bims-muscge 2023-12-03 papers

bims-muscge

Biomed News

on Muscle stem cells and gene therapy

Issue of 2023–12–03
nineteen papers selected by
Chance Bowman, Dartmouth College

Extracellular matrix: the critical contributor to skeletal muscle regeneration-a comprehensive review.
Soluble Factors Associated with Denervation-induced Skeletal Muscle Atrophy.
Challenges of Assessing Exon 53 Skipping of the Human DMD Transcript with Locked Nucleic Acid-Modified Antisense Oligonucleotides in a Mouse Model for Duchenne Muscular Dystrophy.
Persistence of exon 2 skipping and dystrophin expression at 18 months after U7snRNA-mediated therapy in the Dup2 mouse model.
Enhancement of mitochondrial energy metabolism by melatonin promotes vascularized skeletal muscle regeneration in a volumetric muscle loss model.
CRISPR/Cas9 Genome Editing in LGMD2A/R1 Patient-Derived Induced Pluripotent Stem and Skeletal Muscle Progenitor Cells.
Spontaneous Alignment of Myotubes through Myogenic Progenitor Cell Migration.
Muscle miRNAs are influenced by sex at baseline and in response to exercise.
Efficacy of exon-skipping therapy for DMD cardiomyopathy with mutations in actin binding domain 1.
Coordinated Regulation of Myonuclear DNA Methylation, mRNA, and miRNA Levels Associates With the Metabolic Response to Rapid Synergist Ablation-Induced Skeletal Muscle Hypertrophy in Female Mice.
Filamin A cooperates with the androgen receptor in preventing skeletal muscle senescence.
Differentiation-dependent chromosomal organization changes in normal myogenic cells are absent in rhabdomyosarcoma cells.
Detecting early signs in Duchenne muscular dystrophy: comprehensive review and diagnostic implications.
Engineering self-deliverable ribonucleoproteins for genome editing in the brain.
Class IIa HDACs inhibit cell death pathways and protect muscle integrity in response to lipotoxicity.
Designing RNA switches for synthetic biology using inverse-RNA-folding.
Profiling of adenine-derived signaling molecules, cytokinins, in myotubes reveals fluctuations in response to lipopolysaccharide-induced cell stress.
RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis.
Manipulating gene expression levels in mammalian cell factories: an outline of synthetic molecular toolboxes to achieve multiplexed control.

Inflamm Regen. 2023 Nov 27. 43(1): 58

Extracellular matrix: the critical contributor to skeletal muscle regeneration-a comprehensive review.

Khurshid Ahmad, Sibhghatulla Shaikh, Hee Jin Chun, Shahid Ali, Jeong Ho Lim, Syed Sayeed Ahmad, Eun Ju Lee, Inho Choi.

  The regenerative ability of skeletal muscle (SM) in response to damage, injury, or disease is a highly intricate process that involves the coordinated activities of multiple cell types and biomolecular factors. Of these, extracellular matrix (ECM) is considered a fundamental component of SM regenerative ability. This review briefly discusses SM myogenesis and regeneration, the roles played by muscle satellite cells (MSCs), other cells, and ECM components, and the effects of their dysregulations on these processes. In addition, we review the various types of ECM scaffolds and biomaterials used for SM regeneration, their applications, recent advances in ECM scaffold research, and their impacts on tissue engineering and SM regeneration, especially in the context of severe muscle injury, which frequently results in substantial muscle loss and impaired regenerative capacity. This review was undertaken to provide a comprehensive overview of SM myogenesis and regeneration, the stem cells used for muscle regeneration, the significance of ECM in SM regeneration, and to enhance understanding of the essential role of the ECM scaffold during SM regeneration.

Keywords:  Biomaterials; ECM scaffold; Extracellular matrix; Muscle loss; Regeneration; Skeletal muscle

DOI:  https://doi.org/10.1186/s41232-023-00308-z
Curr Protein Pept Sci. 2023 Nov 24.

Soluble Factors Associated with Denervation-induced Skeletal Muscle Atrophy.

Marianny Portal, Claudio Cabello-Verrugio.

