bims-muscge Biomed News
on Muscle stem cells and gene therapy
Issue of 2023‒12‒10
eleven papers selected by
Chance Bowman, Dartmouth College

  1. Skelet Muscle. 2023 Dec 04. 13(1): 20
      Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in DMD gene and loss of the protein dystrophin, which ultimately leads to myofiber membrane fragility and necrosis, with eventual muscle atrophy and contractures. Affected boys typically die in their second or third decade due to either respiratory failure or cardiomyopathy. Among the developed therapeutic strategies for DMD, gene therapy approaches partially restore micro-dystrophin or quasi-dystrophin expression. However, despite extensive attempts to develop definitive therapies for DMD, the standard of care remains corticosteroid, which has only palliative benefits. Animal models have played a key role in studies of DMD pathogenesis and treatment development. The golden retriever muscular dystrophy (GRMD) dog displays a phenotype aligning with the progressive course of DMD. Therefore, canine studies may translate better to humans. Recent studies suggested that nicotinamide adenine dinucleotide (NAD+) cellular content could be a critical determinant for striated muscle function. We showed here that NAD+ content was decreased in the striated muscles of GRMD, leading to an alteration of one of NAD+ co-substrate enzymes, PARP-1. Moreover, we showed that boosting NAD+ content using nicotinamide (NAM), a natural NAD+ precursor, modestly reduces aspects of striated muscle disease. Collectively, our results provide mechanistic insights into DMD.
    Keywords:  Duchenne muscular dystrophy; GRMD; NAD+; Nicotinamide
  2. Biochem J. 2023 Dec 13. 480(23): 1969-1986
      Lysine methylation signaling is well studied for its key roles in the regulation of transcription states through modifications on histone proteins. While histone lysine methylation has been extensively studied, recent discoveries of lysine methylation on thousands of non-histone proteins has broadened our appreciation for this small chemical modification in the regulation of protein function. In this review, we highlight the significance of histone and non-histone lysine methylation signaling in skeletal muscle biology, spanning development, maintenance, regeneration, and disease progression. Furthermore, we discuss potential future implications for its roles in skeletal muscle biology as well as clinical applications for the treatment of skeletal muscle-related diseases.
    Keywords:  epigenetics; lysine methylation; myogenesis
  3. bioRxiv. 2023 Nov 21. pii: 2023.11.21.568117. [Epub ahead of print]
      The conserved Runt-related (RUNX) transcription factor family are well-known master regulators of developmental and regenerative processes. Runx1 and Runx2 are both expressed in satellite cells (SC) and skeletal myotubes. Conditional deletion of Runx1 in adult SC negatively impacted self-renewal and impaired skeletal muscle maintenance. Runx1- deficient SC retain Runx2 expression but cannot support muscle regeneration in response to injury. To determine the unique molecular functions of Runx1 that cannot be compensated by Runx2 we deleted Runx1 in C2C12 that retain Runx2 expression and established that myoblasts differentiation was blocked in vitro due in part to ectopic expression of Mef2c, a target repressed by Runx1 . Structure-function analysis demonstrated that the Ets-interacting MID/EID region of Runx1, absent from Runx2, is critical to regulating myoblasts proliferation, differentiation, and fusion. Analysis of in-house and published ChIP-seq datasets from Runx1 (T-cells, muscle) versus Runx2 (preosteoblasts) dependent tissue identified enrichment for a Ets:Runx composite site in Runx1 -dependent tissues. Comparing ATACseq datasets from WT and Runx1KO C2C12 cells showed that the Ets:Runx composite motif was enriched in peaks open exclusively in WT cells compared to peaks unique to Runx1KO cells. Thus, engagement of a set of targets by the RUNX1/ETS complex define the non-redundant functions of Runx1 .
  4. Dev Cell. 2023 Nov 30. pii: S1534-5807(23)00585-3. [Epub ahead of print]
      Skeletal muscle repair relies on heterogeneous populations of satellite cells (SCs). The mechanisms that regulate SC homeostasis and state transition during activation are currently unknown. Here, we investigated the emerging role of non-genetic micro-heterogeneity, i.e., intrinsic cell-to-cell variability of a population, in this process. We demonstrate that micro-heterogeneity of the membrane protein CRIPTO in mouse-activated SCs (ASCs) identifies metastable cell states that allow a rapid response of the population to environmental changes. Mechanistically, CRIPTO micro-heterogeneity is generated and maintained through a process of intracellular trafficking coupled with active shedding of CRIPTO from the plasma membrane. Irreversible perturbation of CRIPTO micro-heterogeneity affects the balance of proliferation, self-renewal, and myogenic commitment in ASCs, resulting in increased self-renewal in vivo. Our findings demonstrate that CRIPTO micro-heterogeneity regulates the adaptative response of ASCs to microenvironmental changes, providing insights into the role of intrinsic heterogeneity in preserving stem cell population diversity during tissue repair.
