muscle health. Dock3 mKO mice had impaired muscle architecture, reduced locomotor activity, impaired myofiber regeneration, and metabolic dysfunction. We identified a novel DOCK3 interaction with SORBS1 through the C-terminal domain of DOCK3 that may account for its metabolic dysregulation. Together, these findings demonstrate an essential role for DOCK3 in skeletal muscle independent of DOCK3 function in neuronal lineages.

Keywords:  DOCK3; GLUT4 processing; regeneration; skeletal muscle

DOI:  https://doi.org/10.1096/fj.202300386RR
Function (Oxf). 2023 ;4(6): zqad044

Deficiency of phosphatidylethanolamine synthesis: consequences for skeletal muscle.

Robin Elaine Duncan.



Keywords:  NAFLD; Pcyt2/ECT; hepatosteatosis; insulin resistance; myosteatosis; phosphatidylethanolamine; skeletal muscle

DOI:  https://doi.org/10.1093/function/zqad044
Am J Physiol Cell Physiol. 2023 Sep 25.

Extracellular matrix contribution to disease progression and dysfunction in myopathy.

Ashlee M Long, GaHyun Lee, Alexis Demonbreun, Elizabeth M McNally.

  Myopathic processes affect skeletal muscle and heart. In the muscular dystrophies, which are a subset of myopathies, muscle cells are gradually replaced by fibrosis and fat, impairing muscle function as well as regeneration and repair. In addition to skeletal muscle, these genetic disorders often also affect the heart, where fibrofatty infiltration progressively accumulates in the myocardium, impairing heart function. While considerable effort has focused on gene-corrective and gene-replacement approaches to stabilize myofibers and cardiomyocytes, the continual and ongoing deposition of extracellular matrix itself contributes to tissue and organ dysfunction. Transcriptomic and proteomic profiling, along with high resolution imaging and biophysical measurements, have been applied to define extracellular matrix components and their role in contributing to cardiac and skeletal muscle weakness. More recently, decellularization methods have been adapted to an on-slide format to preserve the spatial geography of the extracellular matrix, allowing new insight into matrix remodeling and its direct role in suppressing regeneration in muscle. This review highlights recent literature with focus on the extracellular matrix and molecular mechanisms that contribute to muscle and heart fibrotic disorders. We will also compare how the myopathic matrix differs from healthy matrix, emphasizing how the pathological matrix contributes to disease.

Keywords:  Extracellular matrix; decellularization; fibrosi; heart; muscle

DOI:  https://doi.org/10.1152/ajpcell.00182.2023
Stem Cell Reports. 2023 Sep 20. pii: S2213-6711(23)00313-2. [Epub ahead of print]

Highly contractile 3D tissue engineered skeletal muscles from human iPSCs reveal similarities with primary myoblast-derived tissues.

Erik van der Wal, Alessandro Iuliano, Stijn L M In 't Groen, Anjali P Bholasing, Dominik Priesmann, Preeti Sharma, Bianca den Hamer, Vittorio Saggiomo, Marcus Krüger, W W M Pim Pijnappel, Jessica C de Greef.

  Skeletal muscle research is transitioning toward 3D tissue engineered in vitro models reproducing muscle's native architecture and supporting measurement of functionality. Human induced pluripotent stem cells (hiPSCs) offer high yields of cells for differentiation. It has been difficult to differentiate high-quality, pure 3D muscle tissues from hiPSCs that show contractile properties comparable to primary myoblast-derived tissues. Here, we present a transgene-free method for the generation of purified, expandable myogenic progenitors (MPs) from hiPSCs grown under feeder-free conditions. We defined a protocol with optimal hydrogel and medium conditions that allowed production of highly contractile 3D tissue engineered skeletal muscles with forces similar to primary myoblast-derived tissues. Gene expression and proteomic analysis between hiPSC-derived and primary myoblast-derived 3D tissues revealed a similar expression profile of proteins involved in myogenic differentiation and sarcomere function. The protocol should be generally applicable for the study of personalized human skeletal muscle tissue in health and disease.

Keywords:  3D-tissue engineering; contractile force; drug screening; induced pluripotent stem cells; myoblasts; myofiber; organ-on-a-chip; personalized medicine; satellite cell; skeletal muscle

DOI:  https://doi.org/10.1016/j.stemcr.2023.08.014
Mol Ther Methods Clin Dev. 2023 Sep 14. 30 30-47

Evaluation of an AAV9-mini-dystrophin gene therapy candidate in a rat model of Duchenne muscular dystrophy.

Caroline Le Guiner, Xiao Xiao, Thibaut Larcher, Aude Lafoux, Corinne Huchet, Gilles Toumaniantz, Oumeya Adjali, Ignacio Anegon, Séverine Remy, Josh Grieger, Juan Li, Vahid Farrokhi, Hendrik Neubert, Jane Owens, Maritza McIntyre, Philippe Moullier, R Jude Samulski.