  Skeletal muscle tissue has the critical function of mechanical support protecting the body. In addition, its functions are strongly influenced by the balanced synthesis and degradation processes of structural and regulatory proteins. The inhibition of protein synthesis and/or the activation of catabolism generally determines a pathological state or condition called muscle atrophy, a reduction in muscle mass that results in partial or total loss of function. It has been established that many pathophysiological conditions can cause a decrease in muscle mass. Skeletal muscle innervation involves stable and functional neural interactions with muscles via neuromuscular junctions and is essential for maintaining normal muscle structure and function. Loss of motor innervation induces rapid skeletal muscle fiber degeneration with activation of atrophy-related signaling and subsequent disassembly of sarcomeres, altering normal muscle function. After denervation, an inflammation stage is characterized by the increased expression of pro-inflammatory cytokines that determine muscle atrophy. In this review, we highlighted the impact of some soluble factors on the development of muscle atrophy by denervation.

Keywords:  Muscular atrophy; cytokines; denervation; muscle fiber degeneration; pro-inflammatory cytokines.; protein synthesis; soluble factors

DOI:  https://doi.org/10.2174/0113892037189827231018092036
Nucleic Acid Ther. 2023 Dec;33(6): 348-360

Challenges of Assessing Exon 53 Skipping of the Human DMD Transcript with Locked Nucleic Acid-Modified Antisense Oligonucleotides in a Mouse Model for Duchenne Muscular Dystrophy.

Sarah Engelbeen, Daniel O'Reilly, Davy Van De Vijver, Ingrid Verhaart, Maaike van Putten, Vignesh Hariharan, Matthew Hassler, Anastasia Khvorova, Masad J Damha, Annemieke Aartsma-Rus.

  Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients to restore dystrophin expression by reframing the disrupted open reading frame of the DMD transcript. However, the treatment efficacy of the already conditionally approved AONs remains low. Aiming to optimize AON efficiency, we assessed exon 53 skipping of the DMD transcript with different chemically modified AONs, all with a phosphorothioate backbone: 2'-O-methyl (2'OMe), locked nucleic acid (LNA)-2'OMe, 2'-fluoro (FRNA), LNA-FRNA, αLNA-FRNA, and FANA-LNA-FRNA. Efficient exon 53 skipping was observed with the FRNA, LNA-FRNA, and LNA-2'OMe AONs in human control myoblast cultures. Weekly subcutaneous injections (50 mg/kg AON) for a duration of 6 weeks were well tolerated by hDMDdel52/mdx males. Treatment with the LNA-FRNA and LNA-2'OMe AONs resulted in pronounced exon 53 skip levels in skeletal muscles and heart up to 90%, but no dystrophin restoration was observed. This discrepancy was mainly ascribed to the strong binding nature of LNA modifications to RNA, thereby interfering with the amplification of the unskipped product resulting in artificial overamplification of the exon 53 skip product. Our study highlights that treatment effect on RNA and protein level should both be considered when assessing AON efficiency.

Keywords:  LNA; efficacy; exon skipping; locked nucleic acid; preclinical studies

DOI:  https://doi.org/10.1089/nat.2023.0038
Mol Ther Methods Clin Dev. 2023 Dec 14. 31 101144

Persistence of exon 2 skipping and dystrophin expression at 18 months after U7snRNA-mediated therapy in the Dup2 mouse model.

Liubov V Gushchina, Adrienne J Bradley, Tatyana A Vetter, Jacob W Lay, Natalie L Rohan, Emma C Frair, Nicolas Wein, Kevin M Flanigan.

  Duchenne muscular dystrophy (DMD) is a progressive X-linked disease caused by mutations in the DMD gene that prevent the expression of a functional dystrophin protein. Exon duplications represent 6%-11% of mutations, and duplications of exon 2 (Dup2) are the most common (∼11%) of duplication mutations. An exon-skipping strategy for Dup2 mutations presents a large therapeutic window. Skipping one exon copy results in full-length dystrophin expression, whereas skipping of both copies (Del2) activates an internal ribosomal entry site (IRES) in exon 5, inducing the expression of a highly functional truncated dystrophin isoform. We have previously confirmed the therapeutic efficacy of AAV9.U7snRNA-mediated skipping in the Dup2 mouse model and showed the absence of off-target splicing effects and lack of toxicity in mice and nonhuman primates. Here, we report long-term dystrophin expression data following the treatment of 3-month-old Dup2 mice with the scAAV9.U7.ACCA vector. Significant exon 2 skipping and robust dystrophin expression in the muscles and hearts of treated mice persist at 18 months after treatment, along with the partial rescue of muscle function. These data extend our previous findings and show that scAAV9.U7.ACCA provides long-term protection by restoring the disrupted dystrophin reading frame in the context of exon 2 duplications.

