bims-muscge 2023-10-15 papers

bims-muscge

Biomed News

on Muscle stem cells and gene therapy

Issue of 2023–10–15
twenty-two papers selected by
Chance Bowman, Dartmouth College

Fn14 promotes myoblast fusion during regenerative myogenesis.
Postnatal skeletal muscle myogenesis governed by signal transduction networks: MAPKs and PI3K-Akt control multiple steps.
Pax7 haploinsufficiency impairs muscle stem cell function in Cre-recombinase mice and underscores the importance of appropriate controls.
Skeletal Muscle Extracellular Matrix Structure Under Applied Deformation Observed Using Second Harmonic Generation Microscopy.
Protocol for generation of a time-resolved cellular interactome during tissue remodeling in adult mice.
The Role of Hypoxia and Hypoxia Signaling in Skeletal Muscle Physiology.
The Function and Regulation Mechanism of Non-Coding RNAs in Muscle Development.
Myogenesis Effects of RGX365 to Improve Skeletal Muscle Atrophy.
CRISPR/dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation.
Physical exercise elicits UPRmt in the skeletal muscle: The role of c-Jun N-terminal kinase.
Regeneration of neuromuscular synapses after acute and chronic denervation by inhibiting the gerozyme 15-prostaglandin dehydrogenase.
GADD45A is a mediator of mitochondrial loss, atrophy, and weakness in skeletal muscle.
The mesodermal and myogenic specification of hESCs depend on ZEB1 and are inhibited by ZEB2.
Fiber Type Identification of Human Skeletal Muscle.
Exome Sequencing and Optical Genome Mapping in Molecularly Unsolved Cases of Duchenne Muscular Dystrophy: Identification of a Causative X-Chromosomal Inversion Disrupting the DMD Gene.
From amino-acid to disease: the effects of oxidation on actin-myosin interactions in muscle.
Delivery of gene editing therapeutics.
CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination.
Desmin and its molecular chaperone, the αB-crystallin: How post-translational modifications modulate their functions in heart and skeletal muscles?
Enhancing Antisense Oligonucleotide-Based Therapeutic Delivery with DG9, a Versatile Cell-Penetrating Peptide.
Comparison of pharmaceutical properties and biological activity of prednisolone, deflazacort, and vamorolone in DMD disease models.
Integrated single-cell multiome analysis reveals muscle fiber-type gene regulatory circuitry modulated by endurance exercise.

Life Sci Alliance. 2023 Dec;pii: e202302312. [Epub ahead of print]6(12):

Fn14 promotes myoblast fusion during regenerative myogenesis.

Meiricris Tomaz da Silva, Aniket S Joshi, Micah B Castillo, Tatiana E Koike, Anirban Roy, Preethi H Gunaratne, Ashok Kumar.

Skeletal muscle regeneration involves coordinated activation of an array of signaling pathways. Fibroblast growth factor-inducible 14 (Fn14) is a bona fide receptor for the TWEAK cytokine. Levels of Fn14 are increased in the skeletal muscle of mice after injury. However, the cell-autonomous role of Fn14 in muscle regeneration remains unknown. Here, we demonstrate that global deletion of the Fn14 receptor in mice attenuates muscle regeneration. Conditional ablation of Fn14 in myoblasts but not in differentiated myofibers of mice inhibits skeletal muscle regeneration. Fn14 promotes myoblast fusion without affecting the levels of myogenic regulatory factors in the regenerating muscle. Fn14 deletion in myoblasts hastens initial differentiation but impairs their fusion. The overexpression of Fn14 in myoblasts results in the formation of myotubes having an increased diameter after induction of differentiation. Ablation of Fn14 also reduces the levels of various components of canonical Wnt and calcium signaling both in vitro and in vivo. Forced activation of Wnt signaling rescues fusion defects in Fn14-deficient myoblast cultures. Collectively, our results demonstrate that Fn14-mediated signaling positively regulates myoblast fusion and skeletal muscle regeneration.

DOI: https://doi.org/10.26508/lsa.202302312
Biochem Biophys Res Commun. 2023 Sep 27. pii: S0006-291X(23)01084-7. [Epub ahead of print]682 223-243

Postnatal skeletal muscle myogenesis governed by signal transduction networks: MAPKs and PI3K-Akt control multiple steps.

Takeshi Endo.