  Duchenne muscular dystrophy (DMD) is an X-linked disease caused by loss-of-function mutations in the dystrophin gene and is characterized by muscle wasting and early mortality. Adeno-associated virus-mediated gene therapy is being investigated as a treatment for DMD. In the nonclinical study documented here, we determined the effective dose of fordadistrogene movaparvovec, a clinical candidate adeno-associated virus serotype 9 vector carrying a human mini-dystrophin transgene, after single intravenous injection in a dystrophin-deficient (DMDmdx) rat model of DMD. Overall, we found that transduction efficiency, number of muscle fibers expressing the human mini-dystrophin polypeptide, improvement of the skeletal and cardiac muscle tissue architecture, correction of muscle strength and fatigability, and improvement of diastolic and systolic cardiac function were directly correlated with the amount of vector administered. The effective dose was then tested in older DMDmdx rats with a more dystrophic phenotype similar to the pathology observed in older patients with DMD. Except for a less complete rescue of muscle function in the oldest cohort, fordadistrogene movaparvovec was also found to be therapeutically effective in older DMDmdx rats, suggesting that this product may be appropriate for evaluation in patients with DMD at all stages of disease.

Keywords:  DMDmdx rat; Duchenne muscular dystrophy; age; dose study; gene therapy; mini-dystrophin; recombinant adeno-associated virus

DOI:  https://doi.org/10.1016/j.omtm.2023.05.017
Sci Rep. 2023 09 24. 13(1): 15942

mRNA in situ hybridization exhibits unbalanced nuclear/cytoplasmic dystrophin transcript repartition in Duchenne myogenic cells and skeletal muscle biopsies.

Maria Sofia Falzarano, Martina Mietto, Fernanda Fortunato, Marianna Farnè, Fernanda Martini, Pierpaolo Ala, Rita Selvatici, Francesco Muntoni, Alessandra Ferlini.

To gain insight on dystrophin (DMD) gene transcription dynamics and spatial localization, we assayed the DMD mRNA amount and defined its compartmentalization in myoblasts, myotubes, and skeletal muscle biopsies of Duchenne muscular dystrophy (DMD) patients. Using droplet digital PCR, Real-time PCR, and RNAscope in situ hybridization, we showed that the DMD transcript amount is extremely reduced in both DMD patients' cells and muscle biopsies and that mutation-related differences occur. We also found that, compared to controls, DMD transcript is dramatically reduced in the cytoplasm, as up to 90% of it is localized in nuclei, preferentially at the perinuclear region. Using RNA/protein colocalization experiments, we showed that about 40% of nuclear DMD mRNA is localized in the nucleoli in both control and DMD myogenic cells. Our results clearly show that mutant DMD mRNA quantity is strongly reduced in the patients' myogenic cells and muscle biopsies. Furthermore, mutant DMD mRNA compartmentalization is spatially unbalanced due to a shift in its localization towards the nuclei. This abnormal transcript repartition contributes to the poor abundance and availability of the dystrophin messenger in cytoplasm. This novel finding also has important repercussions for RNA-targeted therapies.

DOI: https://doi.org/10.1038/s41598-023-43134-6
J Tissue Eng. 2023 Jan-Dec;14:14 20417314231201231

Reconstructing vascular networks promotes the repair of skeletal muscle following volumetric muscle loss by pre-vascularized tissue constructs.

Chih-Long Chen, Shih-Yen Wei, Wei-Lin Chen, Ting-Lun Hsu, Ying-Chieh Chen.

  Current treatment for complex and large-scale volumetric muscle loss (VML) injuries remains a limited success and have substantial disadvantages, due to the irreversible loss of muscle mass, slow muscle regeneration, and rapid formation of non-functional fibrosis scars. These VML injuries are accompanied by denervation and the destruction of native vasculature which increases difficulties in the functional restoration of muscle. Here, reconstruction of the vascular network at the injury site was offered as a possible solution for improving the repair of muscle defects through the timely supply of nutrients and oxygen to surrounding cells. A hydrogel-based tissue construct containing various densities of the vascular network was successfully created in the subcutaneous space of mice by manipulating hydrogel properties, and then implanted into the VML injury site. One month after implantation, the mouse treated with the highly vascularized tissue had extensive muscle repair at the injury site and only spent a shorter time completing the inclined plane tests. These findings suggest that the reconstruction of the functional vascular network at the VML injury site accelerated muscle fiber repair through a timely supply of sufficient blood and avoided invasion by host fibroblasts.

Keywords:  Volumetric muscle loss (VML); cell-laden hydrogel; muscle repair; preformed vascular networks; vascular tissue engineering

DOI:  https://doi.org/10.1177/20417314231201231
Biomedicines. 2023 Aug 22. pii: 2339. [Epub ahead of print]11(9):

A Systemically Administered Unconjugated Antisense Oligonucleotide Targeting DUX4 Improves Muscular Injury and Motor Function in FSHD Model Mice.

Tetsuhiro Kakimoto, Akira Ogasawara, Kiyoshi Ishikawa, Takashi Kurita, Kumiko Yoshida, Shuichi Harada, Taeko Nonaka, Yoshimi Inoue, Keiko Uchida, Takashi Tateoka, Tetsuya Ohta, Shinji Kumagai, Takashi Sasaki, Hajime Aihara.