Keywords:  Becker muscular dystrophy; Duchenne muscular dystrophy; U7snRNA; dystrophin; exon skipping; gene therapy; scAAV9.U7.ACCA

DOI:  https://doi.org/10.1016/j.omtm.2023.101144
Free Radic Biol Med. 2023 Nov 25. pii: S0891-5849(23)01122-X. [Epub ahead of print]210 146-157

Enhancement of mitochondrial energy metabolism by melatonin promotes vascularized skeletal muscle regeneration in a volumetric muscle loss model.

Xiaoyang Ge, Chengyue Wang, Guanyu Yang, Dimulati Maimaiti, Mingzhuang Hou, Hao Liu, Huilin Yang, Xi Chen, Yong Xu, Fan He.

  Volumetric muscle loss (VML) is a condition that results in the extensive loss of 20 % or more of skeletal muscle due to trauma or tumor ablation, leading to severe functional impairment and permanent disability. The current surgical interventions have limited functional regeneration of skeletal muscle due to the compromised self-repair mechanism. Melatonin has been reported to protect skeletal muscle from exercise-induced oxidative damage and holds great potential to treat muscle diseases. In this study, we hypothesize that melatonin can enhance myoblast differentiation and promote effective recovery of skeletal muscle following VML. In vitro administration of melatonin resulted in a significant enhancement of myogenesis in C2C12 myoblast cells, as evidenced by the up-regulation of myogenic marker genes in a dose-dependent manner. Further experiments revealed that silent information of regulator type 3 (SIRT3) played a critical role in the melatonin-enhanced myoblast differentiation through enhancement of mitochondrial energy metabolism and activation of mitochondrial antioxidant enzymes such as superoxide dismutase 2 (SOD2). Silencing of Sirt3 completely abrogated the protective effect of melatonin on the mitochondrial function of myoblasts, evidenced by the increased reactive oxygen species, decreased adenosine triphosphate production, and down-regulated myoblast-specific marker gene expression. In order to attain a protracted and consistent release, liposome-encapsuled melatonin was integrated into gelatin methacryloyl hydrogel (GelMA-Lipo@MT). The implantation of GelMA-Lipo@MT into a tibialis anterior muscle defect in a VML model effectively stimulated the formation of myofibers and new blood vessels in situ, while concurrently inhibiting fibrotic collagen deposition. The findings of this study indicate that the incorporation of melatonin with GelMA hydrogel has facilitated the de novo vascularized skeletal muscle regeneration by augmenting mitochondrial energy metabolism. This represents a promising approach for the development of skeletal muscle tissue engineering, which could be utilized for the treatment of VML and other severe muscle injuries.

Keywords:  C2C12; Melatonin; Mitochondrial energy metabolism; SIRT3; Skeletal muscle; Volumetric muscle loss

DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.11.021
Stem Cells Int. 2023 ;2023 9246825

CRISPR/Cas9 Genome Editing in LGMD2A/R1 Patient-Derived Induced Pluripotent Stem and Skeletal Muscle Progenitor Cells.

Lampros Mavrommatis, Abdul Zaben, Urs Kindler, Marie-Cécile Kienitz, Julienne Dietz, Hyun-Woo Jeong, Pierre Böhme, Beate Brand-Saberi, Matthias Vorgerd, Holm Zaehres.

Large numbers of Calpain 3 (CAPN3) mutations cause recessive forms of limb-girdle muscular dystrophy (LGMD2A/LGMDR1) with selective atrophy of the proximal limb muscles. We have generated induced pluripotent stem cells (iPSC) from a patient with two mutations in exon 3 and exon 4 at the calpain 3 locus (W130C, 550delA). Two different strategies to rescue these mutations are devised: (i) on the level of LGMD2A-iPSC, we combined CRISPR/Cas9 genome targeting with a FACS and Tet transactivator-based biallelic selection strategy, which resulted in a new functional chimeric exon 3-4 without the two CAPN3 mutations. (ii) On the level of LGMD2A-iPSC-derived CD82+/Pax7+ myogenic progenitor cells, we demonstrate CRISPR/Cas9 mediated rescue of the highly prevalent exon 4 CAPN3 mutation. The first strategy specifically provides isogenic LGMD2A corrected iPSC for disease modelling, and the second strategy can be further elaborated for potential translational approaches.

DOI: https://doi.org/10.1155/2023/9246825
Tissue Eng Part A. 2023 Nov 29.

Spontaneous Alignment of Myotubes through Myogenic Progenitor Cell Migration.