    Keywords:  CRIPTO; cell fate determination; cell state transition; membrane trafficking; micro-heterogeneity; non-genetic heterogeneity; phospholipase C; satellite cell population dynamics; self-renewal; skeletal muscle regeneration
  5. Arch Pediatr. 2023 Nov;pii: S0929-693X(23)00223-3. [Epub ahead of print]30(8S1): 8S18-8S23
      Gene therapy has emerged as a promising frontier in the pursuit of effective treatments for primary myopathies. This scientific review explores the application of viral vectors and more specifically of recombinant adeno-associated virus (rAAV) vectors as a potent gene delivery tool in the context of primary myopathies, highlighting its transformative potential. Focusing on primary myopathies, including Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophies (LGMDs), X-linked myotubular myopathy (XLMTM), and Pompe disease, we review the ongoing pre-clinical and clinical trials that underscore the therapeutic promise of rAAV-based gene therapies. Recent developments in gene therapy have unveiled innovative gene transfer approaches, particularly with rAAV vectors. These vectors offer a well-tolerated and efficient means of delivering corrective genetic material to diseased muscles, thereby addressing the root causes of primary myopathies. Encouraging data from pre-clinical studies and early clinical trials have demonstrated the potential to ameliorate muscle function, reduce pathological manifestations, and enhance the quality of life for patients afflicted with these devastating diseases. However, the transition from bench to bedside is not without challenges. This review emphasizes the critical need for a comprehensive risk management strategy to better handle potential side effects and immune responses associated with gene therapy. As the field of gene therapy for primary myopathies is advancing, it is imperative to refine and optimize safety measures, ensuring that the transformative potential of these therapies is realized while the risks are minimized. © 2023 Published by Elsevier Masson SAS on behalf of French Society of Pediatrics.
    Keywords:  Gene therapy; Gene transfer; Innovative treatments; Primary myopathies; rAAV
  6. STAR Protoc. 2023 Dec 01. pii: S2666-1667(23)00717-7. [Epub ahead of print]4(4): 102750
      Studying skeletal muscle stem cells (MuSCs) quiescence is challenging as they quickly activate within hours of isolation from muscle. Here, we present a protocol to disassociate and characterize fixed peptides from quiescent MuSCs using trapped ion-mobility time-of-flight mass spectrometry (MS). We describe steps for mouse perfusion, fluorescence-activated cell sorting preparation and sorting, protein extraction, digestion, and liquid chromatography MS analysis. This protocol can be applied to other less-abundant somatic stem cell types using mouse lines with a reporter. For complete details on the use and execution of this protocol, please refer to Zeng et al. (2022, 2023).1,2.
    Keywords:  Mass Spectrometry; Proteomics; Stem Cells
  7. Nucleic Acids Res. 2023 Dec 04. pii: gkad1124. [Epub ahead of print]
      We examined YAP1/TAZ-TEAD signaling pathway activity at neuromuscular junctions (NMJs) of skeletal muscle fibers in adult mice. Our investigations revealed that muscle-specific knockouts of Yap1 or Taz, or both, demonstrate that these transcriptional coactivators regulate synaptic gene expression, the number and morphology of NMJs, and synaptic nuclei. Yap1 or Taz single knockout mice display reduced grip strength, fragmentation of NMJs, and accumulation of synaptic nuclei. Yap1/Taz muscle-specific double knockout mice do not survive beyond birth and possess almost no NMJs, the few detectable show severely impaired morphology and are organized in widened endplate bands; and with motor nerve endings being mostly absent. Myogenic gene expression is significantly impaired in the denervated muscles of knockout mice. We found that Tead1 and Tead4 transcription rates were increased upon incubation of control primary myotubes with AGRN-conditioned medium. Reduced AGRN-dependent acetylcholine receptor clustering and synaptic gene transcription were observed in differentiated primary Tead1 and Tead4 knockout myotubes. In silico analysis of previously reported genomic occupancy sites of TEAD1/4 revealed evolutionary conserved regions of potential TEAD binding motifs in key synaptic genes, the relevance of which was functionally confirmed by reporter assays. Collectively, our data suggest a role for YAP1/TAZ-TEAD1/TEAD4 signaling, particularly through TAZ-TEAD4, in regulating synaptic gene expression and acetylcholine receptor clustering at NMJs.