  Skeletal muscle myogenesis represents one of the most intensively and extensively examined systems of cell differentiation, tissue formation, and regeneration. Muscle regeneration provides an in vivo model system of postnatal myogenesis. It comprises multiple steps including muscle stem cell (or satellite cell) quiescence, activation, migration, myogenic determination, myoblast proliferation, myocyte differentiation, myofiber maturation, and hypertrophy. A variety of extracellular signaling and subsequent intracellular signal transduction pathways or networks govern the individual steps of postnatal myogenesis. Among them, MAPK pathways (the ERK, JNK, p38 MAPK, and ERK5 pathways) and PI3K-Akt signaling regulate multiple steps of myogenesis. Ca2+, cytokine, and Wnt signaling also participate in several myogenesis steps. These signaling pathways often control cell cycle regulatory proteins or the muscle-specific MyoD family and the MEF2 family of transcription factors. This article comprehensively reviews molecular mechanisms of the individual steps of postnatal skeletal muscle myogenesis by focusing on signal transduction pathways or networks. Nevertheless, no or only a partial signaling molecules or pathways have been identified in some responses during myogenesis. The elucidation of these unidentified signaling molecules and pathways leads to an extensive understanding of the molecular mechanisms of myogenesis.

Keywords:  MAPK pathways; Muscle regeneration; MyoD family; PI3K–Akt signaling; Signal transduction; Skeletal muscle myogenesis

DOI:  https://doi.org/10.1016/j.bbrc.2023.09.048
Stem Cell Res Ther. 2023 Oct 13. 14(1): 294

Pax7 haploinsufficiency impairs muscle stem cell function in Cre-recombinase mice and underscores the importance of appropriate controls.

Despoina Mademtzoglou, Perla Geara, Philippos Mourikis, Frederic Relaix.

  Ever since its introduction as a genetic tool, the Cre-lox system has been widely used for molecular genetic studies in vivo in the context of health and disease, as it allows time- and cell-specific gene modifications. However, insertion of the Cre-recombinase cassette in the gene of interest can alter transcription, protein expression, or function, either directly, by modifying the landscape of the locus, or indirectly, due to the lack of genetic compensation or by indirect impairment of the non-targeted allele. This is sometimes the case when Cre-lox is used for muscle stem cell studies. Muscle stem cells are required for skeletal muscle growth, regeneration and to delay muscle disease progression, hence providing an attractive model for stem cell research. Since the transcription factor Pax7 is specifically expressed in all muscle stem cells, tamoxifen-inducible Cre cassettes (CreERT2) have been inserted into this locus by different groups to allow targeted gene recombination. Here we compare the two Pax7-CreERT2 mouse lines that are mainly used to evaluate muscle regeneration and development of pathological features upon deletion of specific factors or pathways. We applied diverse commonly used tamoxifen schemes of CreERT2 activation, and we analyzed muscle repair after cardiotoxin-induced injury. We show that consistently the Pax7-CreERT2 allele targeted into the Pax7 coding sequence (knock-in/knock-out allele) produces an inherent defect in regeneration, manifested as delayed post-injury repair and reduction in muscle stem cell numbers. In genetic ablation studies lacking proper controls, this inherent defect could be misinterpreted as being provoked by the deletion of the factor of interest. Instead, using an alternative Pax7-CreERT2 allele that maintains bi-allelic Pax7 expression or including appropriate controls can prevent misinterpretation of experimental data. The findings presented here can guide researchers establish appropriate experimental design for muscle stem cell genetic studies.

Keywords:  Cre-lox; Mouse molecular genetics; Muscle stem cells; Pax7; Satellite cells; Stem cell research

DOI:  https://doi.org/10.1186/s13287-023-03506-1
Acta Biomater. 2023 Oct 05. pii: S1742-7061(23)00593-7. [Epub ahead of print]

Skeletal Muscle Extracellular Matrix Structure Under Applied Deformation Observed Using Second Harmonic Generation Microscopy.

Niamh Hennessy, Ciaran Simms.

  The mechanical and structural properties of passive skeletal muscle are important for musculoskeletal models in impact biomechanics, rehabilitation engineering and surgical simulation. Passive properties of skeletal muscle depend strongly on the architecture of the extracellular matrix (ECM), but the structure of ECM and its realignment under applied deformation remain poorly understood. We apply second harmonic generation (SHG) microscopy to study muscle ECM in intact muscle samples both under deformation and in the undeformed state. A method for regional relocation was developed, so that the same ECM segment could be viewed before and after applying deformations. Skeletal muscle ECM was viewed at multiple scales and in three states: undeformed, under compression and under tension. Results show that second harmonic generation microscopy provides substantial detail of skeletal muscle ECM over a wide range of length scales, especially the perimysium structure. We present images of individual portions of skeletal muscle ECM both undeformed and subjected to tensile/compressive deformation. We also present data showing the response of the perimysium to a partial thickness cut applied to a section under tensile deformation. STATEMENT OF SIGNIFICANCE: : Second Harmonic Generation (SHG) microscopy is an imaging technique which takes advantage of a non-linear and coherent frequency doubling optical effect that is present in a small number of biological molecules, primarily collagen Type I, II and myosin. Collagen I is the most abundant collagen type in skeletal muscle, making SHG a promising option for visualisation of the skeletal muscle extracellular matrix (ECM). SHG microscopy does not require fixing or staining. This short communication presents the application of SHG microscopy to skeletal muscle ECM to improve our understanding of how collagen fibres reorganise under applied tensile and compression, including microscopic observations of collagen fibre reorganisation for intact samples by using a method to re-identify specific regions in repeated deformation tests.