Lauren Mehanna, Adrianna R Osborne, Charlotte A Peterson, Brad J Berron.

In large volume muscle injuries, widespread damage to muscle fibers and the surrounding connective tissue prevents myogenic progenitor cells (MPCs) from initiating repair. There is a clinical need to rapidly fabricate large muscle tissue constructs for integration at the site of large volume muscle injuries. Most strategies for myotube alignment require microfabricated structures or prolonged orientation times. We utilize the MPC's natural propensity to close gaps across an injury site to guide alignment on collagen I. When MPCs are exposed to an open boundary free of cells, they migrate unidirectionally into the cell-free region and align perpendicular to the original boundary direction. We study the utility of this phenomenon with biotin - streptavidin adhesion to position the cells on the substrate, and then demonstrate the robustness of this strategy with unmodified cells, creating a promising tool for MPC patterning without interrupting their natural function. We pre-position MPCs in straight-line patterns separated with small gaps. This temporary positioning initiates the migratory nature of the MPCs to align and form myotubes across the gaps, similar to how they migrate and align with a single open boundary. There is a directional component to the MPC migration perpendicular (90°) to the original biotin-streptavidin surface patterns. The expression of myosin heavy chain, the motor protein of muscle thick filaments, is confirmed through immunocytochemistry (ICC) in myotubes generated from MPCs in our patterning process, acting as a marker of skeletal muscle differentiation. The rapid and highly specific binding of biotin-streptavidin allows for quick formation of temporary patterns, with MPC alignment based on natural regenerative behavior rather than complex fabrication techniques.

DOI: https://doi.org/10.1089/ten.TEA.2023.0177
BMC Biol. 2023 Nov 27. 21(1): 273

Muscle miRNAs are influenced by sex at baseline and in response to exercise.

Danielle Hiam, Shanie Landen, Macsue Jacques, Sarah Voisin, Séverine Lamon, Nir Eynon.

   BACKGROUND: Sex differences in microRNA (miRNA) expression profiles have been found across multiple tissues. Skeletal muscle is one of the most sex-biased tissues of the body. MiRNAs are necessary for development and have regulatory roles in determining skeletal muscle phenotype and have important roles in the response to exercise in muscle. Yet there is limited research into the role and regulation of miRNAs in the skeletal muscle at baseline and in response to exercise, a well-known modulator of miRNA expression. The aim of this study was to investigate the effect of sex on miRNA expression in the skeletal muscle at baseline and after an acute bout of high-intensity interval exercise. A total of 758 miRNAs were measured using Taqman®miRNA arrays in the skeletal muscle of 42 healthy participants from the Gene SMART study (23 males and 19 females of comparable fitness levels and aged 18-45 years), of which 308 were detected. MiRNAs that differed by sex at baseline and whose change in expression following high-intensity interval exercise differed between the sexes were identified using mixed linear models adjusted for BMI and Wpeak. We performed in silico analyses to identify the putative gene targets of the exercise-induced, sex-specific miRNAs and overrepresentation analyses to identify enriched biological pathways. We performed functional assays by overexpressing two sex-biased miRNAs in human primary muscle cells derived from male and female donors to understand their downstream effects on the transcriptome.
RESULTS: At baseline, 148 miRNAs were differentially expressed in the skeletal muscle between the sexes. Interaction analysis identified 111 miRNAs whose response to an acute bout of high-intensity interval exercise differed between the sexes. Sex-biased miRNA gene targets were enriched for muscle-related processes including proliferation and differentiation of muscle cells and numerous metabolic pathways, suggesting that miRNAs participate in programming sex differences in skeletal muscle function. Overexpression of sex-biased miRNA-30a and miRNA-30c resulted in profound changes in gene expression profiles that were specific to the sex of the cell donor in human primary skeletal muscle cells.
CONCLUSIONS: We uncovered sex differences in the expression levels of muscle miRNAs at baseline and in response to acute high-intensity interval exercise. These miRNAs target regulatory pathways essential to skeletal muscle development and metabolism. Our findings highlight that miRNAs play an important role in programming sex differences in the skeletal muscle phenotype.

Keywords:  Sex differences; Skeletal muscle; Transcriptome; miRNA

DOI:  https://doi.org/10.1186/s12915-023-01755-3
Mol Ther Nucleic Acids. 2023 Dec 12. 34 102060

Efficacy of exon-skipping therapy for DMD cardiomyopathy with mutations in actin binding domain 1.