  Facioscapulohumeral muscular dystrophy (FSHD), one of the most common muscular dystrophies, is caused by an abnormal expression of the DUX4 gene in skeletal muscles, resulting in muscle weakness. In this study, we investigated MT-DUX4-ASO, a novel gapmer antisense oligonucleotide (ASO). MT-DUX4-ASO decreased the expression of DUX4 and its target genes in FSHD patient-derived myoblasts. For the first time, we demonstrated that a systemically administered ASO, even without a ligand for drug delivery, could significantly improve muscle injury and motor function in the ACTA1-MCM/FLExDUX4 (DUX4-TG) mouse model of FSHD. Tamoxifen (TMX) injection transiently induces skeletal-muscle-specific DUX4 expression in DUX4-TG mice, while the skeletal muscles of TMX-untreated DUX4-TG mice have leaky DUX4 expression in a small subset of myofibers similar to those of FSHD patients. Subcutaneous 10 mg/kg of MT-DUX4-ASO at two-week intervals significantly suppressed muscular DUX4 target gene expression, histological muscle injury, and blood muscle injury marker elevation in TMX-untreated DUX4-TG mice. Notably, MT-DUX4-ASO at 10 mg/kg every other week significantly prevented the TMX-induced declines in treadmill test running speed and muscle force in DUX4-TG mice. Thus, the systemically administered unconjugated MT-DUX4-ASO suppressed disease progression in DUX4-TG mice, extending the potential of unconjugated ASOs as a promising FSHD treatment strategy.

Keywords:  DUX4; antisense oligonucleotide; facioscapulohumeral muscular dystrophy; motor function; muscle force; muscle injury

DOI:  https://doi.org/10.3390/biomedicines11092339
Geroscience. 2023 Sep 26.

Bleomycin-treated myoblasts undergo p21-associated cellular senescence and have severely impaired differentiation.

Michael Kamal, Sophie Joanisse, Gianni Parise.

  As we age, the ability to regenerate and repair skeletal muscle damage declines, partially due to increasing dysfunction of muscle resident stem cells-satellite cells (SC). Recent evidence implicates cellular senescence, which is the irreversible arrest of proliferation, as a potentiator of SC impairment during aging. However, little is known about the role of senescence in SC, and there is a large discrepancy in senescence classification within skeletal muscle. The purpose of this study was to develop a model of senescence in skeletal muscle myoblasts and identify how common senescence-associated biomarkers respond. Low-passage C2C12 myoblasts were treated with bleomycin or vehicle and then evaluated for cytological and molecular senescence markers, proliferation status, cell cycle kinetics, and differentiation potential. Bleomycin treatment caused double-stranded DNA breaks, which upregulated p21 mRNA and protein, potentially through NF-κB and senescence-associated super enhancer (SASE) signaling (p < 0.01). Consequently, cell proliferation was abruptly halted due to G2/M-phase arrest (p < 0.01). Bleomycin-treated myoblasts displayed greater senescence-associated β-galactosidase staining (p < 0.01), which increased over several days. These myoblasts remained senescent following 6 days of differentiation and had significant impairments in myotube formation (p < 0.01). Furthermore, our results show that senescence can be maintained despite the lack of p16 gene expression in C2C12 myoblasts. In conclusion, bleomycin treatment provides a valid model of damage-induced senescence that was associated with elevated p21, reduced myoblast proliferation, and aberrant cell cycle kinetics, while confirming that a multi-marker approach is needed for the accurate classification of senescence within skeletal muscle.

Keywords:  Aging; Bleomycin; Myoblast; Senescence; Skeletal muscle

DOI:  https://doi.org/10.1007/s11357-023-00929-9
Bioengineering (Basel). 2023 Sep 09. pii: 1067. [Epub ahead of print]10(9):

Bioprocessing Considerations towards the Manufacturing of Therapeutic Skeletal and Smooth Muscle Cells.

Teresa Franchi-Mendes, Marília Silva, Ana Luísa Cartaxo, Ana Fernandes-Platzgummer, Joaquim M S Cabral, Cláudia L da Silva.

  Tissue engineering approaches within the muscle context represent a promising emerging field to address the current therapeutic challenges related with multiple pathological conditions affecting the muscle compartments, either skeletal muscle or smooth muscle, responsible for involuntary and voluntary contraction, respectively. In this review, several features and parameters involved in the bioprocessing of muscle cells are addressed. The cell isolation process is depicted, depending on the type of tissue (smooth or skeletal muscle), followed by the description of the challenges involving the use of adult donor tissue and the strategies to overcome the hurdles of reaching relevant cell numbers towards a clinical application. Specifically, the use of stem/progenitor cells is highlighted as a source for smooth and skeletal muscle cells towards the development of a cellular product able to maintain the target cell's identity and functionality. Moreover, taking into account the need for a robust and cost-effective bioprocess for cell manufacturing, the combination of muscle cells with biomaterials and the need for scale-up envisioning clinical applications are also approached.

Keywords:  cell manufacturing; skeletal muscle cells; smooth muscle cells; tissue engineering

DOI:  https://doi.org/10.3390/bioengineering10091067
Biomedicines. 2023 Aug 31. pii: 2434. [Epub ahead of print]11(9):

Creation of an iPSC-Based Skeletal Muscle Model of McArdle Disease Harbouring the Mutation c.2392T>C (p.Trp798Arg) in the PYGM Gene.

Victoria Cerrada, Inés García-Consuegra, Joaquín Arenas, M Esther Gallardo.