Keywords:  Extracellular Matrix; Perimysium; Second Harmonic Generation; Skeletal Muscle; Tension-Compression

DOI:  https://doi.org/10.1016/j.actbio.2023.09.047
STAR Protoc. 2023 Oct 11. pii: S2666-1667(23)00605-6. [Epub ahead of print]4(4): 102638

Protocol for generation of a time-resolved cellular interactome during tissue remodeling in adult mice.

Elena Groppa, Lin Wei Tung, Stefania Mattevi, Morten Ritso, Fabio M V Rossi, Paolo Martini.

  Efficient skeletal muscle regeneration necessitates fine-tuned coordination among multiple cell types through an intricate network of intercellular communication. We present a protocol for generation of a time-resolved cellular interactome during tissue remodeling. We describe steps for isolating distinct cell populations from skeletal muscle of adult mice after acute damage and extracting RNA from purified cells prior to the generation of RNA sequencing data. We then detail procedures for generating and deciphering a time- and lineage-resolved model of intercellular crosstalk. For complete details on the use and execution of this protocol, please refer to Groppa et al. (2023).1.

Keywords:  Bioinformatics; Cell Biology; Cell isolation; Computer sciences; Flow Cytometry/Mass Cytometry; Molecular Biology; RNAseq; Sequence analysis; Sequencing; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2023.102638
Adv Biol (Weinh). 2023 Oct 10. e2200300

The Role of Hypoxia and Hypoxia Signaling in Skeletal Muscle Physiology.

Yori Endo, Christina Zhu, Elena Giunta, Cynthia Guo, Danial J Koh, Indranil Sinha.

  Hypoxia and hypoxia signaling play an integral role in regulating skeletal muscle physiology. Environmental hypoxia and tissue hypoxia in muscles cue for their appropriate physiological response and adaptation, and cause an array of cellular and metabolic changes. In addition, muscle stem cells (satellite cells), exist in a hypoxic state, and this intrinsic hypoxic state correlates with their quiescence and stemness. The mechanisms of hypoxia-mediated regulation of satellite cells and myogenesis are yet to be characterized, and their seemingly contradicting effects reported leave their exact roles somewhat perplexing. This review summarizes the recent findings on the effect of hypoxia and hypoxia signaling on the key aspects of muscle physiology, namely, stem cell maintenance and myogenesis with a particular attention given to distinguish the intrinsic versus local hypoxia in an attempt to better understand their respective regulatory roles and how their relationship affects the overall response. This review further describes their mechanistic links and their possible implications on the relevant pathologies and therapeutics.

Keywords:  HIFs; hypoxia signaling; myogenesis; quiescence; satellite cells; skeletal muscle; stem cells

DOI:  https://doi.org/10.1002/adbi.202200300
Int J Mol Sci. 2023 Sep 26. pii: 14534. [Epub ahead of print]24(19):

The Function and Regulation Mechanism of Non-Coding RNAs in Muscle Development.

Yaling Yang, Jian Wu, Wujun Liu, Yumin Zhao, Hong Chen.

  Animal skeletal muscle growth is regulated by a complex molecular network including some non-coding RNAs (ncRNAs). In this paper, we review the non-coding RNAs related to the growth and development of common animal skeletal muscles, aiming to provide a reference for the in-depth study of the role of ncRNAs in the development of animal skeletal muscles, and to provide new ideas for the improvement of animal production performance.

Keywords:  circRNA; lncRNA; miRNA; muscle development

DOI:  https://doi.org/10.3390/ijms241914534
Nutrients. 2023 Oct 09. pii: 4307. [Epub ahead of print]15(19):

Myogenesis Effects of RGX365 to Improve Skeletal Muscle Atrophy.

Hye-Jin Lee, Hui-Ji Choi, Sang-Ah Lee, Dong Hyuk Baek, Jong Beom Heo, Gyu Yong Song, Wonhwa Lee.