Naoko Shiba, Xiao Yang, Mitsuto Sato, Shin Kadota, Yota Suzuki, Masahiro Agata, Kohei Nagamine, Masaki Izumi, Yusuke Honda, Tomoya Koganehira, Hideki Kobayashi, Hajime Ichimura, Shinichiro Chuma, Junichi Nakai, Shugo Tohyama, Keiichi Fukuda, Daigo Miyazaki, Akinori Nakamura, Yuji Shiba.

  Exon-skipping therapy is a promising treatment strategy for Duchenne muscular dystrophy (DMD), which is caused by loss-of-function mutations in the DMD gene encoding dystrophin, leading to progressive cardiomyopathy. In-frame deletion of exons 3-9 (Δ3-9), manifesting a very mild clinical phenotype, is a potential targeted reading frame for exon-skipping by targeting actin-binding domain 1 (ABD1); however, the efficacy of this approach for DMD cardiomyopathy remains uncertain. In this study, we compared three isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing Δ3-9, frameshifting Δ3-7, or intact DMD. RNA sequencing revealed a resemblance in the expression patterns of mechano-transduction-related genes between Δ3-9 and wild-type samples. Furthermore, we observed similar electrophysiological properties between Δ3-9 and wild-type hiPSC-CMs; Δ3-7 hiPSC-CMs showed electrophysiological alterations with accelerated CaMKII activation. Consistently, Δ3-9 hiPSC-CMs expressed substantial internally truncated dystrophin protein, resulting in maintaining F-actin binding and desmin retention. Antisense oligonucleotides targeting exon 8 efficiently induced skipping exons 8-9 to restore functional dystrophin and electrophysiological parameters in Δ3-7 hiPSC-CMs, bringing the cell characteristics closer to those of Δ3-9 hiPSC-CMs. Collectively, exon-skipping targeting ABD1 to convert the reading frame to Δ3-9 may become a promising therapy for DMD cardiomyopathy.

Keywords:  CaMKII; Duchenne muscular dystrophy; MT: oligonucleotides: therapies and applications; actin-binding domain; antisense oligonucleotide-mediated exon skipping; cardiomyopathy; desmin; dystrophin; hiPSC-CMs

DOI:  https://doi.org/10.1016/j.omtn.2023.102060
Function (Oxf). 2024 ;5(1): zqad062

Coordinated Regulation of Myonuclear DNA Methylation, mRNA, and miRNA Levels Associates With the Metabolic Response to Rapid Synergist Ablation-Induced Skeletal Muscle Hypertrophy in Female Mice.

Ahmed Ismaeel, Nicholas T Thomas, Mariah McCashland, Ivan J Vechetti, Sebastian Edman, Johanna T Lanner, Vandré C Figueiredo, Christopher S Fry, John J McCarthy, Yuan Wen, Kevin A Murach, Ferdinand von Walden.

  The central dogma of molecular biology dictates the general flow of molecular information from DNA that leads to a functional cellular outcome. In skeletal muscle fibers, the extent to which global myonuclear transcriptional alterations, accounting for epigenetic and post-transcriptional influences, contribute to an adaptive stress response is not clearly defined. In this investigation, we leveraged an integrated analysis of the myonucleus-specific DNA methylome and transcriptome, as well as myonuclear small RNA profiling to molecularly define the early phase of skeletal muscle fiber hypertrophy. The analysis of myonucleus-specific mature microRNA and other small RNA species provides new directions for exploring muscle adaptation and complemented the methylation and transcriptional information. Our integrated multi-omics interrogation revealed a coordinated myonuclear molecular landscape during muscle loading that coincides with an acute and rapid reduction of oxidative metabolism. This response may favor a biosynthesis-oriented metabolic program that supports rapid hypertrophic growth.

Keywords:  RNA sequencing; RRBS; epigenetics; mitochondrial respiration; oxidative metabolism; small RNA sequencing

DOI:  https://doi.org/10.1093/function/zqad062
Cell Death Discov. 2023 Dec 02. 9(1): 437

Filamin A cooperates with the androgen receptor in preventing skeletal muscle senescence.

Marzia Di Donato, Antimo Moretti, Carmela Sorrentino, Giuseppe Toro, Giulia Gentile, Giovanni Iolascon, Gabriella Castoria, Antimo Migliaccio.