  McArdle disease is a rare autosomal recessive condition caused by mutations in the PYGM gene. This gene encodes the skeletal muscle isoform of glycogen phosphorylase or myophosphorylase. Patients with McArdle disease have an inability to obtain energy from their muscle glycogen stores, which manifests as a marked exercise intolerance. Nowadays, there is no cure for this disorder and recommendations are intended to prevent and mitigate symptoms. There is great heterogeneity among the pathogenic variants found in the PYGM gene, and there is no obvious correlation between genotypes and phenotypes. Here, we present the generation of the first human iPSC-based skeletal muscle model harbouring the second most frequent mutation in PYGM in the Spanish population: NM_005609.4: c.2392T>C (p.Trp798Arg). To this end, iPSCs derived from a McArdle patient and a healthy control were both successfully differentiated into skeletal muscle cells using a small molecule-based protocol. The created McArdle skeletal muscle model was validated by confirming distinctive biochemical aspects of the disease such as the absence of myophosphorylase, the most typical biochemical feature of these patients. This model will be very valuable for use in future high-throughput pharmacological screenings.

Keywords:  McArdle disease; PYGM; disease modelling; iPSCs; induced pluripotent stem cell; skeletal muscle differentiation

DOI:  https://doi.org/10.3390/biomedicines11092434
J Biol Chem. 2023 Sep 22. pii: S0021-9258(23)02309-8. [Epub ahead of print] 105281

The long non-coding RNA lnc-H19 is important for endurance exercise by maintaining slow muscle fiber types.

Yongqi Yue, Yanru Yue, Zeyu Fan, Yingying Meng, Chenglong Wen, Yalong An, Ying Yao, Xiao Li.

  Skeletal muscle consists of different muscle fiber types whose heterogeneity is characterized by different metabolic patterns and expression of MyHC isomers. The transformation of muscle fiber types is regulated by a complex molecular network in which long non-coding RNAs (lncRNAs) play an important role. In this study, we found that lnc-H19 is more enriched in slow muscle fibers. In vitro, interference of lnc-H19 by siRNA significantly promoted the expression of fast muscle fiber gene MyHC IIB and inhibited the expression of the slow muscle fiber gene MyHC I, thereby leading to a fast muscle fiber phenotype. Additionally, interference of lnc-H19 significantly inhibited mRNA expression of the mitochondrial genes, such as COX5A, COX -2, UQCRFSL, FABP3 and CD36. Overexpression of lnc-H19 resulted in an opposite result. In vivo, knockdown of lnc-H19 by AAV-shRNA-H19 suppressed the mRNA expression of the slow muscle fiber gene MyHC I and the protein expression of slow-MyHC. Simultaneously, mitochondria were reduced in number, swollen and vacuolated. The activities of succinate dehydrogenase (SDH), lactic dehydrogenase (LDH), and superoxide dismutase (SOD) were significantly inhibited, and malondialdehyde (MDA) content was significantly increased, indicating that deficiency of lnc-H19 leads to decreased oxidative metabolism and antioxidant capacity in muscle. Further, inhibition of lnc-H19 decreased the weight-bearing swimming time and limb suspension time of mice. In conclusion, our results revealed the role of lnc-H19 in maintaining slow muscle fiber types and maintaining exercise endurance, which may help to further improve the regulatory network of lnc-H19 in muscle function.

Keywords:  Lnc-H19; endurance exercise; mitochondria; myofiber type

DOI:  https://doi.org/10.1016/j.jbc.2023.105281
JCI Insight. 2023 Sep 26. pii: e165974. [Epub ahead of print]

Dimethyl fumarate modulates the dystrophic disease program following short-term treatment.

Cara A Timpani, Stephanie Kourakis, Danielle A Debruin, Dean G Campelj, Nancy Pompeani, Narges Dargahi, Angelo Patrick R Bautista, Ryan M Bagaric, Elya J Ritenis, Lauren Sahakian, Didier Debrincat, Nicole Stupka, Patricia Hafner, Peter G Arthur, Jessica R Terrill, Vasso Apostolopoulos, Judy B De Haan, Nuri Gueven, Dirk Fischer, Emma Rybalka.

  New medicines are urgently required to treat the fatal neuromuscular disease, Duchenne muscular dystrophy (DMD). Dimethyl fumarate (DMF) is a potent immunomodulatory small molecule nuclear erythroid 2-related factor 2 (Nrf2) activator with current clinical utility in the treatment of multiple sclerosis and psoriasis that could be effective for DMD and rapidly translatable. Here, we tested two weeks of daily 100mg/kg DMF versus 5mg/kg standard care prednisone (PRED) treatment in juvenile mdx mice with early symptomatic DMD. Both drugs modulated seed genes driving the DMD disease program and improved force production in fast-twitch muscle. However, only DMF showed pro-mitochondrial effects, protected contracting muscles from fatigue, improved histopathology and augmented clinically compatible muscle function tests. DMF may be a more selective modulator of the DMD disease program than PRED warranting follow-up longitudinal studies to evaluate disease modifying impact.

Keywords:  Drug therapy; Muscle Biology; Neuromuscular disease; Skeletal muscle; Therapeutics

DOI:  https://doi.org/10.1172/jci.insight.165974
Dev Biol. 2023 Sep 25. pii: S0012-1606(23)00164-1. [Epub ahead of print]

Tenascin-C-EGFR activation induces functional human satellite cell proliferation and promotes wound-healing of skeletal muscles via oleanic acid.

Hao Zhang, Lin Zhou, Huihao Wang, Wei Gu, Zhiqiang Li, Jun Sun, Xiaoen Wei, Yuxin Zheng.