  Age-related skeletal muscle atrophy and weakness not only reduce the quality of life of those afflicted, but also worsen the prognosis of underlying diseases. We evaluated the effect of RGX365, a protopanaxatriol-type rare ginsenoside mixture, on improving skeletal muscle atrophy. We investigated the myogenic effect of RGX365 on mouse myoblast cells (C2C12) and dexamethasone (10 µM)-induced atrophy of differentiated C2C12. RGX365-treated myotube diameters and myosin heavy chain (MyHC) expression levels were analyzed using immunofluorescence. We evaluated the myogenic effects of RGX365 in aging sarcopenic mice. RGX365 increased myoblast differentiation and MyHC expression, and attenuated the muscle atrophy-inducing F-box (Atrogin-1) and muscle RING finger 1 (MuRF1) expression. Notably, one month of oral administration of RGX365 to 23-month-old sarcopenic mice improved muscle fiber size and the expression of skeletal muscle regeneration-associated molecules. In conclusion, rare ginsenosides, agonists of steroid receptors, can ameliorate skeletal muscle atrophy during long-term administration.

Keywords:  RGX365; myogenesis; myosin heavy chain; rare ginsenosides; sarcopenia

DOI:  https://doi.org/10.3390/nu15194307
Int J Mol Sci. 2023 Oct 03. pii: 14865. [Epub ahead of print]24(19):

CRISPR/dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation.

Ruijie Cai, Runyu Lv, Xin'e Shi, Gongshe Yang, Jianjun Jin.

  CRISPR/Cas9-mediated cleavage of DNA, which depends on the endonuclease activity of Cas9, has been widely used for gene editing due to its excellent programmability and specificity. However, the changes to the DNA sequence that are mediated by CRISPR/Cas9 affect the structures and stability of the genome, which may affect the accuracy of results. Mutations in the RuvC and HNH regions of the Cas9 protein lead to the inactivation of Cas9 into dCas9 with no endonuclease activity. Despite the loss of endonuclease activity, dCas9 can still bind the DNA strand using guide RNA. Recently, proteins with active/inhibitory effects have been linked to the end of the dCas9 protein to form fusion proteins with transcriptional active/inhibitory effects, named CRISPRa and CRISPRi, respectively. These CRISPR tools mediate the transcription activity of protein-coding and non-coding genes by regulating the chromosomal modification states of target gene promoters, enhancers, and other functional elements. Here, we highlight the epigenetic mechanisms and applications of the common CRISPR/dCas9 tools, by which we hope to provide a reference for future related gene regulation, gene function, high-throughput target gene screening, and disease treatment.

Keywords:  CRISPR activation; CRISPR inhibition; DNA methylation; histone modification; transcription regulation

DOI:  https://doi.org/10.3390/ijms241914865
Mol Metab. 2023 Oct 10. pii: S2212-8778(23)00150-3. [Epub ahead of print] 101816

Physical exercise elicits UPRmt in the skeletal muscle: The role of c-Jun N-terminal kinase.

Rodrigo Stellzer Gaspar, Carlos Kiyoshi Katashima, Barbara Moreira Crisol, Fernanda Silva Carneiro, Igor Sampaio, Leonardo Dos Reis Silveira, Adelino Sanchez Ramos da Silva, Dennys Esper Cintra, José Rodrigo Pauli, Eduardo Rochete Ropelle.

   OBJECTIVE: The mitochondrial unfolded protein response (UPRmt) is an adaptive cellular response to stress to ensure mitochondrial proteostasis and function. Here we explore the capacity of physical exercise to induce UPRmt in the skeletal muscle.
METHODS: Therefore, we combined mouse models of exercise (swimming and treadmill running), pharmacological intervention, and bioinformatics analyses.
RESULTS: Firstly, RNA sequencing and Western blotting analysis revealed that an acute aerobic session stimulated several mitostress-related genes and protein content in muscle, including the UPRmt markers. Conversely, using a large panel of isogenic strains of BXD mice, we identified that BXD73a and 73b strains displayed low levels of several UPRmt-related genes in the skeletal muscle, and this genotypic feature was accompanied by body weight gain, lower locomotor activity, and aerobic capacity. Finally, we identified that c-Jun N-terminal kinase (JNK) activation was critical in exercise-induced UPRmt in the skeletal muscle since pharmacological JNK pathway inhibition blunted exercise-induced UPRmt markers in mice muscle.
CONCLUSION: Our findings provide new insights into how exercise triggers mitostress signals toward the oxidative capacity in the skeletal muscle.

Keywords:  JNK; Mitochondria; Physical exercise; Skeletal muscle; UPR(mt)

DOI:  https://doi.org/10.1016/j.molmet.2023.101816
Sci Transl Med. 2023 Oct 11. 15(717): eadg1485

Regeneration of neuromuscular synapses after acute and chronic denervation by inhibiting the gerozyme 15-prostaglandin dehydrogenase.