Aging induces a slow and progressive decrease in muscle mass and function, causing sarcopenia. Androgens control muscle trophism and exert important anabolic functions through the binding to the androgen receptor. Therefore, analysis of the androgen receptor-mediated actions in skeletal muscle might provide new hints for a better understanding of sarcopenia pathogenesis. In this study, we report that expression of the androgen receptor in skeletal muscle biopsies from 20 subjects is higher in young, as compared with old subjects. Co-immunoprecipitation experiments reveal that the androgen receptor is complexed with filamin A mainly in young, that in old subjects. Therefore, we have in depth analyzed the role of such complex using C2C12 myoblasts that express a significant amount of the androgen receptor. In these cells, hormone stimulation rapidly triggers the assembly of the androgen receptor/filamin A complex. Such complex prevents the senescence induced by oxidative stress in C2C12 cells, as disruption of the androgen receptor/filamin A complex by Rh-2025u stapled peptide re-establishes the senescent phenotype in C2C12 cells. Simultaneously, androgen stimulation of C2C12 cells rapidly triggers the activation of various signaling effectors, including Rac1, focal adhesion kinase, and mitogen-activated kinases. Androgen receptor blockade by bicalutamide or perturbation of androgen receptor/filamin A complex by Rh-2025u stapled peptide both reverse the hormone activation of signaling effectors. These findings further reinforce the role of the androgen receptor and its extranuclear partners in the rapid hormone signaling that controls the functions of C2C12 cells. Further investigations are needed to promote clinical interventions that might ameliorate muscle cell function as well the clinical outcome of age-related frailty.

DOI: https://doi.org/10.1038/s41420-023-01737-y
Front Cell Dev Biol. 2023 ;11 1293891

Differentiation-dependent chromosomal organization changes in normal myogenic cells are absent in rhabdomyosarcoma cells.

Joe Ibarra, Tyler Hershenhouse, Luay Almassalha, David Walterhouse, Vadim Backman, Kyle L MacQuarrie.

  Myogenesis, the progression of proliferating skeletal myoblasts to terminally differentiated myotubes, regulates thousands of target genes. Uninterrupted linear arrays of such genes are differentially associated with specific chromosomes, suggesting chromosome specific regulatory roles in myogenesis. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle, shares common features with normal muscle cells. We hypothesized that RMS and myogenic cells possess differences in chromosomal organization related to myogenic gene arrangement. We compared the organizational characteristics of chromosomes 2 and 18, chosen for their difference in myogenic gene arrangement, in cultured RMS cell lines and normal myoblasts and myotubes. We found chromosome-specific differences in organization during normal myogenesis, with increased area occupied and a shift in peripheral localization specifically for chromosome 2. Most strikingly, we found a differentiation-dependent difference in positioning of chromosome 2 relative to the nuclear axis, with preferential positioning along the major nuclear axis present only in myotubes. RMS cells demonstrated no preference for such axial positioning, but induced differentiation through transfection of the pro-myogenic miRNA miR-206 resulted in an increase of major axial positioning of chromosome 2. Our findings identify both a differentiation-dependent, chromosome-specific change in organization in normal myogenesis, and highlight the role of chromosomal spatial organization in myogenic differentiation.

Keywords:  chromosome; differentiation; myogenesis; nuclear organization; rhabdomyosarcoma

DOI:  https://doi.org/10.3389/fcell.2023.1293891
Front Pediatr. 2023 ;11 1276144

Detecting early signs in Duchenne muscular dystrophy: comprehensive review and diagnostic implications.

Eugenio Mercuri, Marika Pane, Gianpaolo Cicala, Claudia Brogna, Emma Ciafaloni.

  Despite the early onset of clinical signs suggestive of Duchenne muscular dystrophy (DMD), a diagnosis is often not made until four years of age or older, with a diagnostic delay of up to two years from the appearance of the first symptoms. As disease-modifying therapies for DMD become available that are ideally started early before irreversible muscle damage occurs, the importance of avoiding diagnostic delay increases. Shortening the time to a definite diagnosis in DMD allows timely genetic counseling and assessment of carrier status, initiation of multidisciplinary standard care, timely initiation of appropriate treatments, and precise genetic mutation characterization to assess suitability for access to drugs targeted at specific mutations while reducing the emotional and psychological family burden of the disease. This comprehensive literature review describes the early signs of impairment in DMD and highlights the bottlenecks related to the different diagnostic steps. In summary, the evidence suggests that the best mitigation strategy for improving the age at diagnosis is to increase awareness of the early symptoms of DMD and encourage early clinical screening with an inexpensive and sensitive serum creatine kinase test in all boys who present signs of developmental delay and specific motor test abnormality at routine pediatrician visits.

