bims-muscge 2023-09-10 papers

bims-muscge

Biomed News

on Muscle stem cells and gene therapy

Issue of 2023–09–10
28 papers selected by
Chance Bowman, Dartmouth College

Evolving Therapeutic Options for the Treatment of Duchenne Muscular Dystrophy.
Applications and Research Advances in the Delivery of CRISPR/Cas9 Systems for the Treatment of Inherited Diseases.
Radiation induces long-term muscle fibrosis and promotes a fibrotic phenotype in fibro-adipogenic progenitors.
Lineage tracing of newly accrued nuclei in skeletal myofibers uncovers distinct transcripts and interplay between nuclear populations.
DMD antisense oligonucleotide mediated exon skipping efficiency correlates with flanking intron retention time and target position within the exon.
Alignment, cross-linking, and beyond: A collagen architect's guide to the skeletal muscle extracellular matrix.
Decoding the transcriptome of Duchenne muscular dystrophy to the single nuclei level reveals clinical-genetic correlations.
Mitochondrial Properties in Skeletal Muscle Fiber.
Reduced Expression of Septin7 Hinders Skeletal Muscle Regeneration.
Efficient mRNA delivery using lipid nanoparticles modified with fusogenic coiled-coil peptides.
Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.
PAX7, a Key for Myogenesis Modulation in Muscular Dystrophies through Multiple Signaling Pathways: A Systematic Review.
Androgen Action on Myogenesis Throughout the Lifespan; Comparison with Neurogenesis.
Caveolin-3 loss linked with the P104L LGMD-1C mutation modulates skeletal muscle mTORC1 signalling and cholesterol homeostasis.
The metabolomic plasma profile of patients with Duchenne muscular dystrophy: providing new evidence for its pathogenesis.
Downregulation of Zebrafish Cytosolic Sialidase Neu3.2 Affects Skeletal Muscle Development.
CRISPR applications in cancer diagnosis and treatment.
Expression and function of Four AAV-based constructs for dystrophin restoration in the mdx mouse model of duchenne muscular dystrophy.
In vitro-generated human muscle reserve cells are heterogeneous for Pax7 with distinct molecular states and metabolic profiles.
Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase.
Effects of Aging on Collagen in the Skeletal Muscle of Mice.
Nonsense-mediated mRNA decay suppresses injury-induced muscle regeneration via inhibiting MyoD transcriptional activity.
Dual Adeno-Associated Virus 9 with Codon-Optimized DYSF Gene Promotes In Vivo Muscle Regeneration and May Decrease Inflammatory Response in Limb Girdle Muscular Dystrophy Type R2.
Cell Derived Extracellular Matrix Fiber Scaffolds Improve Recovery from Volumetric Muscle Loss.
Prolonged mechanical muscle loading increases mechanosensor gene and protein levels and causes a moderate fast-to-slow fiber type switch in mice.
The transcriptional regulator KLF15 is necessary for myoblast differentiation and muscle regeneration by activating FKBP5.
CircLRRFIP1 promotes the proliferation and differentiation of chicken skeletal muscle satellite cells by sponging the miR-15 family via activating AKT3-mTOR/p70S6K signaling pathway.
Evolutionary conserved circular MEF2A RNAs regulate myogenic differentiation and skeletal muscle development.

Neurotherapeutics. 2023 Sep 06.

Evolving Therapeutic Options for the Treatment of Duchenne Muscular Dystrophy.

Eleonora S D'Ambrosio, Jerry R Mendell.

  Duchenne muscular dystrophy (DMD) is the most common childhood form of muscular dystrophy. It is caused by mutations in the DMD gene, leading to reduced or absent expression of the dystrophin protein. Clinically, this results in loss of ambulation, cardiomyopathy, respiratory failure, and eventually death. In the past decades, the use of corticosteroids has slowed down the disease progression. More recently, the development of genetically mediated therapies has emerged as the most promising treatment for DMD. These strategies include exon skipping with antisense oligonucleotides, gene replacement therapy with adeno-associated virus, and gene editing with CRISPR (clustered regularly interspaced short palindromic repeats) technology. In this review, we highlight the most up-to-date therapeutic progresses in the field, with emphasis on past and recent experiences, as well as the latest clinical results of DMD micro-dystrophin gene therapy. Additionally, we discuss the lessons learned along the way and the challenges encountered, all of which have helped advance the field, with the potential to finally alleviate such a devastating disease.

Keywords:  Corticosteroids; Duchenne muscular dystrophy; Exon skipping; Micro-dystrophin; Revertant fibers; Viral vectors

DOI:  https://doi.org/10.1007/s13311-023-01423-y
Int J Mol Sci. 2023 Aug 25. pii: 13202. [Epub ahead of print]24(17):

Applications and Research Advances in the Delivery of CRISPR/Cas9 Systems for the Treatment of Inherited Diseases.

Xinyue Lu, Miaomiao Zhang, Ge Li, Shixin Zhang, Jingbo Zhang, Xiaoge Fu, Fengying Sun.

  The rapid advancements in gene therapy have opened up new possibilities for treating genetic disorders, including Duchenne muscular dystrophy, thalassemia, cystic fibrosis, hemophilia, and familial hypercholesterolemia. The utilization of the clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has revolutionized the field of gene therapy by enabling precise targeting of genes. In recent years, CRISPR/Cas9 has demonstrated remarkable efficacy in treating cancer and genetic diseases. However, the susceptibility of nucleic acid drugs to degradation by nucleic acid endonucleases necessitates the development of functional vectors capable of protecting the nucleic acids from enzymatic degradation while ensuring safety and effectiveness. This review explores the biomedical potential of non-viral vector-based CRISPR/Cas9 systems for treating genetic diseases. Furthermore, it provides a comprehensive overview of recent advances in viral and non-viral vector-based gene therapy for genetic disorders, including preclinical and clinical study insights. Additionally, the review analyzes the current limitations of these delivery systems and proposes avenues for developing novel nano-delivery platforms.

Keywords:  CRISPR/Cas9; gene editing; nano delivery system; non-viral vectors; viral vectors

DOI:  https://doi.org/10.3390/ijms241713202
J Cachexia Sarcopenia Muscle. 2023 Sep 06.

Radiation induces long-term muscle fibrosis and promotes a fibrotic phenotype in fibro-adipogenic progenitors.

Nicolas Collao, Donna D'Souza, Laura Messeiller, Evan Pilon, Jessica Lloyd, Jillian Larkin, Matthew Ngu, Alexanne Cuillerier, Alexander E Green, Keir J Menzies, Yan Burelle, Michael De Lisio.