Mohsen A Bakooshli, Yu Xin Wang, Elena Monti, Shiqi Su, Peggy Kraft, Minas Nalbandian, Ludmila Alexandrova, Joshua R Wheeler, Hannes Vogel, Helen M Blau.

To date, there are no approved treatments for the diminished strength and paralysis that result from the loss of peripheral nerve function due to trauma, heritable neuromuscular diseases, or aging. Here, we showed that denervation resulting from transection of the sciatic nerve triggered a marked increase in the prostaglandin-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in skeletal muscle in mice, providing evidence that injury drives early expression of this aging-associated enzyme or gerozyme. Treating mice with a small-molecule inhibitor of 15-PGDH promoted regeneration of motor axons and formation of neuromuscular synapses leading to an acceleration in recovery of force after an acute nerve crush injury. In aged mice with chronic denervation of muscles, treatment with the 15-PGDH inhibitor increased motor neuron viability and restored neuromuscular junctions and function. These presynaptic changes synergized with previously reported muscle tissue remodeling to result in a marked increase in the strength of aged muscles. We further found that 15-PGDH aggregates defined the target fibers that are histopathologic hallmarks of human neurogenic myopathies, suggesting that the gerozyme may be involved in their etiology. Our data suggest that inhibition of 15-PGDH may constitute a therapeutic strategy to physiologically boost prostaglandin E2, restore neuromuscular connectivity, and promote recovery of strength after acute or chronic denervation due to injury, disease, or aging.

DOI: https://doi.org/10.1126/scitranslmed.adg1485
JCI Insight. 2023 Oct 10. pii: e171772. [Epub ahead of print]

GADD45A is a mediator of mitochondrial loss, atrophy, and weakness in skeletal muscle.

George R Marcotte, Matthew J Miller, Hawley E Kunz, Zachary C Ryan, Matthew D Strub, Patrick M Vanderboom, Carrie J Heppelmann, Sarah Chau, Zachary D Von Ruff, Sean P Kilroe, Andrew T McKeen, Jason M Dierdorff, Jennifer I Stern, Karl A Nath, Chad E Grueter, Vitor A Lira, Andrew R Judge, Blake B Rasmussen, K Sreekumaran Nair, Ian R Lanza, Scott M Ebert, Christopher M Adams.

  Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle-specific expression of Growth Arrest and DNA Damage Inducible Alpha (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MEKK4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.

Keywords:  Metabolism; Mitochondria; Muscle Biology; Skeletal muscle

DOI:  https://doi.org/10.1172/jci.insight.171772
Cell Rep. 2023 Oct 10. pii: S2211-1247(23)01234-2. [Epub ahead of print]42(10): 113222

The mesodermal and myogenic specification of hESCs depend on ZEB1 and are inhibited by ZEB2.

Chiara Ninfali, Laura Siles, Anna Esteve-Codina, Antonio Postigo.

  Human embryonic stem cells (hESCs) can differentiate into any cell lineage. Here, we report that ZEB1 and ZEB2 promote and inhibit mesodermal-to-myogenic specification of hESCs, respectively. Knockdown and/or overexpression experiments of ZEB1, ZEB2, or PAX7 in hESCs indicate that ZEB1 is required for hESC Nodal/Activin-mediated mesodermal specification and PAX7+ human myogenic progenitor (hMuP) generation, while ZEB2 inhibits these processes. ZEB1 downregulation induces neural markers, while ZEB2 downregulation induces mesodermal/myogenic markers. Mechanistically, ZEB1 binds to and transcriptionally activates the PAX7 promoter, while ZEB2 binds to and activates the promoter of the neural OTX2 marker. Transplanting ZEB1 or ZEB2 knocked down hMuPs into the muscles of a muscular dystrophy mouse model, showing that hMuP engraftment and generation of dystrophin-positive myofibers depend on ZEB1 and are inhibited by ZEB2. The mouse model results suggest that ZEB1 expression and/or downregulating ZEB2 in hESCs may also enhance hESC regenerative capacity for human muscular dystrophy therapy.

Keywords:  CP: Developmental biology; CP: Stem cell research; EMT; OTX2; PAX7; ZEB1 and ZEB2; cellular therapy; epithelial-to-mesenchymal transition; hESC; hESCs differentiation; human embryonic stem cells; mesodermal precursors; myogenic precursors

DOI:  https://doi.org/10.1016/j.celrep.2023.113222
J Vis Exp. 2023 Sep 22.

Fiber Type Identification of Human Skeletal Muscle.