Keywords:  Duchenne muscular dystrophy; delayed diagnosis; developmental milestones; physical milestones; time to diagnosis

DOI:  https://doi.org/10.3389/fped.2023.1276144
bioRxiv. 2023 Nov 15. pii: 2023.11.15.567251. [Epub ahead of print]

Engineering self-deliverable ribonucleoproteins for genome editing in the brain.

Kai Chen, Elizabeth C Stahl, Min Hyung Kang, Bryant Xu, Ryan Allen, Marena Trinidad, Jennifer A Doudna.

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects 1 . However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineered self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identified potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins identified a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibited substantially improved editing efficacy compared to other constructs. We found that self-deliverable Cas9 RNPs generated robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo .

DOI: https://doi.org/10.1101/2023.11.15.567251
Cell Death Dis. 2023 Dec 01. 14(12): 787

Class IIa HDACs inhibit cell death pathways and protect muscle integrity in response to lipotoxicity.

Sheree D Martin, Timothy Connor, Andrew Sanigorski, Kevin A McEwen, Darren C Henstridge, Brunda Nijagal, David De Souza, Dedreia L Tull, Peter J Meikle, Greg M Kowalski, Clinton R Bruce, Paul Gregorevic, Mark A Febbraio, Fiona M Collier, Ken R Walder, Sean L McGee.

Lipotoxicity, the accumulation of lipids in non-adipose tissues, alters the metabolic transcriptome and mitochondrial metabolism in skeletal muscle. The mechanisms involved remain poorly understood. Here we show that lipotoxicity increased histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5), which reduced the expression of metabolic genes and oxidative metabolism in skeletal muscle, resulting in increased non-oxidative glucose metabolism. This metabolic reprogramming was also associated with impaired apoptosis and ferroptosis responses, and preserved muscle cell viability in response to lipotoxicity. Mechanistically, increased HDAC4 and 5 decreased acetylation of p53 at K120, a modification required for transcriptional activation of apoptosis. Redox drivers of ferroptosis derived from oxidative metabolism were also reduced. The relevance of this pathway was demonstrated by overexpression of loss-of-function HDAC4 and HDAC5 mutants in skeletal muscle of obese db/db mice, which enhanced oxidative metabolic capacity, increased apoptosis and ferroptosis and reduced muscle mass. This study identifies HDAC4 and HDAC5 as repressors of skeletal muscle oxidative metabolism, which is linked to inhibition of cell death pathways and preservation of muscle integrity in response to lipotoxicity.

DOI: https://doi.org/10.1038/s41419-023-06319-5
Trends Biotechnol. 2023 Nov 30. pii: S0167-7799(23)00327-X. [Epub ahead of print]

Designing RNA switches for synthetic biology using inverse-RNA-folding.

Sumit Mukherjee, Danny Barash.

  RNA switches respond to specific ligands to control gene expression. They are widely used in synthetic biology applications and hold potential for future RNA-based therapeutic breakthroughs. However, the crux is their precise design. Here, we will discuss how inverse-RNA-folding could be utilized for the accurate design of RNA switches.

Keywords:  RNA switch; RNA-based therapeutics; inverse-RNA-folding; riboswitch; ribozyme; synthetic biology

DOI:  https://doi.org/10.1016/j.tibtech.2023.11.005
Physiol Rep. 2023 Dec;11(23): e15870

Profiling of adenine-derived signaling molecules, cytokinins, in myotubes reveals fluctuations in response to lipopolysaccharide-induced cell stress.

Stephanie W Tobin, Dev Seneviratne, Lorna Phan, Mark Seegobin, Alexander L Rico, Beth Westby, Anna Kisiala, Sanela Martic, Craig R Brunetti, R J Neil Emery.

  Cytokinins (CTKs) are a diverse collection of evolutionarily conserved adenine-derived signaling molecules classically studied as phytohormones; however, their roles and production have been less studied in mammalian systems. Skeletal muscles are sensitive to cellular cues such as inflammation and in response, alter their secretome to regulate the muscle stem cell and myofiber niche. Using cultured C2C12 muscle cells, we profiled CTK levels to understand (1) whether CTKs are part of the muscle secretome and (2) whether CTKs are responsive to cellular stress. To induce cellular stress, C2C12 myotubes were treated with lipopolysaccharides (LPS) for 24 h and then media and cell fractions were collected for ultra high-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-(ESI+)-HRMS/MS) for metabolomics and CTK profiling. Across LPS-treated and control cells, 11 CTKs were detected in the extracellular space while 6 were detected intracellularly. We found that muscle cells are enriched in isopentenyladenine (iP) species (from free base, riboside to nucleotide forms), and that extracellular levels are increased after LPS treatment. Our study establishes that muscle cells express various forms of CTKs, and that CTK levels are responsive to LPS-induced cell stress, suggesting a role for CTKs in intra- and extracellular signaling of mammalian cells.