  Human satellite cells (HuSCs) have been deemed to be the potential cure to treat muscular atrophy diseases such as Duchenne muscular dystrophy. However, the clinical trials of HuSCs were restricted to the inadequacy of donors because of that freshly isolated HuSCs quickly lost the Pax7 expression and myogenesis capacity in vivo after a few days of culture. Here we found that oleanic acid, a kind of triterpenoid endowed with diverse biological functions with treatment potential, could efficiently promote HuSCs proliferation. The HuSCs cultured in the medium supplement with oleanic acid could maintain a high expression level of Pax7 and retain the ability to differentiate into myotubes as well as facilitate muscle regeneration in injured muscles of recipient mice. We further revealed that Tenascin-C acts as the core mechanism to activate the EGFR signaling pathway followed by HuSCs proliferation. Taken together, our data provide an efficient method to expand functional HuSCs and a novel mechanism that controls HuSCs proliferation, which sheds light on the HuSCs-based therapy to treat muscle diseases.

Keywords:  Cell proliferation; EGFR; HuSCs; Oleanic acid; Tenascin-C

DOI:  https://doi.org/10.1016/j.ydbio.2023.09.010
Nat Struct Mol Biol. 2023 Sep 28.

Cryo-EM structures of Myomaker reveal a molecular basis for myoblast fusion.

Tao Long, Yichi Zhang, Linda Donnelly, Hui Li, Yu-Chung Pien, Ning Liu, Eric N Olson, Xiaochun Li.

The fusion of mononucleated myoblasts produces multinucleated muscle fibers leading to the formation of skeletal muscle. Myomaker, a skeletal muscle-specific membrane protein, is essential for myoblast fusion. Here we report the cryo-EM structures of mouse Myomaker (mMymk) and Ciona robusta Myomaker (cMymk). Myomaker contains seven transmembrane helices (TMs) that adopt a G-protein-coupled receptor-like fold. TMs 2-4 form a dimeric interface, while TMs 3 and 5-7 create a lipid-binding site that holds the polar head of a phospholipid and allows the alkyl tails to insert into Myomaker. The similarity of cMymk and mMymk suggests a conserved Myomaker-mediated cell fusion mechanism across evolutionarily distant species. Functional analyses demonstrate the essentiality of the dimeric interface and the lipid-binding site for fusogenic activity, and heterologous cell-cell fusion assays show the importance of transcellular interactions of Myomaker protomers for myoblast fusion. Together, our findings provide structural and functional insights into the process of myoblast fusion.

DOI: https://doi.org/10.1038/s41594-023-01110-8
Am J Sports Med. 2023 Sep 25. 3635465231195850

Therapeutic Myogenesis Induced by Ultrasound Exposure in a Volumetric Skeletal Muscle Loss Injury Model.

Farina Mohamad Yusoff, Ayumu Nakashima, Masato Kajikawa, Shinji Kishimoto, Tatsuya Maruhashi, Yukihito Higashi.

   BACKGROUND: Low-intensity pulsed ultrasound (LIPUS) irradiation has been shown to induce various responses in different cells. It has been shown that LIPUS activates extracellular signal-regulated kinase 1/2 (ERK1/2) through integrin.
PURPOSE: To study the effects of LIPUS on myogenic regulatory factors and other related myogenesis elements in a volumetric skeletal muscle loss injury model.
STUDY DESIGN: Controlled laboratory study.
METHODS: C57BL/6J mice were subjected to full-thickness muscle defect injury of the quadriceps and treated with direct application of LIPUS 20 min/d or non-LIPUS treatment (control) for 3, 7, and 14 days. LIPUS was also applied to C2C12 cells in culture in the presence of low and high doses of lipopolysaccharides. The expression levels of myogenic regulatory factors and the expression levels of myokine-related and angiogenic-related proteins of the control and LIPUS groups were analyzed.
RESULTS: Muscle volume in the injury site was restored at day 14 with LIPUS treatment. Paired-box protein 7, myogenic factor 5, myogenin, and desmin expressions were significantly different between control and LIPUS groups at days 7 and 14. Myokine and angiogenic cytokine-related factors were significantly increased in the LIPUS group at day 3 and decreased with no significant difference between the groups by day 14. LIPUS induced different responses of myogenic regulatory factors in C2C12 cells with low and high doses of lipopolysaccharides. LIPUS promoted myogenesis through short-lived increase in interleukin-6 and heme oxygenase 1, together with activation of ERK1/2.
CONCLUSION: LIPUS had a constant effect on the variables of tissue damage, from macrotrauma to microtrauma, leading to efficient muscle regeneration.
CLINICAL RELEVANCE: The focus of therapeutic strategies with LIPUS has been not only for microvascular regeneration but also for skeletal muscle and related local tissue recovery from acute or chronic damage.

Keywords:  low-intensity pulsed ultrasound; myogenesis; volumetric skeletal muscle loss injury

DOI:  https://doi.org/10.1177/03635465231195850
Cytoskeleton (Hoboken). 2023 Sep 28.

A-Band assembly in avian skeletal muscles observed with super-resolution microscopy.

Matthew Welchons, Jushuo Wang, Yingli Fan, Jean M Sanger, Joseph W Sanger.