   BACKGROUND: Radiation-induced muscle pathology, characterized by muscle atrophy and fibrotic tissue accumulation, is the most common debilitating late effect of therapeutic radiation exposure particularly in juvenile cancer survivors. In healthy muscle, fibro/adipogenic progenitors (FAPs) are required for muscle maintenance and regeneration, while in muscle pathology FAPs are precursors for exacerbated extracellular matrix deposition. However, the role of FAPs in radiation-induced muscle pathology has not previously been explored.
METHODS: Four-week-old Male CBA or C57Bl/6J mice received a single dose (16 Gy) of irradiation (IR) to a single hindlimb with the shielded contralateral limb (CLTR) serving as a non-IR control. Mice were sacrificed 3, 7, 14 (acute IR response), and 56 days post-IR (long-term IR response). Changes in skeletal muscle morphology, myofibre composition, muscle niche cellular dynamics, DNA damage, proliferation, mitochondrial respiration, and metabolism and changes in progenitor cell fate where assessed.
RESULTS: Juvenile radiation exposure resulted in smaller myofibre cross-sectional area, particularly in type I and IIA myofibres (P < 0.05) and reduced the proportion of type I myofibres (P < 0.05). Skeletal muscle fibrosis (P < 0.05) was evident at 56 days post-IR. The IR-limb had fewer endothelial cells (P < 0.05) and fibro-adipogenic progenitors (FAPs) (P < 0.05) at 56 days post-IR. Fewer muscle satellite (stem) cells were detected at 3 and 56 days in the IR-limb (P < 0.05). IR induced FAP senescence (P < 0.05), increased their fibrogenic differentiation (P < 0.01), and promoted their glycolytic metabolism. Further, IR altered the FAP secretome in a manner that impaired muscle satellite (stem) cell differentiation (P < 0.05) and fusion (P < 0.05).
CONCLUSIONS: Our study suggests that following juvenile radiation exposure, FAPs contribute to long-term skeletal muscle atrophy and fibrosis. These findings provide rationale for investigating FAP-targeted therapies to ameliorate the negative late effects of radiation exposure in skeletal muscle.

Keywords:  Atrophy; Differentiation; Extracellular matrix; Mesenchymal progenitors; Metabolism; Myofibroblast; Skeletal muscle

DOI:  https://doi.org/10.1002/jcsm.13320
bioRxiv. 2023 Aug 25. pii: 2023.08.24.554609. [Epub ahead of print]

Lineage tracing of newly accrued nuclei in skeletal myofibers uncovers distinct transcripts and interplay between nuclear populations.

Chengyi Sun, Casey O Swoboda, Michael J Petrany, Sreeja Parameswaran, Andrew VonHandorf, Matthew T Weirauch, Christoph Lepper, Douglas P Millay.

Multinucleated skeletal muscle cells have an obligatory need to acquire additional nuclei through fusion with activated skeletal muscle stem cells when responding to both developmental and adaptive growth stimuli. A fundamental question in skeletal muscle biology has been the reason underlying this need for new nuclei in syncytial cells that already harbor hundreds of nuclei. To begin to answer this long-standing question, we utilized nuclear RNA-sequencing approaches and developed a lineage tracing strategy capable of defining the transcriptional state of recently fused nuclei and distinguishing this state from that of pre-existing nuclei. Our findings reveal the presence of conserved markers of newly fused nuclei both during development and after a hypertrophic stimulus in the adult. However, newly fused nuclei also exhibit divergent gene expression that is determined by the myogenic environment to which they fuse. Moreover, accrual of new nuclei through fusion is required for nuclei already resident in adult myofibers to mount a normal transcriptional response to a load-inducing stimulus. We propose a model of mutual regulation in the control of skeletal muscle development and adaptations, where newly fused and pre-existing myonuclear populations influence each other to maintain optimal functional growth.

DOI: https://doi.org/10.1101/2023.08.24.554609
RNA Biol. 2023 Jan;20(1): 693-702

DMD antisense oligonucleotide mediated exon skipping efficiency correlates with flanking intron retention time and target position within the exon.

Remko Goossens, Nisha Verwey, Yavuz Ariyurek, Fred Schnell, Annemieke Aartsma-Rus.

  Mutations in the DMD gene are causative for Duchenne muscular dystrophy (DMD). Antisense oligonucleotide (AON) mediated exon skipping to restore disrupted dystrophin reading frame is a therapeutic approach that allows production of a shorter but functional protein. As DMD causing mutations can affect most of the 79 exons encoding dystrophin, a wide variety of AONs are needed to treat the patient population. Design of AONs is largely guided by trial-and-error, and it is yet unclear what defines the skippability of an exon. Here, we use a library of phosphorodiamidate morpholino oligomer (PMOs) AONs of similar physical properties to test the skippability of a large number of DMD exons. The DMD transcript is non-sequentially spliced, meaning that certain introns are retained longer in the transcript than downstream introns. We tested whether the relative intron retention time has a significant effect on AON efficiency, and found that targeting an out-of-frame exon flanked at its 5'-end by an intron that is retained in the transcript longer ('slow' intron) leads to overall higher exon skipping efficiency than when the 5'-end flanking intron is 'fast'. Regardless of splicing speed of flanking introns, we find that positioning an AON closer to the 5'-end of the target exon leads to higher exon skipping efficiency opposed to targeting an exons 3'-end. The data enclosed herein can be of use to guide future target selection and preferential AON binding sites for both DMD and other disease amenable by exon skipping therapies.

Keywords:  AON; ASO; Antisense oligonucleotides; DMD; Duchenne Muscular Dystrophy; Dystrophin; Exon skipping

DOI:  https://doi.org/10.1080/15476286.2023.2254041
Am J Physiol Cell Physiol. 2023 Sep 04.

Alignment, cross-linking, and beyond: A collagen architect's guide to the skeletal muscle extracellular matrix.

Ross P Wohlgemuth, Sarah E Brashear, Lucas R Smith.

  The muscle extracellular matrix (ECM) forms a complex network of collagens, proteoglycans, and other proteins that produce a favorable environment for muscle regeneration, protect the sarcolemma from contraction-induced damage, and provide a pathway for the lateral transmission of contractile force. In each of these functions, the structure and organization of the muscle ECM plays an important role. Many aspects of collagen architecture, including collagen alignment, cross-linking, and packing density affect the regenerative capacity, passive mechanical properties, and contractile force transmission pathways of skeletal muscle. The balance between fortifying the muscle ECM and maintaining ECM turnover and compliance is highly dependent on the integrated organization, or architecture, of the muscle matrix-especially related to collagen. While muscle ECM remodeling patterns in response to exercise and disease are similar, in that collagen synthesis can increase in both cases, one outcome leads to a stronger muscle and the other leads to fibrosis. In this review we provide a comprehensive analysis of the architectural features of each layer of muscle ECM-epimysium, perimysium, and endomysium. Further, we detail the importance of muscle ECM architecture to biomechanical function in the context of exercise or fibrosis, including disease, injury, and aging. We describe how collagen architecture is linked to active and passive muscle biomechanics, and which architectural features are acutely dynamic and adapt over time. Future studies should investigate the significance of collagen architecture in muscle stiffness, ECM turnover, and lateral force transmission in the context of health and fibrosis.