Keywords:  cytokinin; muscle; myokine

DOI:  https://doi.org/10.14814/phy2.15870
iScience. 2023 Nov 17. 26(11): 108258

RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis.

Hannah J Wiedner, R Eric Blue, Matheus Sadovsky, C Allie Mills, Xander H T Wehrens, Laura E Herring, Jimena Giudice.

  Alternative splicing is a prevalent gene-regulatory mechanism, with over 95% of multi-exon human genes estimated to be alternatively spliced. Here, we describe a tissue-specific, developmentally regulated, highly conserved, and disease-associated alternative splicing event in exon 7 of the eyes absent homolog 3 (Eya3) gene. We discovered that EYA3 expression is vital to the proliferation and differentiation of myoblasts. Genome-wide transcriptomic analysis and mass spectrometry-based proteomic studies identified SIX homeobox 4 (SIX4) and zinc finger and BTB-domain containing 1 (ZBTB1), as major transcription factors that interact with EYA3 to dictate gene expression. EYA3 isoforms differentially regulate transcription, indicating that splicing aids in temporal control of gene expression during muscle cell differentiation. Finally, we identified RNA-binding fox-1 homolog 2 (RBFOX2) as the main regulator of EYA3 splicing. Together, our findings illustrate the interplay between alternative splicing and transcription during myogenesis.

Keywords:  Molecular biology; Omics; Physiology

DOI:  https://doi.org/10.1016/j.isci.2023.108258
N Biotechnol. 2023 Nov 29. pii: S1871-6784(23)00066-3. [Epub ahead of print]

Manipulating gene expression levels in mammalian cell factories: an outline of synthetic molecular toolboxes to achieve multiplexed control.

Peter Eisenhut, Nicolas Marx, Giulia Borsi, Maja Papež, Caterina Ruggeri, Martina Baumann, Nicole Borth.

  Cells, both of prokaryotic and eukaryotic origin, have developed dedicated molecular mechanisms to tightly control expression levels of their genes where the specific transcriptomic signature across all genes eventually determines the cell phenotype. Modulating cellular phenotypes is of major interest, either to study their role in disease or to reprogram cells for the manufacture of recombinant products, such as biopharmaceuticals. For the latter, cells of mammalian origin, such as Chinese hamster ovary (CHO) and Human embryonic kidney 293 (HEK293) cells, are most commonly employed to produce therapeutic proteins. Altering their phenotype is often achieved randomly by subcloning and selection of appropriate behavior or by genetic engineering. In both cases, the objective is to obtain expression systems that generate the desired product with the highest possible quality and quantity. Early genetic engineering approaches have often been attempted by "uncontrolled" overexpression or knock-down/-out of specific genetic factors. Many studies in the past years, however, have highlighted that a controlled manipulation of transgene expression, by rationally regulating and fine-tuning the strength of overexpression or knock-down to an optimum level, can adjust phenotypic traits with much greater precision than such "uncontrolled" approaches. To control and (fine-)tune the expression level of one or multiple transgenes or endogenous genes, synthetic biology tools inspired by naturally occurring gene regulation mechanisms have been generated to develop novel, molecular toolboxes that enable (fine-)tunable and/or inducible control of gene expression. In this review, we discuss various molecular tools that have been established in mammalian cell lines and group them by their mode of action: transcriptional, post-transcriptional, translational and post-translational regulation. Major emphasis is placed on studies in which such tools were employed to engineer recombinant protein production in CHO or other mammalian cell factories. We discuss the advantages and disadvantages of using these tools for each cell regulatory layer and with respect to cell line engineering approaches. This review highlights the existence of a plethora of synthetic toolboxes that could be employed, alone or in combination, to optimize cellular systems and eventually gain enhanced control over the cellular phenotype to equip mammalian cell factories with the tools required for efficient production of emerging, more difficult-to-express biologics formats.

Keywords:  CHO cells; Cell line engineering; Gene expression; Synthetic Biology

DOI:  https://doi.org/10.1016/j.nbt.2023.11.003