  Myofibrils in vertebrate skeletal muscle are organized in aligned arrays of filaments formed from multiple protein components. Despite considerable information describing individual proteins, how they assemble de novo into mature myofibrils is still a challenge. Studies in our lab of sarcomeric protein localization during myofibril assembly led us to propose a three-step progression: premyofibrils to nascent myofibrils, culminating in mature myofibrils. Premyofibrils, forming at the spreading edges of muscle cells, are composed of minisarcomeres containing small bands of non-muscle myosin II filaments alternating with muscle-specific α-actinin Z-Bodies attached to barbed ends of actin filaments, establishing bipolar F-actin arrangements in sarcomeres. Assembly of nascent myofibrils occurs with addition of muscle-specific myosin II, F-actin, titin, and the alignment of Z-Bodies in adjacent fibrils to form beaded Z-Bands. Muscle-specific myosin II filaments in nascent myofibrils appear in an overlapping arrangement when viewed with wide-field and confocal microscopes. In mature myofibrils, non-muscle myosin II is absent, and M-Band proteins localize to the muscle myosin II filaments, aiding their alignment by cross-linking them into A-Bands. Super-resolution microscopy (SIM and STED) revealed muscle myosin II in mini-A-Bands in nascent myofibrils. In contrast to previous reports that vertebrate muscle myosin thick filaments form at their final 1.6 μm lengths, mini-A-Bands are first detected at a length of about 0.4 μm, and gradually increase four-fold in length to 1.6 μm in mature myofibrils. These new discoveries in avian skeletal muscle cells share a common characteristic with invertebrate muscles where some A-Bands can grow to lengths reaching 25 μm.

Keywords:  SIM; STED; mini-A-Bands; myofibrillogenesis; nascent myofibrils

DOI:  https://doi.org/10.1002/cm.21792
Front Cell Dev Biol. 2023 ;11 1246998

Regulation of adult stem cell function by ketone bodies.

Ole Emil Andersen, Jens Vase Poulsen, Jean Farup, Antoine de Morree.

  Adult stem cells play key roles in tissue homeostasis and regeneration. Recent evidence suggests that dietary interventions can significantly impact adult stem cell function. Some of these effects depend on ketone bodies. Adult stem cells could therefore potentially be manipulated through dietary regimens or exogenous ketone body supplementation, a possibility with significant implications for regenerative medicine. In this review we discuss recent findings of the mechanisms by which ketone bodies could influence adult stem cells, including ketogenesis in adult stem cells, uptake and transport of circulating ketone bodies, receptor-mediated signaling, and changes to cellular metabolism. We also discuss the potential effects of ketone bodies on intracellular processes such as protein acetylation and post-transcriptional control of gene expression. The exploration of mechanisms underlying the effects of ketone bodies on stem cell function reveals potential therapeutic targets for tissue regeneration and age-related diseases and suggests future research directions in the field of ketone bodies and stem cells.

Keywords:  adult stem cells; beta-hydroxybutyrylation; cellular metabolism; ketone bodies; regenerative medicine; stem cell function; tissue homeostasis

DOI:  https://doi.org/10.3389/fcell.2023.1246998
Antioxidants (Basel). 2023 Sep 07. pii: 1738. [Epub ahead of print]12(9):

Redox Profile of Skeletal Muscles: Implications for Research Design and Interpretation.

Olga Vasileiadou, George G Nastos, Panagiotis N Chatzinikolaou, Dimitrios Papoutsis, Dimitra I Vrampa, Spyridon Methenitis, Nikos V Margaritelis.

  Mammalian skeletal muscles contain varying proportions of Type I and II fibers, which feature different structural, metabolic and functional properties. According to these properties, skeletal muscles are labeled as 'red' or 'white', 'oxidative' or 'glycolytic', 'slow-twitch' or 'fast-twitch', respectively. Redox processes (i.e., redox signaling and oxidative stress) are increasingly recognized as a fundamental part of skeletal muscle metabolism at rest, during and after exercise. The aim of the present review was to investigate the potential redox differences between slow- (composed mainly of Type I fibers) and fast-twitch (composed mainly of Type IIa and IIb fibers) muscles at rest and after a training protocol. Slow-twitch muscles were almost exclusively represented in the literature by the soleus muscle, whereas a wide variety of fast-twitch muscles were used. Based on our analysis, we argue that slow-twitch muscles exhibit higher antioxidant enzyme activity compared to fast-twitch muscles in both pre- and post-exercise training. This is also the case between heads or regions of fast-twitch muscles that belong to different subcategories, namely Type IIa (oxidative) versus Type IIb (glycolytic), in favor of the former. No safe conclusion could be drawn regarding the mRNA levels of antioxidant enzymes either pre- or post-training. Moreover, slow-twitch skeletal muscles presented higher glutathione and thiol content as well as higher lipid peroxidation levels compared to fast-twitch. Finally, mitochondrial hydrogen peroxide production was higher in fast-twitch muscles compared to slow-twitch muscles at rest. This redox heterogeneity between different muscle types may have ramifications in the analysis of muscle function and health and should be taken into account when designing exercise studies using specific muscle groups (e.g., on an isokinetic dynamometer) or isolated muscle fibers (e.g., electrical stimulation) and may deliver a plausible explanation for the conflicting results about the ergogenic potential of antioxidant supplements.