Keywords:  Collagen; Crosslinking; Extracellular Matrix; Fibrosis; Skeletal muscle

DOI:  https://doi.org/10.1152/ajpcell.00287.2023
Cell Death Dis. 2023 09 07. 14(9): 596

Decoding the transcriptome of Duchenne muscular dystrophy to the single nuclei level reveals clinical-genetic correlations.

Xavier Suárez-Calvet, Esther Fernández-Simón, Daniel Natera, Cristina Jou, Patricia Pinol-Jurado, Elisa Villalobos, Carlos Ortez, Alexandra Monceau, Marianela Schiava, Anna Codina, José Verdu-Díaz, James Clark, Zoe Laidler, Priyanka Mehra, Rasya Gokul-Nath, Jorge Alonso-Perez, Chiara Marini-Bettolo, Giorgio Tasca, Volker Straub, Michela Guglieri, Andrés Nascimento, Jordi Diaz-Manera.

Duchenne muscular dystrophy is a genetic disease produced by mutations in the dystrophin gene characterized by early onset muscle weakness leading to severe and irreversible disability. The cellular and molecular consequences of the lack of dystrophin in humans are only partially known, which is crucial for the development of new therapies aiming to slow or stop the progression of the disease. Here we have analyzed quadriceps muscle biopsies of seven DMD patients aged 2 to 4 years old and five age and gender matched controls using single nuclei RNA sequencing (snRNAseq) and correlated the results obtained with clinical data. SnRNAseq identified significant differences in the proportion of cell population present in the muscle samples, including an increase in the number of regenerative fibers, satellite cells, and fibro-adipogenic progenitor cells (FAPs) and a decrease in the number of slow fibers and smooth muscle cells. Muscle samples from the younger patients with stable mild weakness were characterized by an increase in regenerative fibers, while older patients with moderate and progressive weakness were characterized by loss of muscle fibers and an increase in FAPs. An analysis of the gene expression profile in muscle fibers identified a strong regenerative signature in DMD samples characterized by the upregulation of genes involved in myogenesis and muscle hypertrophy. In the case of FAPs, we observed upregulation of genes involved in the extracellular matrix regeneration but also several signaling pathways. Indeed, further analysis of the potential intercellular communication profile showed a dysregulation of the communication profile in DMD samples identifying FAPs as a key regulator of cell signaling in DMD muscle samples. In conclusion, our study has identified significant differences at the cellular and molecular levels in the different cell populations present in skeletal muscle samples of patients with DMD compared to controls.

DOI: https://doi.org/10.1038/s41419-023-06103-5
Cells. 2023 Aug 30. pii: 2183. [Epub ahead of print]12(17):

Mitochondrial Properties in Skeletal Muscle Fiber.

Han Dong, Shih-Yin Tsai.

  Mitochondria are the primary source of energy production and are implicated in a wide range of biological processes in most eukaryotic cells. Skeletal muscle heavily relies on mitochondria for energy supplements. In addition to being a powerhouse, mitochondria evoke many functions in skeletal muscle, including regulating calcium and reactive oxygen species levels. A healthy mitochondria population is necessary for the preservation of skeletal muscle homeostasis, while mitochondria dysregulation is linked to numerous myopathies. In this review, we summarize the recent studies on mitochondria function and quality control in skeletal muscle, focusing mainly on in vivo studies of rodents and human subjects. With an emphasis on the interplay between mitochondrial functions concerning the muscle fiber type-specific phenotypes, we also discuss the effect of aging and exercise on the remodeling of skeletal muscle and mitochondria properties.

Keywords:  mitochondria; skeletal muscle physiology

DOI:  https://doi.org/10.3390/cells12172183
Int J Mol Sci. 2023 Aug 31. pii: 13536. [Epub ahead of print]24(17):

Reduced Expression of Septin7 Hinders Skeletal Muscle Regeneration.

László Szabó, Andrea Telek, János Fodor, Nóra Dobrosi, Klaudia Dócs, Zoltán Hegyi, Mónika Gönczi, László Csernoch, Beatrix Dienes.

  Septins are considered the fourth component of the cytoskeleton with the septin7 isoform playing a critical role in the formation of diffusion barriers in phospholipid bilayers and intra- and extracellular scaffolds. While its importance has already been confirmed in different intracellular processes, very little is known about its role in skeletal muscle. Muscle regeneration was studied in a Sept7 conditional knock-down mouse model to prove the possible role of septin7 in this process. Sterile inflammation in skeletal muscle was induced which was followed by regeneration resulting in the upregulation of septin7 expression. Partial knock-down of Sept7 resulted in an increased number of inflammatory cells and myofibers containing central nuclei. Taken together, our data suggest that partial knock-down of Sept7 hinders the kinetics of muscle regeneration, indicating its crucial role in skeletal muscle functions.

Keywords:  central nuclei; muscle injury; regeneration; septin7; skeletal muscle

DOI:  https://doi.org/10.3390/ijms241713536
Nanoscale. 2023 Sep 06.

Efficient mRNA delivery using lipid nanoparticles modified with fusogenic coiled-coil peptides.

Ye Zeng, Mengjie Shen, Roy Pattipeiluhu, Xuequan Zhou, Yun Zhang, Thomas Bakkum, Thomas H Sharp, Aimee L Boyle, Alexander Kros.

Gene delivery has great potential in modulating protein expression in specific cells to treat diseases. Such therapeutic gene delivery demands sufficient cellular internalization and endosomal escape. Of various nonviral nucleic acid delivery systems, lipid nanoparticles (LNPs) are the most advanced, but still, are very inefficient as the majority are unable to escape from endosomes/lysosomes. Here, we develop a highly efficient gene delivery system using fusogenic coiled-coil peptides. We modified LNPs, carrying EGFP-mRNA, and cells with complementary coiled-coil lipopeptides. Coiled-coil formation between these lipopeptides induced fast nucleic acid uptake and enhanced GFP expression. The cellular uptake of coiled-coil modified LNPs is likely driven by membrane fusion thereby omitting typical endocytosis pathways. This direct cytosolic delivery circumvents the problems commonly observed with the limited endosomal escape of mRNA. Therefore fusogenic coiled-coil peptide modification of existing LNP formulations to enhance nucleic acid delivery efficiency could be beneficial for several gene therapy applications.