Heidy K Latchman, Stefan G Wette, Dion J Ellul, Robyn M Murphy, Noni T Frankenberg.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy. The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples. These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

DOI: https://doi.org/10.3791/65750
Int J Mol Sci. 2023 Sep 28. pii: 14716. [Epub ahead of print]24(19):

Exome Sequencing and Optical Genome Mapping in Molecularly Unsolved Cases of Duchenne Muscular Dystrophy: Identification of a Causative X-Chromosomal Inversion Disrupting the DMD Gene.

Leoni S Erbe, Sabine Hoffjan, Sören Janßen, Moritz Kneifel, Karsten Krause, Wanda M Gerding, Kristina Döring, Anne-Katrin Güttsches, Andreas Roos, Elena Buena Atienza, Caspar Gross, Thomas Lücke, Hoa Huu Phuc Nguyen, Matthias Vorgerd, Cornelia Köhler.

  Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease that mainly affects boys due to X-linked recessive inheritance. In most affected individuals, MLPA or sequencing-based techniques detect deletions, duplications, or point mutations in the dystrophin-encoding DMD gene. However, in a small subset of patients clinically diagnosed with DMD, the molecular cause is not identified with these routine methods. Evaluation of the 60 DMD patients in our center revealed three cases without a known genetic cause. DNA samples of these patients were analyzed using whole-exome sequencing (WES) and, if unconclusive, optical genome mapping (OGM). WES led to a diagnosis in two cases: one patient was found to carry a splice mutation in the DMD gene that had not been identified during previous Sanger sequencing. In the second patient, we detected two variants in the fukutin gene (FKTN) that were presumed to be disease-causing. In the third patient, WES was unremarkable, but OGM identified an inversion disrupting the DMD gene (~1.28 Mb) that was subsequently confirmed with long-read sequencing. These results highlight the importance of reanalyzing unsolved cases using WES and demonstrate that OGM is a useful method for identifying large structural variants in cases with unremarkable exome sequencing.

Keywords:  DMD; Duchenne muscular dystrophy; FKTN; OGM; dystrophin; fukutin; inversion; long-read sequencing; optical genome mapping; whole-exome sequencing

DOI:  https://doi.org/10.3390/ijms241914716
J Muscle Res Cell Motil. 2023 Oct 08.

From amino-acid to disease: the effects of oxidation on actin-myosin interactions in muscle.

Daren Elkrief, Oleg Matusovsky, Yu-Shu Cheng, Dilson E Rassier.

  Actin-myosin interactions form the basis of the force-producing contraction cycle within the sarcomere, serving as the primary mechanism for muscle contraction. Post-translational modifications, such as oxidation, have a considerable impact on the mechanics of these interactions. Considering their widespread occurrence, the explicit contributions of these modifications to muscle function remain an active field of research. In this review, we aim to provide a comprehensive overview of the basic mechanics of the actin-myosin complex and elucidate the extent to which oxidation influences the contractile cycle and various mechanical characteristics of this complex at the single-molecule, myofibrillar and whole-muscle levels. We place particular focus on amino acids shown to be vulnerable to oxidation in actin, myosin, and some of their binding partners. Additionally, we highlight the differences between in vitro environments, where oxidation is controlled and limited to actin and myosin and myofibrillar or whole muscle environments, to foster a better understanding of oxidative modification in muscle. Thus, this review seeks to encompass a broad range of studies, aiming to lay out the multi layered effects of oxidation in in vitro and in vivo environments, with brief mention of clinical muscular disorders associated with oxidative stress.

Keywords:  Actin-myosin interaction; Force generation; Muscle aging and disease; Myofibrillar weakness; Oxidation; Skeletal and cardiac muscle

DOI:  https://doi.org/10.1007/s10974-023-09658-0
Nanomedicine. 2023 Oct 07. pii: S1549-9634(23)00062-X. [Epub ahead of print] 102711

Delivery of gene editing therapeutics.

Bhavesh D Kevadiya, Farhana Islam, Pallavi Deol, Lubaba A Zaman, Dina A Mosselhy, Md Ashaduzzaman, Neha Bajwa, Nanda Kishore Routhu, Preet Amol Singh, Shilpa Dawre, Lalitkumar K Vora, Sumaiya Nahid, Deepali Mathur, Mohammad Ullah Nayan, Ashish Baldi, Ramesh Kothari, Tapan A Patel, Jitender Madan, Zahra Gounani, Jitender Bariwal, Kenneth S Hettie, Howard E Gendelman.