Keywords:  antioxidants; enzymes; fibers; oxidative stress; redox; skeletal muscle

DOI:  https://doi.org/10.3390/antiox12091738
Int J Mol Sci. 2023 Sep 20. pii: 14335. [Epub ahead of print]24(18):

FLII Modulates the Myogenic Differentiation of Progenitor Cells via Actin Remodeling-Mediated YAP1 Regulation.

Mai Thi Nguyen, Quoc Kiet Ly, Hyun-Jung Kim, Wan Lee.

  The dynamic rearrangement of the actin cytoskeleton plays an essential role in myogenesis, which is regulated by diverse mechanisms, such as mechanotransduction, modulation of the Hippo signaling pathway, control of cell proliferation, and the influence of morphological changes. Despite the recognized importance of actin-binding protein Flightless-1 (FLII) during actin remodeling, the role played by FLII in the differentiation of myogenic progenitor cells has not been explored. Here, we investigated the roles of FLII in the proliferation and differentiation of myoblasts. FLII was found to be enriched in C2C12 myoblasts, and its expression was stable during the early stages of differentiation but down-regulated in fully differentiated myotubes. Knockdown of FLII in C2C12 myoblasts resulted in filamentous actin (F-actin) accumulation and inhibited Yes-associated protein 1 (YAP1) phosphorylation, which triggers its nuclear translocation from the cytoplasm. Consequently, the expressions of YAP1 target genes, including PCNA, CCNB1, and CCND1, were induced, and the cell cycle and proliferation of myoblasts were promoted. Moreover, FLII knockdown significantly inhibited the expression of myogenic regulatory factors, i.e., MyoD and MyoG, thereby impairing myoblast differentiation, fusion, and myotube formation. Thus, our findings demonstrate that FLII is crucial for the differentiation of myoblasts via modulation of the F-actin/YAP1 axis and suggest that FLII is a putative novel therapeutic target for muscle wasting.

Keywords:  FLII; actin remodeling; differentiation; mechanotransduction; myogenesis

DOI:  https://doi.org/10.3390/ijms241814335
Biomolecules. 2023 Aug 28. pii: 1319. [Epub ahead of print]13(9):

Advances in Dystrophinopathy Diagnosis and Therapy.

Fawzy A Saad, Gabriele Siciliano, Corrado Angelini.

  Dystrophinopathies are x-linked muscular disorders which emerge from mutations in the Dystrophin gene, including Duchenne and Becker muscular dystrophy, and dilated cardiomyopathy. However, Duchenne muscular dystrophy interconnects with bone loss and osteoporosis, which are exacerbated by glucocorticoids therapy. Procedures for diagnosing dystrophinopathies include creatine kinase assay, haplotype analysis, Southern blot analysis, immunological analysis, multiplex PCR, multiplex ligation-dependent probe amplification, Sanger DNA sequencing, and next generation DNA sequencing. Pharmacological therapy for dystrophinopathies comprises glucocorticoids (prednisone, prednisolone, and deflazacort), vamorolone, and ataluren. However, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and β-blockers are the first-line to prevent dilated cardiomyopathy in dystrophinopathy patients. Duchenne muscular dystrophy gene therapy strategies involve gene transfer, exon skipping, exon reframing, and CRISPR gene editing. Eteplirsen, an antisense-oligonucleotide drug for skipping exon 51 from the Dystrophin gene, is available on the market, which may help up to 14% of Duchenne muscular dystrophy patients. There are various FDA-approved exon skipping drugs including ExonDys-51 for exon 51, VyonDys-53 and Viltolarsen for exon 53 and AmonDys-45 for exon 45 skipping. Other antisense oligonucleotide drugs in the pipeline include casimersen for exon 45, suvodirsen for exon 51, and golodirsen for exon 53 skipping. Advances in the diagnosis and therapy of dystrophinopathies offer new perspectives for their early discovery and care.

Keywords:  Becker muscular dystrophy; CRISPR gene editing; Duchenne muscular dystrophy; dilated cardiomyopathy; dystrophinopathies; gene drugs; gene therapy; pharmacological therapy; prime gene editing; serum creatine kinase

DOI:  https://doi.org/10.3390/biom13091319
Int J Mol Sci. 2023 Sep 18. pii: 14233. [Epub ahead of print]24(18):

Evolution of CRISPR/Cas Systems for Precise Genome Editing.

Magdalena Hryhorowicz, Daniel Lipiński, Joanna Zeyland.

  The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges remain, such as the risk of immunological reactions or off-target effects. To overcome these problems, many new methods and CRISPR/Cas-based tools have been developed. In this review, we describe the current classification of CRISPR systems and new precise genome-editing technologies, summarize the latest applications of this technique in several fields of research, and, finally, discuss CRISPR/Cas system limitations, ethical issues, and challenges.

Keywords:  CRISPR classification; CRISPR/Cas9 system; Cas12a nuclease; Cas13a nuclease; Cas9 nuclease; base editors; genome editing; prime editors

DOI:  https://doi.org/10.3390/ijms241814233
bioRxiv. 2023 Sep 15. pii: 2023.09.13.557204. [Epub ahead of print]

A Novel Imaging Method (FIM-ID) Reveals that Myofibrillogenesis Plays a Major Role in the Mechanically Induced Growth of Skeletal Muscle.