DOI: https://doi.org/10.1039/d3nr02175k
Nucleic Acids Res. 2023 Sep 08. pii: gkad676. [Epub ahead of print]

Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.

Jakob Haldrup, Sofie Andersen, Alexander Rafael LaVilla Labial, Jonas Holst Wolff, Frederik Plum Frandsen, Thomas Wisbech Skov, Anne Bruun Rovsing, Ian Nielsen, Thomas Stax Jakobsen, Anne Louise Askou, Martin K Thomsen, Thomas J Corydon, Emil Aagaard Thomsen, Jacob Giehm Mikkelsen.

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.

DOI: https://doi.org/10.1093/nar/gkad676
Int J Mol Sci. 2023 Aug 22. pii: 13051. [Epub ahead of print]24(17):

PAX7, a Key for Myogenesis Modulation in Muscular Dystrophies through Multiple Signaling Pathways: A Systematic Review.

Nor Idayu A Rahman, Chung Liang Lam, Nadiah Sulaiman, Nur Atiqah Haizum Abdullah, Fazlina Nordin, Shahrul Hisham Zainal Ariffin, Muhammad Dain Yazid.

  Muscular dystrophy is a heterogenous group of hereditary muscle disorders caused by mutations in the genes responsible for muscle development, and is generally defined by a disastrous progression of muscle wasting and massive loss in muscle regeneration. Pax7 is closely associated with myogenesis, which is governed by various signaling pathways throughout a lifetime and is frequently used as an indicator in muscle research. In this review, an extensive literature search adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines was performed to identify research that examined signaling pathways in living models, while quantifying Pax7 expression in myogenesis. A total of 247 articles were retrieved from the Web of Science (WoS), PubMed and Scopus databases and were thoroughly examined and evaluated, resulting in 19 articles which met the inclusion criteria. Admittedly, we were only able to discuss the quantification of Pax7 carried out in research affecting various type of genes and signaling pathways, rather than the expression of Pax7 itself, due to the massive differences in approach, factor molecules and signaling pathways analyzed across the research. However, we highlighted the thorough evidence for the alteration of the muscle stem cell precursor Pax7 in multiple signaling pathways described in different living models, with an emphasis on the novel approach that could be taken in manipulating Pax7 expression itself in dystrophic muscle, towards the discovery of an effective treatment for muscular dystrophy. Therefore, we believe that this could be applied to the potential gap in muscle research that could be filled by tuning the well-established marker expression to improve dystrophic muscle.

Keywords:  Pax7; muscular dystrophy; myogenesis; signaling pathways

DOI:  https://doi.org/10.3390/ijms241713051
Front Neuroendocrinol. 2023 Sep 03. pii: S0091-3022(23)00049-3. [Epub ahead of print] 101101

Androgen Action on Myogenesis Throughout the Lifespan; Comparison with Neurogenesis.

Sabrina Tzivia Barsky, Douglas Ashley Monks.

  Androgens' pleiotropic actions in promoting sex differences present not only a challenge to providing a comprehensive account of their function, but also an opportunity to gain insights by comparing androgenic actions across organ systems. Although often overlooked by neuroscientists, skeletal muscle is another androgen-responsive organ system which shares with the nervous system properties of electrochemical excitability, behavioral relevance, and remarkable capacity for adaptive plasticity. Here we review androgenic regulation of mitogenic plasticity in skeletal muscle with the goal of identifying areas of interest to those researching androgenic mechanisms mediating sexual differentiation of neurogenesis. We use an organizational-activational framework to relate broad areas of similarity and difference between androgen effects on mitogenesis in muscle and brain throughout the lifespan, from early organogenesis, through pubertal organization, adult activation, and aging. The focus of the review is androgenic regulation of muscle-specific stem cells (satellite cells), which share with neural stem cells essential functions in development, plasticity, and repair, albeit with distinct, muscle-specific features. Also considered are areas of paracrine and endocrine interaction between androgen action on muscle and nervous system, including mediation of neural plasticity of innervating and distal neural populations by muscle-produced trophic factors.

Keywords:  Satellite cell; androgen receptor; myogenesis; neurogenesis; neuromuscular system

DOI:  https://doi.org/10.1016/j.yfrne.2023.101101
J Cachexia Sarcopenia Muscle. 2023 Sep 06.

Caveolin-3 loss linked with the P104L LGMD-1C mutation modulates skeletal muscle mTORC1 signalling and cholesterol homeostasis.

Dinesh S Shah, Raid B Nisr, Gabriela Krasteva-Christ, Harinder S Hundal.

   BACKGROUND: Caveolins are the principal structural components of plasma membrane caveolae. Dominant pathogenic mutations in the muscle-specific caveolin-3 (Cav3) gene isoform, such as the limb girdle muscular dystrophy type 1C (LGMD-1C) P104L mutation, result in dramatic loss of the Cav3 protein and pathophysiological muscle weakness/wasting. We hypothesize that such muscle degeneration may be linked to disturbances in signalling events that impact protein turnover. Herein, we report studies assessing the effects of Cav3 deficiency on mammalian or mechanistic target of rapamycin complex 1 (mTORC1) signalling in skeletal muscle cells.
METHODS: L6 myoblasts were stably transfected with Cav3P104L or expression of native Cav3 was abolished by CRISPR/Cas9 genome editing (Cav3 knockout [Cav3KO]) prior to performing subcellular fractionation and immunoblotting, analysis of real-time mitochondrial respiration or fixed cell immunocytochemistry. Skeletal muscle from wild-type and Cav3-/- mice was processed for immunoblot analysis of downstream mTORC1 substrate phosphorylation.
RESULTS: Cav3 was detected in lysosomal-enriched membranes isolated from L6 myoblasts and observed by confocal microscopy to co-localize with lysosomal-specific markers. Cav3P104L expression, which results in significant (~95%) loss of native Cav3, or CRISPR/Cas9-mediated Cav3KO, reduced amino acid-dependent mTORC1 activation. The decline in mTORC1-directed signalling was detected by immunoblot analysis of L6 muscle cells and gastrocnemius Cav3-/- mouse muscle as judged by reduced phosphorylation of mTORC1 substrates that play key roles in the initiation of protein synthesis (4EBP1S65 and S6K1T389 ). S6K1T389 and 4EBP1S65 phosphorylation reduced by over 75% and 80% in Cav3KO muscle cells and by over 90% and 30% in Cav3-/- mouse skeletal muscle, respectively. The reduction in protein synthetic capacity in L6 muscle cells was confirmed by analysis of puromycylated peptides using the SUnSET assay. Cav3 loss was also associated with a 26% increase in lysosomal cholesterol, and pharmacological manipulation of lysosomal cholesterol was effective in replicating the reduction in mTORC1 activity observed in Cav3KO cells. Notably, re-expression of Cav3 in Cav3KO myoblasts normalized lysosomal cholesterol content, which coincided with a recovery in protein translation and an associated increase in mTORC1-directed phosphorylation of downstream targets.
CONCLUSIONS: Our findings indicate that Cav3 can localize on lysosomal membranes and is a novel regulator of mTORC1 signalling in muscle. Cav3 deficiency associated with the Cav3P104L mutation impairs mTORC1 activation and protein synthetic capacity in skeletal muscle cells, which may be linked to disturbances in lysosomal cholesterol trafficking and contribute to the pathology of LGMD-1C.