  8. Mol Ther Methods Clin Dev. 2023 Dec 14. 31 101147
      The first generation of adeno-associated virus (AAV) vectors composed of the naturally occurring capsids and genomes, although effective in some instances, are unlikely to be optimal for gene therapy in humans. The use of the first generation of two different AAV serotype vectors (AAV9 and AAVrh74) in four separate clinical trials failed to be effective in patients with Duchenne muscular dystrophy, although some efficacy was observed in a subset of patients with AAVrh74 vectors leading to US Food and Drug Administration approval (Elevidys). In two trials with the first generation of AAV9 vectors, several serious adverse events were observed, including the death of a patient in one trial, and more recently, in the death of a second patient in an N-of-1 clinical trial. In a fourth trial with the first generation of AAVrh74 vectors, myositis and myocarditis were also observed. Here, we report that capsid- and genome-modified optimized AAVrh74 vectors are significantly more efficient in transducing primary human skeletal muscle cells in vitro and in all major muscle tissues in vivo following systemic administration in a murine model. The availability of optimized AAVrh74 vectors promises to be safe and effective in the potential gene therapy of muscle diseases in humans.
    Keywords:  AAV vectors; gene expression; gene therapy; gene transfer; muscle diseases, capsid-modifications, genome-modifications
  9. Front Physiol. 2023 ;14 1289537
      Skeletal muscles underpin myriad human activities, maintaining an intricate balance between protein synthesis and degradation crucial to muscle mass preservation. Historically, disruptions in this balance-where degradation overshadows synthesis-have marked the onset of muscle atrophy, a condition diminishing life quality and, in grave instances, imperiling life itself. While multiple protein degradation pathways exist-including the autophagy-lysosome, calcium-dependent calpain, and cysteine aspartate protease systems-the ubiquitin-proteasome pathway emerges as an especially cardinal avenue for intracellular protein degradation, wielding pronounced influence over the muscle atrophy trajectory. This paper ventures a panoramic view of predominant muscle atrophy types, accentuating the ubiquitin-proteasome pathway's role therein. Furthermore, by drawing from recent scholarly advancements, we draw associations between the ubiquitin-proteasome pathway and specific pathological conditions linked to muscle atrophy. Our exploration seeks to shed light on the ubiquitin-proteasome pathway's significance in skeletal muscle dynamics, aiming to pave the way for innovative therapeutic strategies against muscle atrophy and affiliated muscle disorders.
    Keywords:  cachexia; muscle disuse; sarcopenia; skeletal muscle atrophy; ubiquitin-proteasom
  10. Nat Biomed Eng. 2023 Dec 06.
      Spinal muscular atrophy (SMA) is caused by mutations in SMN1. SMN2 is a paralogous gene with a C•G-to-T•A transition in exon 7, which causes this exon to be skipped in most SMN2 transcripts, and results in low levels of the protein survival motor neuron (SMN). Here we show, in fibroblasts derived from patients with SMA and in a mouse model of SMA that, irrespective of the mutations in SMN1, adenosine base editors can be optimized to target the SMN2 exon-7 mutation or nearby regulatory elements to restore the normal expression of SMN. After optimizing and testing more than 100 guide RNAs and base editors, and leveraging Cas9 variants with high editing fidelity that are tolerant of different protospacer-adjacent motifs, we achieved the reversion of the exon-7 mutation via an A•T-to-G•C edit in up to 99% of fibroblasts, with concomitant increases in the levels of the SMN2 exon-7 transcript and of SMN. Targeting the SMN2 exon-7 mutation via base editing or other CRISPR-based methods may provide long-lasting outcomes to patients with SMA.
  11. Adv Physiol Educ. 2023 Dec 07.
      Given the recently proposed three-filament theory of muscle contraction, we present a low-cost physical sarcomere model aimed at illustrating the role of titin in the production of active force in skeletal muscle. Using inexpensive materials, it is possible to illustrate actin-myosin cross-bridge interactions between the thick and thin filaments and demonstrate the two different mechanisms by which titin is thought to contribute to active and passive muscle force. Specifically, the model illustrates how titin, a molecule with spring-like properties, may increase its stiffness by binding free calcium upon muscle activation and reducing its extensible length by attaching itself to actin, resulting in the greater force generating capacity after an active than a passive elongation that has been observed experimentally. The model is simple to build and manipulate, and demonstration to high school students was shown to result in positive perception and improved understanding of the otherwise complex titin related mechanisms of force production in skeletal and cardiac muscles.
    Keywords:  cross-bridge theory; learning tools; muscle contraction; muscle stretching; three-filament sarcomere model