  For the past decades, gene editing demonstrated the potential of attenuating each of the root causes of genetic, infectious, immune, cancerous, and degenerative disorders. More recently, Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 (CRISPR-Cas9) editing proved effective for editing genomic, cancerous, or microbial DNA to limit disease onset or spread. However, the strategies to deliver CRISPR-Cas9 cargos and elicit protective immune responses requires safe delivery to disease targeted cells and tissues. While viral vector-based systems and viral particles demonstrate high efficiency and stable transgene expression, each are limited in their packaging capacities and secondary untoward immune responses. In contrast, the nonviral vector lipid nanoparticles were successfully used for as vaccine and therapeutic deliverables. Herein, we highlight each available gene delivery systems for the treatment and prevention of a broad range of infectious, inflammatory, genetic, and degenerative diseases. STATEMENT OF SIGNIFICANCE: CRISPR-Cas9 gene editing for disease treatment and prevention is an emerging field that can change the outcome of many chronic debilitating disorders.

Keywords:  CRISPR-Cas9; Gene editing; Lipid nanoparticles; Viral vector delivery

DOI:  https://doi.org/10.1016/j.nano.2023.102711
Int J Mol Sci. 2023 Sep 28. pii: 14701. [Epub ahead of print]24(19):

CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination.

Tahmina Tabassum, Giovanni Pietrogrande, Michael Healy, Ernst J Wolvetang.

  DNA repair in mammalian cells involves the coordinated action of a range of complex cellular repair machinery. Our understanding of these DNA repair processes has advanced to the extent that they can be leveraged to improve the efficacy and precision of Cas9-assisted genome editing tools. Here, we review how the fusion of CRISPR-Cas9 to functional domains of proteins that directly or indirectly impact the DNA repair process can enhance genome editing. Such studies have allowed the development of diverse technologies that promote efficient gene knock-in for safer genome engineering practices.

Keywords:  CRISPR-Cas9; DNA repair; gene editing; homologous recombination

DOI:  https://doi.org/10.3390/ijms241914701
Biochimie. 2023 Oct 10. pii: S0300-9084(23)00256-0. [Epub ahead of print]

Desmin and its molecular chaperone, the αB-crystallin: How post-translational modifications modulate their functions in heart and skeletal muscles?

Charlotte Claeyssen, Nathan Bulangalire, Bruno Bastide, Onnik Agbulut, Caroline Cieniewski-Bernard.

  Maintenance of the highly organized striated muscle tissue requires a cell-wide dynamic network through protein-protein interactions providing an effective mechanochemical integrator of morphology and function. Through a continuous and complex trans-cytoplasmic network, desmin intermediate filaments ensure this essential role in heart and in skeletal muscle. Besides their role in the maintenance of cell shape and architecture (permitting contractile activity efficiency and conferring resistance towards mechanical stress), desmin intermediate filaments are also key actors of cell and tissue homeostasis. Desmin participates to several cellular processes such as differentiation, apoptosis, intracellular signalisation, mechanotransduction, vesicle trafficking, organelle biogenesis and/or positioning, calcium homeostasis, protein homeostasis, cell adhesion, metabolism and gene expression. Desmin intermediate filaments assembly requires αB-crystallin, a small heat shock protein. Over its chaperone activity, αB-crystallin is involved in several cellular functions such as cell integrity, cytoskeleton stabilization, apoptosis, autophagy, differentiation, mitochondria function or aggresome formation. Importantly, both proteins are known to be strongly associated to the aetiology of several cardiac and skeletal muscles pathologies related to desmin filaments disorganization and a strong disturbance of desmin interactome. Note that these key proteins of cytoskeleton architecture are extensively modified by post-translational modifications that could affect their functional properties. Therefore, we reviewed in the herein paper the impact of post-translational modifications on the modulation of cellular functions of desmin and its molecular chaperone, the αB-crystallin.

Keywords:  Desmin; Intermediate filaments; O-GlcNAcylation; Phosphorylation; Striated muscles; αB-Crystallin

DOI:  https://doi.org/10.1016/j.biochi.2023.10.002
Cells. 2023 Oct 02. pii: 2395. [Epub ahead of print]12(19):

Enhancing Antisense Oligonucleotide-Based Therapeutic Delivery with DG9, a Versatile Cell-Penetrating Peptide.

Umme Sabrina Haque, Toshifumi Yokota.

  Antisense oligonucleotide-based (ASO) therapeutics have emerged as a promising strategy for the treatment of human disorders. Charge-neutral PMOs have promising biological and pharmacological properties for antisense applications. Despite their great potential, the efficient delivery of these therapeutic agents to target cells remains a major obstacle to their widespread use. Cellular uptake of naked PMO is poor. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular uptake and intracellular delivery of oligonucleotide-based drugs. Among these, the DG9 peptide has been identified as a versatile CPP with remarkable potential for enhancing the delivery of ASO-based therapeutics due to its unique structural features. Notably, in the context of phosphorodiamidate morpholino oligomers (PMOs), DG9 has shown promise in enhancing delivery while maintaining a favorable toxicity profile. A few studies have highlighted the potential of DG9-conjugated PMOs in DMD (Duchenne Muscular Dystrophy) and SMA (Spinal Muscular Atrophy), displaying significant exon skipping/inclusion and functional improvements in animal models. The article provides an overview of a detailed understanding of the challenges that ASOs face prior to reaching their targets and continued advances in methods to improve their delivery to target sites and cellular uptake, focusing on DG9, which aims to harness ASOs' full potential in precision medicine.