Keywords:  LGMD-1C; amino acid; caveolin-3; caveolinopathy; lysosome; mTORC1; skeletal muscle

DOI:  https://doi.org/10.1002/jcsm.13317
Orphanet J Rare Dis. 2023 Sep 05. 18(1): 273

The metabolomic plasma profile of patients with Duchenne muscular dystrophy: providing new evidence for its pathogenesis.

Huayan Xu, Xiaotang Cai, Ke Xu, Qihong Wu, Bei Xu.

   BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal genetic muscle-wasting disease that affects 1 in 5000 male births with no current cure. Despite great progress has been made in the research of DMD, its underlying pathological mechanism based on the metabolomics is still worthy of further study. Therefore, it is necessary to gain a deeper understanding of the mechanisms or pathogenesis underlying DMD, which may reveal potential therapeutic targets and/or biomarkers.
RESULTS: Plasma samples from 42 patients with DMD from a natural history study and 40 age-matched healthy volunteers were subjected to a liquid chromatography-mass spectrometry-based non-targeted metabolomics approach. Acquired metabolic data were evaluated by principal component analysis, partial least squares-discriminant analysis, and metabolic pathway analysis to explore distinctive metabolic patterns in patients with DMD. Differentially expressed metabolites were identified using publicly available and integrated databases. By comparing the DMD and healthy control groups, 25 differential metabolites were detected, including amino acids, unsaturated fatty acids, carnitine, lipids, and metabolites related to the gut microbiota. Correspondingly, linoleic acid metabolism, D-glutamine and D-glutamate metabolism, glycerophospholipid metabolism, and alanine, aspartate, and glutamate metabolism were significantly altered in patients with DMD, compared with those of healthy volunteers.
CONCLUSIONS: Our study demonstrated the abnormal metabolism of amino acids, energy, and lipids in patients with DMD, consistent with pathological features, such as recurrent muscle necrosis and regeneration, interstitial fibrosis, and fat replacement. Additionally, we found that metabolites of intestinal flora were disordered in DMD patients, providing support for treatment of intestinal microbia disturbance in DMD diseases. Our study provides a new research strategy for understanding the pathogenesis of DMD.

Keywords:  Duchenne muscular dystrophy; Mass spectrometry; Metabonomics; Plasma

DOI:  https://doi.org/10.1186/s13023-023-02885-1
Int J Mol Sci. 2023 Sep 01. pii: 13578. [Epub ahead of print]24(17):

Downregulation of Zebrafish Cytosolic Sialidase Neu3.2 Affects Skeletal Muscle Development.

Daniela Zizioli, Silvia Codenotti, Giuliana Benaglia, Marta Manzoni, Elena Massardi, Alessandro Fanzani, Giuseppe Borsani, Eugenio Monti.

  Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish neu3.2, mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1-4-cell-stage embryos injected with neu3.2 splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior-posterior axis formation. Myog and myod1 expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of Neu2 mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases.

Keywords:  muscle development; muscle differentiation; myoblast differentiation; sialic acid; sialidases; somite formation; zebrafish

DOI:  https://doi.org/10.3390/ijms241713578
Cell Mol Biol Lett. 2023 Sep 06. 28(1): 73

CRISPR applications in cancer diagnosis and treatment.

Mingxia Wang, Menghui Chen, Xia Wu, Xinbo Huang, Bo Yu.

  Cancer remains a significant global health challenge, necessitating the exploration of novel and more precise therapeutic options beyond conventional treatments. In this regard, clustered regularly interspaced short palindromic repeats (CRISPR) systems have emerged as highly promising tools for clinical gene editing applications. The CRISPR family encompasses diverse CRISPR-associated (Cas) proteins that possess the ability to recognize specific target sequences. The initial CRISPR system consisted of the Cas9 protein and a single-guide RNA, which guide Cas9 to the desired target sequence, facilitating precise double-stranded cleavage. In addition to the traditional cis-cleavage activity, the more recently discovered Cas12 and Cas13 proteins exhibit trans-cleavage activity, which expands their potential applications in cancer diagnosis. In this review, we provide an overview of the functional characteristics of Cas9, Cas12, and Cas13. Furthermore, we highlight the latest advancements and applications of these CRISPR systems in cancer gene therapy and molecular diagnosis. We also emphasize the importance of understanding the strengths and limitations of each CRISPR system to maximize their clinical utility. By providing a comprehensive overview of the current state of CRISPR technology in cancer research, we aim to inspire further exploration and innovation in this rapidly evolving field.

Keywords:  Cancer; Cas12; Cas13; Cas9; Clinical trials; Diagnostic tools; Gene therapy

DOI:  https://doi.org/10.1186/s11658-023-00483-4
Biol Open. 2023 Sep 05. pii: bio.059797. [Epub ahead of print]

Expression and function of Four AAV-based constructs for dystrophin restoration in the mdx mouse model of duchenne muscular dystrophy.

Rachael A Potter, Danielle A Griffin, Kristin N Heller, Jerry R Mendell, Louise R Rodino-Klapac.

  Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several cassettes are being tested in clinical trials, this study compared efficacy of the following four shortened dystrophin-promoter combinations with implications for outcomes in clinical trials: MHCK7 or MCK promoter with a shortened dystrophin transgene containing the N-terminus and spectrin repeats R1, R2, R3 and R24 (rAAVrh74.MHCK7.micro-dystrophin and rAAVrh74.MCK.micro-dystrophin, respectively); shortened dystrophin construct containing the neuronal nitric oxide (nNOS) binding site (rAAVrh74.MHCK7.DV.mini-dystrophin); and shortened dystrophin containing the C-terminus (rAAVrh74.MHCK7.micro-dystrophin.Cterm). Functional and histological benefit were examined at 4 weeks following intramuscular delivery in mdx mice. rAAVrh74.MHCK7.micro-dystrophin provided the most robust transgene expression and significantly increased specific force output in the tibialis anterior muscle. Muscle environment was normalized (i.e., reductions in central nucleation), indicating functional and histological advantages of rAAVrh74.MHCK7.micro-dystrophin. Thus, promoter choice and transgene design are critical for optimal dystrophin expression/distribution for maximal functional improvement.