Kent W Jorgenson, Jamie E Hibbert, Ramy K A Sayed, Anthony N Lange, Joshua S Godwin, Paulo H C Mesquita, Bradley A Ruple, Mason C McIntosh, Andreas N Kavazis, Michael D Roberts, Troy A Hornberger.

An increase in mechanical loading, such as that which occurs during resistance exercise, induces radial growth of muscle fibers (i.e., an increase in cross-sectional area). Muscle fibers are largely composed of myofibrils, but whether radial growth is mediated by an increase in the size of the myofibrils (i.e., myofibril hypertrophy) and/or the number of myofibrils (i.e., myofibrillogenesis) is not known. Electron microscopy (EM) can provide images with the level of resolution that is needed to address this question, but the acquisition and subsequent analysis of EM images is a time- and cost-intensive process. To overcome this, we developed a novel method for visualizing myofibrils with a standard fluorescence microscope (FIM-ID). Images from FIM-ID have a high degree of resolution and contrast, and these properties enabled us to develop pipelines for automated measurements of myofibril size and number. After extensively validating the automated measurements, we used both mouse and human models of increased mechanical loading to discover that the radial growth of muscle fibers is largely mediated by myofibrillogenesis. Collectively, the outcomes of this study offer insight into a foundationally important topic in the field of muscle growth and provide future investigators with a time- and cost-effective means to study it.

DOI: https://doi.org/10.1101/2023.09.13.557204
Stem Cell Res. 2023 Sep 18. pii: S1873-5061(23)00193-9. [Epub ahead of print]72 103207

Generation of two induced pluripotent stem cell lines from Duchenne muscular dystrophy patients.

Wenqiang Liu, Wenshu Zeng, Xiaohui Kong, Min Htet, Rebecca Yu, Matthew Wheeler, John W Day, Joseph C Wu.

  Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that leads to death in early adulthood. Patients with DMD have null mutations leading to loss of functional dystrophin protein. Here we generated two DMD induced pluripotent stem cell (iPSC) lines, one with deletion of exon 51 and the other with a single nucleotide nonsense mutation (c.10171C > T). Both lines expressed high levels of pluripotency markers, had the capability of differentiating into derivatives of the three germ layers, and possessed normal karyotypes. These iPSC lines can serve as powerful tools to model DMD in vitro and as a platform for therapeutic development.

Keywords:  Duchenne muscular dystrophy; Human induced pluripotent stem cells

DOI:  https://doi.org/10.1016/j.scr.2023.103207
J Control Release. 2023 Sep 21. pii: S0168-3659(23)00619-3. [Epub ahead of print]

Protein-based delivery systems for RNA delivery.

Haichao Zhu, Hong Luo, Ruilong Chang, Yifan Yang, Dingkang Liu, Yue Ji, Hai Qin, Haibo Rong, Jun Yin.

  RNA-based therapeutics have emerged as promising approaches to modulate gene expression and generate therapeutic proteins or antigens capable of inducing immune responses to treat a variety of diseases, such as infectious diseases, cancers, immunologic disorders, and genetic disorders. However, the efficient delivery of RNA molecules into cells poses significant challenges due to their large molecular weight, negative charge, and susceptibility to degradation by RNase enzymes. To overcome these obstacles, viral and non-viral vectors have been developed, including lipid nanoparticles, viral vectors, proteins, dendritic macromolecules, among others. Among these carriers, protein-based delivery systems have garnered considerable attention due to their potential to address specific issues associated with nanoparticle-based systems, such as liver accumulation and immunogenicity. This review provides an overview of currently marketed RNA drugs, underscores the significance of RNA delivery vector development, delineates the essential characteristics of an ideal RNA delivery vector, and introduces existing protein carriers for RNA delivery. By offering valuable insights, this review aims to serve as a reference for the future development of protein-based delivery vectors for RNA therapeutics.

Keywords:  Delivery vectors; Protein carriers; RNA delivery challenges; RNA drug; RNA-based therapeutics

DOI:  https://doi.org/10.1016/j.jconrel.2023.09.032
PLoS Biol. 2023 Sep 25. 21(9): e3002317

The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs.

Danielle C Hamm, Ellen M Paatela, Sean R Bennett, Chao-Jen Wong, Amy E Campbell, Cynthia L Wladyka, Andrew A Smith, Sujatha Jagannathan, Andrew C Hsieh, Stephen J Tapscott.

Translational control is critical for cell fate transitions during development, lineage specification, and tumorigenesis. Here, we show that the transcription factor double homeobox protein 4 (DUX4), and its previously characterized transcriptional program, broadly regulates translation to change the cellular proteome. DUX4 is a key regulator of zygotic genome activation in human embryos, whereas misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and is associated with MHC-I suppression and immune evasion in cancer. We report that translation initiation and elongation factors are disrupted downstream of DUX4 expression in human myoblasts. Genome-wide translation profiling identified mRNAs susceptible to DUX4-induced translation inhibition, including those encoding antigen presentation factors and muscle lineage proteins, while DUX4-induced mRNAs were robustly translated. Endogenous expression of DUX4 in human FSHD myotubes and cancer cell lines also correlated with reduced protein synthesis and MHC-I presentation. Our findings reveal that DUX4 orchestrates translational reprogramming by suppressing the cellular proteome while maintaining translation of DUX4-induced mRNAs to promote an early developmental program.

DOI: https://doi.org/10.1371/journal.pbio.3002317