Keywords:  DG9 peptide; antisense oligonucleotides; cell penetrating peptides; delivery; phosphorodiamidate morpholino oligomers (PMO)

DOI:  https://doi.org/10.3390/cells12192395
Hum Mol Genet. 2023 Oct 11. pii: ddad173. [Epub ahead of print]

Comparison of pharmaceutical properties and biological activity of prednisolone, deflazacort, and vamorolone in DMD disease models.

Grace Liu, Philip Lipari, Anna Mollin, Stephen Jung, Irina Teplova, Wencheng Li, Lanqing Ying, Vijay More, William Lennox, Shirley Yeh, Eric McGann, Young-Choon Moon, Cari Rice, Eduardo Huarte, Barbara Gruszka, Balmiki Ray, Elizabeth Goodwin, Patricia Buckendahl, Edward Yurkow, Bruce Braughton, Jana Narasimhan, Ellen Welch, Gregory Voronin, Marla Weetall.

  Duchenne muscular dystrophy (DMD) is a progressive disabling X-linked recessive disorder that causes gradual and irreversible loss of muscle, resulting in early death. The corticosteroids prednisone/prednisolone and deflazacort are used to treat DMD as the standard of care; however, only deflazacort is FDA approved for DMD. The novel atypical corticosteroid vamorolone is being investigated for treatment of DMD. We compared the pharmaceutical properties as well as the efficacy and safety of the three corticosteroids across multiple doses in the B10-mdx DMD mouse model. Pharmacokinetic studies in the mouse and evaluation of p-glycoprotein (P-gP) efflux in a cellular system demonstrated that vamorolone is not a strong P-gp substrate resulting in measurable central nervous system (CNS) exposure in the mouse. In contrast, deflazacort and prednisolone are strong P-gp substrates. All three corticosteroids showed efficacy, but also side effects at efficacious doses. After dosing mdx mice for two weeks, all three corticosteroids induced changes in gene expression in the liver and the muscle, but prednisolone and vamorolone induced more changes in the brain than did deflazacort. Both prednisolone and vamorolone induced depression-like behavior. All three corticosteroids reduced endogenous corticosterone levels, increased glucose levels, and reduced osteocalcin levels. Using micro-computed tomography, femur bone density was decreased, reaching significance with prednisolone. The results of these studies indicate that efficacious doses of vamorolone, are associated with similar side effects as seen with other corticosteroids. Further, because vamorolone is not a strong P-gp substrate, vamorolone distributes into the CNS increasing the potential CNS side-effects.

Keywords:  PK-PD; corticosteroids; deflazacort; duchenne muscular dystrophy; prednisolone

DOI:  https://doi.org/10.1093/hmg/ddad173
bioRxiv. 2023 Oct 09. pii: 2023.09.26.558914. [Epub ahead of print]

Integrated single-cell multiome analysis reveals muscle fiber-type gene regulatory circuitry modulated by endurance exercise.

Aliza B Rubenstein, Gregory R Smith, Zidong Zhang, Xi Chen, Toby L Chambers, Frederique Ruf-Zamojski, Natalia Mendelev, Wan Sze Cheng, Michel Zamojski, Mary Anne S Amper, Venugopalan D Nair, Andrew R Marderstein, Stephen B Montgomery, Olga G Troyanskaya, Elena Zaslavsky, Todd Trappe, Scott Trappe, Stuart C Sealfon.

Endurance exercise is an important health modifier. We studied cell-type specific adaptations of human skeletal muscle to acute endurance exercise using single-nucleus (sn) multiome sequencing in human vastus lateralis samples collected before and 3.5 hours after 40 min exercise at 70% VO2max in four subjects, as well as in matched time of day samples from two supine resting circadian controls. High quality same-cell RNA-seq and ATAC-seq data were obtained from 37,154 nuclei comprising 14 cell types. Among muscle fiber types, both shared and fiber-type specific regulatory programs were identified. Single-cell circuit analysis identified distinct adaptations in fast, slow and intermediate fibers as well as LUM-expressing FAP cells, involving a total of 328 transcription factors (TFs) acting at altered accessibility sites regulating 2,025 genes. These data and circuit mapping provide single-cell insight into the processes underlying tissue and metabolic remodeling responses to exercise.

DOI: https://doi.org/10.1101/2023.09.26.558914