Keywords:  Adeno-associated virus; Duchenne muscular dystrophy; Dystrophin; Gene therapy; Mdx mouse model; Micro-dystrophin

DOI:  https://doi.org/10.1242/bio.059797
Stem Cell Res Ther. 2023 Sep 08. 14(1): 243

In vitro-generated human muscle reserve cells are heterogeneous for Pax7 with distinct molecular states and metabolic profiles.

Axelle Bouche, Benoit Borner, Chloé Richard, Ysaline Grand, Didier Hannouche, Thomas Laumonier.

   BACKGROUND: The capacity of skeletal muscles to regenerate relies on Pax7+ muscle stem cells (MuSC). While in vitro-amplified MuSC are activated and lose part of their regenerative capacity, in vitro-generated human muscle reserve cells (MuRC) are very similar to quiescent MuSC with properties required for their use in cell-based therapies.
METHODS: In the present study, we investigated the heterogeneity of human MuRC and characterized their molecular signature and metabolic profile.
RESULTS: We observed that Notch signaling is active and essential for the generation of quiescent human Pax7+ MuRC in vitro. We also revealed, by immunofluorescence and flow cytometry, two distinct subpopulations of MuRC distinguished by their relative Pax7 expression. After 48 h in differentiation medium (DM), the Pax7High subpopulation represented 35% of the total MuRC pool and this percentage increased to 61% after 96 h in DM. Transcriptomic analysis revealed that Pax7High MuRC were less primed for myogenic differentiation as compared to Pax7Low MuRC and displayed a metabolic shift from glycolysis toward fatty acid oxidation. The bioenergetic profile of human MuRC displayed a 1.5-fold decrease in glycolysis, basal respiration and ATP-linked respiration as compared to myoblasts. We also observed that AMPKα1 expression was significantly upregulated in human MuRC that correlated with an increased phosphorylation of acetyl-CoA carboxylase (ACC). Finally, we showed that fatty acid uptake was increased in MuRC as compared to myoblasts, whereas no changes were observed for glucose uptake.
CONCLUSIONS: Overall, these data reveal that the quiescent MuRC pool is heterogeneous for Pax7 with a Pax7High subpopulation being in a deeper quiescent state, less committed to differentiation and displaying a reduced metabolic activity. Altogether, our data suggest that human Pax7High MuRC may constitute an appropriate stem cell source for potential therapeutic applications in skeletal muscle diseases.

Keywords:  AMPK; Fatty acid oxidation; Human muscle reserve cell; Metabolism; Pax7 heterogeneity; Quiescence

DOI:  https://doi.org/10.1186/s13287-023-03483-5
Mol Ther Nucleic Acids. 2023 Sep 12. 33 890-897

Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase.

Ting Lan, Huangyao Chen, Chengcheng Tang, Yuhui Wei, Yang Liu, Jizeng Zhou, Zhenpeng Zhuang, Quanjun Zhang, Min Chen, Xiaoqing Zhou, Yue Chi, Jinling Wang, Yu He, Liangxue Lai, Qingjian Zou.

  Prime editor (PE) is a versatile genome editing tool that does not need extra DNA donors or inducing double-strand breaks. However, in vivo implementation of PE remains a challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) with the F155Y mutation to keep gene editing efficiency. We discovered the most efficient gene editing variants of MMLV RT with the smallest size. After optimization of the pegRNAs and incorporation with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited the mouse Hsf1 gene in the mouse retina precisely after delivery with adeno-associated viruses (AAVs), although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE and exploiting its therapeutic potential against human genetic diseases.

Keywords:  AAV; CjCas9; MT: RNA/DNA Editing; miniature; prime editor; truncated MMLV

DOI:  https://doi.org/10.1016/j.omtn.2023.08.018
Int J Mol Sci. 2023 Aug 23. pii: 13121. [Epub ahead of print]24(17):

Effects of Aging on Collagen in the Skeletal Muscle of Mice.

Yuji Kanazawa, Ryo Miyachi, Takashi Higuchi, Hiaki Sato.

  Aging affects several tissues in the body, including skeletal muscle. Multiple types of collagens are localized in the skeletal muscle and contribute to the maintenance of normal muscle structure and function. Since the effects of aging on muscle fibers vary by muscle fiber type, it is expected that the effects of aging on intramuscular collagen might be influenced by muscle fiber type. In this study, we examined the effect of aging on collagen levels in the soleus (slow-twitch muscle) and gastrocnemius (fast-twitch muscle) muscles of 3-, 10-, 24-, and 28-month-old male C57BL/6J mice using molecular and morphological analysis. It was found that aging increased collagen I, III, and VI gene expression and immunoreactivity in both slow- and fast-twitch muscles and collagen IV expression in slow-twitch muscles. However, collagen IV gene expression and immunoreactivity in fast-twitch muscle were unaffected by aging. In contrast, the expression of the collagen synthesis marker heat shock protein 47 in both slow- and fast-twitch muscles decreased with aging, while the expression of collagen degradation markers increased with aging. Overall, these results suggest that collagen gene expression and immunoreactivity are influenced by muscle fiber type and collagen type and that the balance between collagen synthesis and degradation tends to tilt toward degradation with aging.

Keywords:  aging; collagen; fast-twitch muscle; skeletal muscle; slow-twitch muscle

DOI:  https://doi.org/10.3390/ijms241713121
J Cell Physiol. 2023 Sep 08.

Nonsense-mediated mRNA decay suppresses injury-induced muscle regeneration via inhibiting MyoD transcriptional activity.

Yanjie Tan, Jing Zhang, Yi Jin.

  Skeletal muscle regeneration is a crucial physiological process that occurs in response to injury or disease. As an important transcriptome surveillance system that regulates tissue development, the role of nonsense-mediated mRNA decay (NMD) in muscle regeneration remains unclear. Here, we found that NMD inhibits myoblast differentiation by targeting the phosphoinositide-3-kinase regulatory subunit 5 gene, which leads to the suppression of the transcriptional activity of myogenic differentiation (MyoD), a key regulator of myoblast differentiation. This disruption of MyoD transcriptional activity subsequently affects the expression levels of myogenin and myosin heavy chain, crucial markers of myoblast differentiation. Additionally, through up-frameshift protein 1 knockdown experiments, we observed that inhibiting NMD can accelerate muscle regeneration in vivo. These findings highlight the potential of NMD as a novel therapeutic target for the treatment of muscle-related injuries and diseases.

Keywords:  MyoD transcriptional activity; muscle regeneration; myoblast differentiation; nonsense-mediated mRNA decay

DOI:  https://doi.org/10.1002/jcp.31118
Int J Mol Sci. 2023 Aug 31. pii: 13551. [Epub ahead of print]24(17):

Dual Adeno-Associated Virus 9 with Codon-Optimized DYSF Gene Promotes In Vivo Muscle Regeneration and May Decrease Inflammatory Response in Limb Girdle Muscular Dystrophy Type R2.

Ivan A Yakovlev, Aleksei M Emelin, Yana S Slesarenko, Igor S Limaev, Iuliia A Vetrova, Liliya D Belikova, Ekaterina N Grafskaia, Pavel A Bobrovsky, Mikhail V Pokrovsky, Elena V Kuzubova, Vladimir M Pokrovsky, Pyotr A Lebedev, Sergei N Bardakov, Artur A Isaev, Roman V Deev.

  Dysferlinopathy treatment is an active area of investigation. Gene therapy is one potential approach. We studied muscle regeneration and inflammatory response after injection of an AAV-9 with a codon-optimized DYSF gene. A dual-vector system AAV.DYSF.OVERLAP with overlapping DYSF cDNA sequences was generated. Two AAV vectors were separately assembled by a standard triple-transfection protocol from plasmids carrying parts of the DYSF gene. Artificial myoblasts from dysferlin-deficient fibroblasts were obtained by MyoD overexpression. RT-PCR and Western blot were used for RNA and protein detection in vitro. A dysferlinopathy murine model (Bla/J) was used for in vivo studies. Histological assay, morphometry, and IHC were used for the muscle tissue analysis. Dysferlin was detected in vitro and in vivo at subphysiological levels. RT-PCR and Western Blot detected dysferlin mRNA and protein in AAV.DYSF.OVERLAP-transduced cells, and mRNA reached a 7-fold elevated level compared to the reference gene (GAPDH). In vivo, the experimental group showed intermediate median values for the proportion of necrotic muscle fibers, muscle fibers with internalized nuclei, and cross-sectional area of muscle fibers compared to the same parameters in the control groups of WT and Bla/J mice, although the differences were not statistically significant. The inverse relationship between the dosage and the severity of inflammatory changes in the muscles may be attributed to the decrease in the number of necrotic fibers. The share of transduced myofibers reached almost 35% in the group with the highest dose. The use of two-vector systems based on AAV is justified in terms of therapeutic efficacy. The expression of dysferlin at a subphysiological level, within a short observation period, is capable of inducing the restoration of muscle tissue structure, reducing inflammatory activity, and mitigating necrotic processes. Further research is needed to provide a more detailed assessment of the impact of the transgene and viral vector on the inflammatory component, including longer observation periods.

Keywords:  LGMD R2; dual AAV vector; dysferlin; gene transfer; immune response; muscle regeneration

DOI:  https://doi.org/10.3390/ijms241713551
Tissue Eng Part A. 2023 Sep 02.

Cell Derived Extracellular Matrix Fiber Scaffolds Improve Recovery from Volumetric Muscle Loss.

Cassandra Reed, Tai Huynh, Jacob Schluns, Payton Phelps, Jamie Hestekin, Jeff Wolchok.

Skeletal muscle is capable of robust self-repair following mild trauma, yet in cases of traumatic volumetric muscle loss (VML), where more than 20% of a muscle's mass is lost, this capacity is overwhelmed. Current soft tissue repair techniques and traditional rehabilitation have not been able to reverse the pathological changes that occur following VML injury. There are currently no surgical guidelines that effectively address the treatment of VML injuries which has motivated the exploration of implantable scaffolding strategies. In this study, the use of an allogenic scaffold fabricated using fibers built from the extracellular matrix (ECM) collected from muscle fibroblast cells during growth in culture was explored as a repair strategy using a lower-limb VLM injury (tibialis anterior muscle) in a rat model. Recovery outcomes (8 weeks) were explored in comparison to unrepaired controls as well previously examined allogenic scaffolds prepared from decellularized skeletal muscle (DSM) tissue (n=9 / sample group). At 8-week follow-up we found that the repair of VML injuries using ECM fiber scaffolds in combination with an autogenic mince muscle (MM) paste significantly increased recovery of peak contractile force (79±13%) of normal contralateral muscle) when compared to unrepaired VML controls (57±13%). Similar significant improvements were measured for muscle mass restoration (93±10%) in response to ECM fiber + MM repair when compared to unrepaired VML controls (73±13%). Of particular note, mass and contractile strength recovery outcomes for ECM fiber scaffolds were not significantly different from DSM + MM repair controls. These in-vivo findings support the further exploration of cell derived ECM fiber scaffolds as promising strategy for the repair of VML injury with recovery outcomes that compare favorably to current state of the field tissue derived ECM scaffolds. Furthermore, while the therapeutic potential of ECM fibers as a treatment strategy for muscle injury was explored in this study, they could be adapted for high throughput fabrication methods developed and routinely used by the textile industry to create a broad range of woven implants (ex. hernia meshes) for even greater impact.

DOI: https://doi.org/10.1089/ten.TEA.2022.0227
J Appl Physiol (1985). 2023 Sep 07.

Prolonged mechanical muscle loading increases mechanosensor gene and protein levels and causes a moderate fast-to-slow fiber type switch in mice.

Mathias Vanmunster, Ana Victoria Rojo-Garcia, Alexander Pacolet, Ilse Jonkers, Katrien Koppo, Rik Lories, Frank Suhr.

  Mechanosensing and subsequent mechanotransduction are indispensable for muscle plasticity. Nevertheless, a scarcity of literature exists regarding an all-encompassing understanding of the muscle mechanosensing machinery's response to prolonged loading, especially in conditions that resemble a natural physiological state of skeletal muscle. This study aimed to comprehensively explore the effects of prolonged mechanical loading on mechanosensitive components, skeletal muscle characteristics, and metabolism-related gene clusters. Twenty male C57BL/6J mice were randomly divided into two groups: control and prolonged mechanical loading. To induce prolonged mechanical loading on the triceps brachii (TRI) and biceps brachii (BIC) muscles, a 14-day period of tail suspension was implemented. In TRI only, prolonged mechanical loading caused a mild fast-to-slow fiber type shift together with increased mechanosensor gene and protein levels. It also increased transcription factors associated with slow muscle fibers while decreasing those related to fast-type muscle gene expression. Succinate dehydrogenase activity, a marker of muscle oxidative capacity, and genes involved in oxidative and mitochondrial turnover increased, while glycolytic-related genes decreased. Moreover, prolonged mechanical loading stimulated markers of muscle protein synthesis. Taken together, our data show a collective muscle-specific increase in mechanosensor gene and protein levels upon a period of prolonged mechanical loading in conditions that reflect a more natural physiological state of skeletal muscle in mice. We provide additional proof-of-concept that prolonged tail suspension-induced loading of the forelimbs triggers a muscle-specific fast-to-slow fiber type switch, and this coincides with increased protein synthesis-related signaling.

Keywords:  fiber types; mechanical loading; mechanosensors; muscle metabolism; skeletal muscle

DOI:  https://doi.org/10.1152/japplphysiol.00204.2023
J Biol Chem. 2023 Sep 04. pii: S0021-9258(23)02254-8. [Epub ahead of print] 105226

The transcriptional regulator KLF15 is necessary for myoblast differentiation and muscle regeneration by activating FKBP5.

Shijuan Gao, Shan Huang, Yanhong Zhang, Guangming Fang, Yan Liu, Congcong Zhang, Yulin Li, Jie Du.

  Successful muscle regeneration following injury is essential for functional homeostasis of skeletal muscles. Krüppel-like factor 15 (KLF15) is a metabolic transcriptional regulator in the muscles. However, little is known regarding its function in muscle regeneration. Here, we examined microarray datasets from the Gene Expression Omnibus (GEO) database, which indicated downregulated KLF15 in muscles from patients with various muscle diseases. Additionally, we found that Klf15 knockout (Klf15KO) impaired muscle regeneration following injury in mice. Furthermore, KLF15 expression was robustly induced during myoblast differentiation. Myoblasts with KLF15 deficiency showed a marked reduction in their fusion capacity. Unbiased transcriptome analysis of muscles on day 7 post-injury revealed downregulated genes involved in cell differentiation and metabolic processes in Klf15KO muscles. The FK506-binding protein 51 (FKBP5), a positive regulator of myoblast differentiation, was ranked as one of the most strongly downregulated genes in the Klf15KO group. A mechanistic search revealed that KLF15 binds directly to the promoter region of FKBP5 and activates FKBP5 expression. Local delivery of FKBP5 rescued the impaired muscle regeneration in Klf15KO mice. Our findings reveal a positive regulatory role of KLF15 in myoblast differentiation and muscle regeneration by activating FKBP5 expression. KLF15 signaling may be a novel therapeutic target for muscle disorders associated with injuries or diseases.

Keywords:  FKBP5; KLF15; muscle regeneration; myoblast differentiation

DOI:  https://doi.org/10.1016/j.jbc.2023.105226
Poult Sci. 2023 Aug 21. pii: S0032-5791(23)00569-2. [Epub ahead of print]102(11): 103050

CircLRRFIP1 promotes the proliferation and differentiation of chicken skeletal muscle satellite cells by sponging the miR-15 family via activating AKT3-mTOR/p70S6K signaling pathway.

Yamei Wu, Jing Zhao, Xiyu Zhao, Haorong He, Can Cui, Yao Zhang, Qing Zhu, Huadong Yin, Shunshun Han.

  Skeletal muscle is important for animal meat production, regulating movements, and maintaining homeostasis. Circular RNAs (circRNAs) have been founded to play vital role in myogenesis. However, the effects of the numerous circRNAs on growth and development of the skeletal muscle are yet to be uncovered. Herein, we identified circLRRFIP1, which is a novel circular RNA that is preferentially expressed in the skeletal muscle. To study the role of circLRRFIP1 in the skeletal muscle, the skeletal muscle satellite cells (SMSCs) was used to silenced or overexpressed circLRRFIP1. The results obtained in this study showed that circLRRFIP1 play a positive role in the proliferation and differentiation of SMSCs. The SMSCs were generated with stable knockdown and overexpression of circLRRFIP1, and the results showed that circLRRFIP1 exerts a stimulatory effect on the proliferation and differentiation of SMSCs. We further generated SMSCs with stable knockdown and overexpression of circLRRFIP1, and the results revealed that circLRRFIP1 exerts a stimulatory effect on the proliferation and differentiation of SMSCs. Mechanistically, circLRRFIP1 targets the myogenic inhibitory factor-miR-15 family to release the suppression of the miR-15 family to AKT3. The knockdown of AKT inhibits SMSC differentiation through the mTOR/p70S6K pathway. Taken together, the results obtained in this present study revealed the important role and the regulatory mechanisms of circLRRFIP1 in the development of chicken skeletal muscle. Therefore, this study provides an attractive target for molecular breeding to enhance meat production in the chicken industry.

Keywords:  AKT3; circLRRFIP1; differentiation; miRNA-15 family; proliferation

DOI:  https://doi.org/10.1016/j.psj.2023.103050
PLoS Genet. 2023 Sep 07. 19(9): e1010923

Evolutionary conserved circular MEF2A RNAs regulate myogenic differentiation and skeletal muscle development.

Xiaoxu Shen, Xiyu Zhao, Haorong He, Jing Zhao, Yuanhang Wei, Yuqi Chen, Shunshun Han, Yifeng Zhu, Yao Zhang, Qing Zhu, Huadong Yin.

Circular RNAs (circRNAs) have been recognized as critical regulators of skeletal muscle development. Myocyte enhancer factor 2A (MEF2A) is an evolutionarily conserved transcriptional factor that regulates myogenesis. However, it remains unclear whether MEF2A produces functional circRNAs. In this study, we identified two evolutionarily conserved circular MEF2A RNAs (circMEF2As), namely circMEF2A1 and circMEF2A2, in chicken and mouse muscle stem cells. Our findings revealed that circMEF2A1 promotes myogenesis by regulating the miR-30a-3p/PPP3CA/NFATC1 axis, whereas circMEF2A2 facilitates myogenic differentiation by targeting the miR-148a-5p/SLIT3/ROBO2/β-catenin signaling pathway. Furthermore, in vivo experiments demonstrated that circMEF2As both promote skeletal muscle growth. We also discovered that the linear MEF2A mRNA-derived MEF2A protein binds to its own promoter region, accelerating the transcription of MEF2A and upregulating the expression of both linear MEF2A and circMEF2As, forming a MEF2A autoregulated positive feedback loop. Moreover, circMEF2As positively regulate the expression of linear MEF2A by adsorbing miR-30a-3p and miR-148a-5p, which directly contribute to the MEF2A autoregulated feedback loop. Importantly, we found that mouse circMEF2As are essential for the myogenic differentiation of C2C12 cells. Collectively, our results demonstrated the evolution, function, and underlying mechanisms of circMEF2As in animal myogenesis, which may provide novel insight for both the farm animal meat industry and human medicine.

DOI: https://doi.org/10.1371/journal.pgen.1010923